Synthetic DNA sequence having enhanced insecticidal activity in maize

DNA sequences optimized for expression in plants are disclosed. The DNA sequences preferably encode for an insecticidal polypeptides, particularly insecticidal proteins from Bacillus thuringiensis. Plant promoters, particular tissue-specific and tissue-preferred promoters are also provided. Additionally disclosed are transformation vectors comprising said DNA sequences. The transformation vectors demonstrate high levels of insecticidal activity when transformed into maize.

FIELD OF THE INVENTION 
The present invention relates to DNA sequences encoding insecticidal 
proteins, and expression of these sequences in plants. 
BACKGROUND OF THE INVENTION 
Expression of the insecticidal protein (IP) genes derived from Bacillus 
thuringiensis (Bt) in plants has proven extremely difficult. Attempts have 
been made to express chimeric promoter/Bt IP gene combinations in plants. 
Typically, only low levels of protein have been obtained in transgenic 
plants. See, for example, Vaeck et al., Nature 328:33-37, 1987; Barton et 
al., Plant Physiol. 85:1103-1109, 1987; Fischoff et al., Bio/Technology 
5:807-813, 1987. 
One postulated explanation for the cause of low expression is that 
fortuitious transcription processing sites produce aberrant forms of Bt IP 
mRNA transcript. These aberrantly processed transcripts are non-functional 
in a plant, in terms of producing an insecticidal protein. Possible 
processing sites include polyadenylation sites, intron splicing sites, 
transcriptional termination signals and transport signals. Most genes do 
not contain sites that will deleteriously affect gene expression in that 
gene's normal host organism. However, the fortuitous occurrence of such 
processing sites in a coding region might complicate the expression of 
that gene in transgenic hosts. For example, the coding region for the Bt 
insecticidal crystal protein gene derived from Bacillus thuringiensis 
strain kurstaki (GENBANK BTHKURHD, accession M15271, B. thuringiensis var. 
kurstaki, HD-1; Geiser et al. Gene 48:109-118 (1986)) as derived directly 
from Bacillus thuringiensis, might contain sites which prevent this gene 
from being properly processed in plants. 
Further difficulties exist when attempting to express Bacillus 
thuringiensis protein in an organism such as a plant. It has been 
discovered that the codon usage of a native Bt IP gene is significantly 
different from that which is typical of a plant gene. In particular, the 
codon usage of a native Bt IP gene is very different from that of a maize 
gene. As a result, the mRNA from this gene may not be efficiently 
utilized. Codon usage might influence the expression of genes at the level 
of translation or transcription or mRNA processing. To optimize an 
insecticidal gene for expression in plants, attempts have been made to 
alter the gene to resemble, as much as possible, genes naturally contained 
within the host plant to be transformed. 
Adang et al., EP 0359472 (1990), relates to a synthetic Bacillus 
thuringiensis tenebrionis (Btt) gene which is 85% homologous to the native 
Btt gene and which is designed to have an A+T content approximating that 
found in plants in general. Table 1 of Adang et al. show the codon 
sequence of a synthetic Btt gene which was made to resemble more closely 
the normal codon distribution of dicot genes. Adang et al. state that a 
synthetic gene coding for IP can be optimized for enhanced expression in 
monocot plants through similar methods, presenting the frequency of codon 
usage of highly expressed monocot proteins in Table 1. At page 9, Adang et 
al. state that the synthetic Btt gene is designed to have an A+T content 
of 55% (and, by implication, a G+C content of 45%). At page 20, Adang et 
al. disclose that the synthetic gene is designed by altering individual 
amino acid codons in the native Bt gene to reflect the overall 
distribution of codons preferred by dicot genes for each amino acid within 
the coding region of the gene. Adang et al. further state that only some 
of the native Btt gene codons will be replaced by the most preferred plant 
codon for each amino acid, such that the overall distribution of codons 
used in dicot proteins is preserved. 
Fischhoff et al., EP 0 385 962 (1990), relates to plant genes encoding the 
crystal protein toxin of Bacillus thuringiensis. At table V, Fischhoff et 
al. disclose percent usages for codons for each amino acid. At page 8, 
Fischoff et al. suggest modifying a native Bt gene by removal of putative 
polyadenylation signals and ATTTA sequences. Fischoff et al. further 
suggest scanning the native Bt gene sequence for regions with greater than 
four consecutive adenine or thymine nucleotides to identify putative plant 
polyadenylation signals. Fischoff et al. state that the nucleotide 
sequence should be altered if more than one putative polyadenylation 
signal is identified within ten nucleotides of each other. At page 9, 
Fischoff et al. state that efforts should be made to select codons to 
preferably adjust the G+C content to about 50%. 
Perlak et al., PNAS USA, 88:3324-3328 (1991), relates to modified coding 
sequences of the Bacillus thuringiensis cryIA(b) gene, similar to those 
shown in Fischoff et al. As shown in table 1 at page 3325, the partially 
modified cryIA(b) gene of Perlak et al. is approximately 96% homologous to 
the native cryIA(b) gene (1681 of 1743 nucleotides), with a G+C content of 
41%, number of plant polyadenylation signal sequences (PPSS) reduced from 
18 to 7 and number of ATTTA sequences reduced from 13 to 7. The fully 
modified cryIA(b) gene of Perlak et al. is disclosed to be fully synthetic 
(page 3325, column 1). This gene is approximately 79% homologous to the 
native cryIA(b) gene (1455 of 1845 nucleotides), with a G+C content of 
49%, number of plant polyadenylation signal sequences (PPSS) reduced to 1 
and all ATTTA sequences removed. 
Barton et al., EP 0431 829 (1991), relates to the expression of 
insecticidal toxins in plants. At column 10, Barton et al. describe the 
construction of a synthetic AaIT insect toxin gene encoding a scorpion 
toxin using the most preferred codon for each amino acid according to the 
chart shown in FIG. 1 of the document. 
SUMMARY OF THE INVENTION 
The present invention is drawn to methods for enhancing expression of 
heterologous genes in plant cells. Generally, a gene or coding region of 
interest is constructed to provide a plant specific preferred codon 
sequence. In this manner, codon usage for a particular protein is altered 
to increase expression in a particular plant. Such plant optimized coding 
sequences can be operably linked to promoters capable of directing 
expression of the coding seqence in a plant cell. 
Specifically, it is one of the objects of the present invention to provide 
synthetic insecticidal protein genes which have been optimized for 
expression in plants. 
It is another object of the present invention to provide synthetic Bt 
insecticidal protein genes to maximize the expression of Bt proteins in a 
plant, preferably in a maize plant. It is one feature of the present 
invention that a synthetic Bt IP gene is constructed using the most 
preferred maize codons, except for alterations necessary to provide 
ligation sites for construction of the full synthetic gene. 
According to the above objects, we have synthesized Bt insecticidal crystal 
protein genes in which the codon usage has been altered in order to 
increase expression in plants, particularly maize. However, rather than 
alter the codon usage to resemble a maize gene in terms of overall codon 
distribution, we have optimized the codon usage by using the codons which 
are most preferred in maize (maize preferred codons) in the synthesis of 
the synthetic gene. The optimized maize preferred codon usage is effective 
for expression of high levels of the Bt insecticidal protein. This might 
be the result of maximizing the amount of Bt insecticidal protein 
translated from a given population of messenger RNAs. The synthesis of a 
Bt IP gene using maize preferred codons also tends to eliminate fortuitous 
processing sites that might occur in the native coding sequence. The 
expression of this synthetic gene is significantly higher in maize cells 
than that of the native IP Bt gene. 
Preferred synthetic, maize optimized DNA sequences of the present invention 
derive from the protein encoded by the cryIA(b) gene in Bacillus 
thuringiensis var. kurstaki, HD-1; Geiser et al., Gene, 48:109-118 (1986) 
or the cryIB gene (AKA Crya4 gene) described by Brizzard and Whiteley, 
Nuc. Acids. Res., 16:2723 (1988). The DNA sequence of the native kurstaki 
HD-1 cryIA(b) gene is shown as SEQ ID NO:1. These proteins are active 
against various lepidopteran insects, including Ostrinia nubilalis, the 
European Corn Borer. 
While the present invention has been exemplified by the synthesis of maize 
optimized Bt protein genes, it is recognized that the method can be 
utilized to optimize expression of any protein in plants. 
The instant optimized genes can be fused with a variety of promoters, 
including constitutive, inducible, temporally regulated, developmentally 
regulated, tissue-preferred and tissue-specific promoters to prepare 
recombinant DNA molecules, i.e., chimeric genes. The maize optimized gene 
(coding sequence) provides substantially higher levels of expression in a 
transformed plant, when compared with a non-maize optimized gene. 
Accordingly, plants resistant to Coleopteran or Lepidopteran pests, such 
as European corn borer and sugarcane borer, can be produced. 
It is another object of the present invention to provide tissue-preferred 
and tissue-specific promoters which drive the expression of an operatively 
associated structural gene of interest in a specific part or parts of a 
plant to the substantial exclusion of other parts. 
It is another object of the present invention to provide pith-preferred 
promoters. By "pith-preferred," it is intended that the promoter is 
capable of directing the expression of an operatively associated 
structural gene in greater abundance in the pith of a plant than in the 
roots, outer sheath, and brace roots, and with substantially no expression 
in seed. 
It is yet another object of this invention to provide pollen-specific 
promoters. By "pollen-specific," it is intended that the promoter is 
capable of directing the expression of an operatively associated 
structural gene of interest substantially exclusively in the pollen of a 
plant, with negligible expression in any other plant part. By 
"negligible," it is meant functionally insignificant. 
It is yet another object of the present invention to provide recombinant 
DNA molecules comprising a tissue-preferred promoter or tissue-specific 
promoter operably associated or linked to a structural gene of interest, 
particularly a structural gene encoding an insecticidal protein, and 
expression of the recombinant molecule in a plant. 
It is a further object of the present invention to provide transgenic 
plants which express at least one structural gene of interest operatively 
in a tissue-preferred or tissue-specific expression pattern. 
In one specific embodiment of the invention disclosed and claimed herein, 
the tissue-preferred or tissue-specific promoter is operably linked to a 
structural gene encoding an insecticidal protein, and a plant is stably 
transformed with at least one such recombinant molecule. The resultant 
plant will be resistant to particular insects which feed on those parts of 
the plant in which the gene(s) is(are) expressed. Preferred structural 
genes encode B.t. insecticidal proteins. More preferred are maize 
optimized B.t. IP genes.

DETAILED DESCRIPTION OF THE INVENTION 
The following definitions are provided in order to provide clarity with 
respect to the terms as they are used in the specification and claims to 
describe the present invention. 
Maize preferred codon: Preferred codon refers to the preference exhibited 
by a specific host cell in the usage of nucleotide codons to specify a 
given amino acid. The preferred codon for an amino acid for a particular 
host is the single codon which most frequently encodes that amino acid in 
that host. The maize preferred codon for a particular amino acid may be 
derived from known gene sequences from maize. For example, maize codon 
usage for 28 genes from maize plants are listed in Table 4 of Murray et 
al., Nucleic Acids Research, 17:477-498 (1989), the disclosure of which is 
incorporated herein by reference. For instance, the maize preferred codon 
for alanine is GCC, since, according to pooled sequences of 26 maize genes 
in Murray et al., supra, that codon encodes alanine 36% of the time, 
compared to GCG (24%), GCA (13%), and GCT (27%). 
Pure maize optimized sequence: An optimized gene or DNA sequence refers to 
a gene in which the nucleotide sequence of a native gene has been modified 
in order to utilize preferred codons for maize. For example, a synthetic 
maize optimized Bt cryIA(b) gene is one wherein the nucleotide sequence of 
the native Bt cryIA(b) gene has been modified such that the codons used 
are the maize preferred codons, as described above. A pure maize optimized 
gene is one in which the nucleotide sequence comprises 100 percent of the 
maize preferred codon sequences for a particular polypeptide. For example, 
the pure maize optimized Bt cryIA(b) gene is one in which the nucleotide 
sequence comprises 100 percent maize preferred codon sequences and encodes 
a polypeptide with the same amino acid sequence as that produced by the 
native Bt cryIA(b) gene. The pure nucleotide sequence of the optimized 
gene may be varied to permit manipulation of the gene, such as by altering 
a nucleotide to create or eliminate restriction sites. The pure nucleotide 
sequence of the optimized gene may also be varied to eliminate potentially 
deleterious processing sites, such as potential polyadenylation sites or 
intron recognition sites. 
It is recognized that "partially maize optimized," sequences may also be 
utilized. By partially maize optimized, it is meant that the coding region 
of the gene is a chimeric (hybrid), being comprised of sequences derived 
from a native insecticidal gene and sequences which have been optimized 
for expression in maize. A partially optimized gene expresses the 
insecticidal protein at a level sufficient to control insect pests, and 
such expression is at a higher level than achieved using native sequences 
only. Partially maize optimized sequences include those which contain at 
least about 5% optimized sequences. 
Full-length Bt Genes: Refers to DNA sequences comprising the full 
nucleotide sequence necessary to encode the polypeptide produced by a 
native Bt gene. For example, the native Bt cryIA(b) gene is approximately 
3.5 Kb in length and encodes a polypeptide which is approximately 1150 
amino acids in length. A full-length synthetic cryIA(b) Bt gene would be 
at least approximately 3.5 Kb in length. 
Truncated Bt Genes: Refers to DNA sequences comprising less than the full 
nucleotide sequence necessary to encode the polypeptide produced by a 
native Bt gene, but which encodes the active toxin portion of the 
polypeptide. For example, a truncated synthetic Bt gene of approximately 
1.9 Kb encodes the active toxin portion of the polypeptide such that the 
protein product exhibits insecticidal activity. 
Tissue-preferred promoter: The term "tissue-preferred promoter" is used to 
indicate that a given regulatory DNA sequence will promote a higher level 
of transcription of an associated structural gene or DNA coding sequence, 
or of expression of the product of the associated gene as indicated by any 
conventional RNA or protein assay, or that a given DNA sequence will 
demonstrate some differential effect; i.e., that the transcription of the 
associated DNA sequences or the expression of a gene product is greater in 
some tissue than in all other tissues of the plant. 
"Tissue-specific promoter" is used to indicate that a given regulatory DNA 
sequence will promote transcription of an associated coding DNA sequence 
essentially entirely in one or more tissues of a plant, or in one type of 
tissue, e.g. green tissue, while essentially no transcription of that 
associated coding DNA sequence will occur in all other tissues or types of 
tissues of the plant. 
The present invention provides DNA sequences optimized for expression in 
plants, especially in maize plants. In a preferred embodiment of the 
present invention, the DNA sequences encode the production of an 
insecticidal toxin, preferably a polypeptide sharing substantially the 
amino acid sequence of an insecticidal crystal protein toxin normally 
produced by Bacillus thuringiensis. The synthetic gene may encode a 
truncated or full-length insecticidal protein. Especially preferred are 
synthetic DNA sequences which encode a polypeptide effective against 
insects of the order Lepidoptera and Coleoptera, and synthetic DNA 
sequences which encode a polypeptide having an amino acid sequence 
essentially the same as one of the crystal protein toxins of Bacillus 
thuringiensis variety kurstaki, HD-1. 
The present invention provides synthetic DNA sequences effective to yield 
high expression of active insecticidal proteins in plants, preferably 
maize protoplasts, plant cells and plants. The synthetic DNA sequences of 
the present invention have been modified to resemble a maize gene in terms 
of codon usage and G+C content. As a result of these modifications, the 
synthetic DNA sequences of the present invention do not contain the 
potential processing sites which are present in the native gene. The 
resulting synthetic DNA sequences (synthetic Bt IP coding sequences) and 
plant transformation vectors containing this synthetic DNA sequence 
(synthetic Bt IP genes) result in surprisingly increased expression of the 
synthetic Bt IP gene, compared to the native Bt IP gene, in terms of 
insecticidal protein production in plants, particularly maize. The high 
level of expression results in maize cells and plants that exhibit 
resistance to lepidopteran insects, preferably European Corn Borer and 
Diatrea saccharalis, the Sugarcane Borer. 
The synthetic DNA sequences of the present invention are designed to encode 
insecticidal proteins from Bacillus thuringiensis, but are optimized for 
expression in maize in terms of G+C content and codon usage. For example, 
the maize codon usage table described in Murray et al., supra, is used to 
reverse translate the amino acid sequence of the toxin produced by the 
Bacillus thuringiensis subsp. kurstaki HD-1 cryIA(b) gene, using only the 
most preferred maize codons. The reverse translated DNA sequence is 
referred to as the pure maize optimized sequence and is shown as SEQ ID 
NO:4. This sequence is subsequently modified to eliminate unwanted 
restriction endonuclease sites, and to create desired restriction 
endonuclease sites. These modifications are designed to facilitate cloning 
of the gene without appreciably altering the codon usage or the maize 
optimized sequence. During the cloning procedure, in order to facilitate 
cloning of the gene, other modifications are made in a region that appears 
especially susceptible to errors induced during cloning by the polymerase 
chain reaction (PCR). The final sequence of the maize optimized synthetic 
Bt IP gene is shown in Sequence 2. A comparision of the maize optimized 
synthetic Bt IP gene with the native kurstaki cryIA(b) Bt gene is shown in 
FIG. 1. 
In a preferred embodiment of the present invention, the protein produced by 
the synthetic DNA sequence is effective against insects of the order 
Lepidoptera or Coleoptera. In a more preferred embodiment, the polypeptide 
encoded by the synthetic DNA sequence consists essentially of the 
full-length or a truncated amino acid sequence of an insecticidal protein 
normally produced by Bacillus thuringiensis var. kurstaki HD-1. In a 
particular embodiment, the synthetic DNA sequence encodes a polypeptide 
consisting essentially of a truncated amino acid sequence of the Bt 
CryIA(b) protein. 
The insecticidal proteins of the invention are expressed in a plant in an 
amount sufficient to control insect pests, i.e. insect controlling 
amounts. It is recognized that the amount of expression of insecticidal 
protein in a plant necessary to control insects may vary depending upon 
species of plant, type of insect, environmental factors and the like. 
Generally, the insect population will be kept below the economic threshold 
which varies from plant to plant. For example, to control European corn 
borer in maize, the economic threshold is .5 eggmass/plant which 
translates to about 10 larvae/plant. 
The methods of the invention are useful for controlling a wide variety of 
insects including but not limited to rootworms, cutworms, armyworms, 
particularly fall and beet armyworms, wireworms, aphids, corn borers, 
particularly European corn borers, sugarcane borer, lesser corn stalk 
borer, Southwestern corn borer, etc. 
In a preferred embodiment of the present invention, the synthetic coding 
DNA sequence optimized for expression in maize comprises a G+C percentage 
greater than that of the native cryIA(b) gene. It is preferred that the 
G+C percentage be at least about 50 percent, and more preferably at least 
about 60 percent. It is especially preferred that the G+C percent be about 
64 percent. 
In another preferred embodiment of the present invention, the synthetic 
coding DNA sequence optimized for expression in maize comprises a 
nucleotide sequence having at least about 90 percent homology with the 
"pure" maize optimized nucleotide sequence of the native Bacillus 
thuringiensis cryIA(b) protein, more preferably at least about 95 percent 
homology, and most preferably at least about 98 percent. 
Other preferred embodiments of the present invention include synthetic DNA 
sequences having essentially the DNA sequence of SEQ ID NO:4, as well as 
mutants or variants thereof; transformation vectors comprising essentially 
the DNA sequence of SEQ ID NO:4; and isolated DNA sequences derived from 
the plasmids pCIB4406, pCIB4407, pCIB4413, pCIB4414, pCIB4416, pCIB4417, 
pCIB4418, pCIB4419, pCIB4420, pCIB4421, pCIB4423, pCIB4434, pCIB4429, 
pCIB4431, pCIB4433. Most preferred are isolated DNA sequences derived from 
the plasmids pCIB4418 and pCIB4420, pCIB4434, pCIB4429, pCIB4431, and 
pCIB4433. 
In order to construct one of the maize optimized DNA sequences of the 
present invention, synthetic DNA oligonucleotides are made with an average 
length of about 80 nucleotides. These oligonucleotides are designed to 
hybridize to produce fragments comprising the various quarters of the 
truncated toxin gene. The oligonucleotides for a given quarter are 
hybridized and amplified using PCR. The quarters are then cloned and the 
cloned quarters are sequenced to find those containing the desired 
sequences. In one instance, the fourth quarter, the hybridized 
oligonucleotides are cloned directly without PCR amplification. Once all 
clones of four quarters are found which contain open reading frames, an 
intact gene encoding the active insecticidal protein is assembled. The 
assembled gene may then be tested for insecticidal activity against any 
insect of interest including the European Corn Borer (ECB) and the 
sugarcane borer. (Examples 5A and 5B, respectively). When a fully 
functional gene is obtained, it is again sequenced to confirm its primary 
structure. The fully functional gene is found to give 100% mortality when 
bioassayed against ECB. The fully functional gene is also modified for 
expression in maize. 
The maize optimized gene is tested in a transient expression assay, e.g. a 
maize transient expression assay. The native Bt cryIA(b) coding sequence 
for the active insecticidal toxin is not expressed at a detectable level 
in a maize transient expression system. Thus, the level of expression of 
the synthesized gene can be determined. By the present methods, expression 
of a protein in a transformed plant can be increased at least about 100 
fold to about 50,000 fold, more specifically at least about 1,000 fold to 
at least about 20,000 fold. 
Increasing expression of an insecticial gene to an effective level does not 
require manipulation of a native gene along the entire sequence. Effective 
expression can be achieved by manipulating only a portion of the sequences 
necessary to obtain increased expression. A full-length, maize optimized 
CryIA(b) gene may be prepared which contains a protein of the native 
CryIA(b) sequence. For example, FIG. 7 illustrates a full-length, maize 
optimized CryIA(b) gene which is a synthetic-native hybrid. That is, about 
2kb of the gene (nucleotides 1-1938 of SEQ ID NO:8) is maize optimized, 
i.e. synthetic. The remainder, C-terminal nucleotides 647-1155 of SEQ ID 
NO:8, are identical to the corresponding sequence native of the CryIA(b) 
gene. Construction of the illustrated gene is described in Example 6, 
below. 
It is recognized that by using the methods described herein, a variety of 
synthetic/native hybrids may be constructed and tested for expression. The 
important aspect of hybrid construction is that the protein is produced in 
sufficient amounts to control insect pests. In this manner, critical 
regions of the gene may be identified and such regions synthesized using 
preferred codons. The synthetic sequences can be linked with native 
sequences as demonstrated in the Examples below. Generally, N-terminal 
portions or processing sites can be synthesized and substituted in the 
native coding sequence for enhanced expression in plants. 
In another embodiment of the present invention, the maize optimized genes 
encoding cryIA(b) protein may be manipulated to render the encoded protein 
more heat stable or temperature stable compared to the native cryIA(b) 
protein. It has been shown that the cryIA(b) gene found in Bacillus 
thuringiensis kurstaki HD-1 contains a 26 amino acid deletion, when 
compared with the cryIA(a) and cryIA(c) proteins, in the --COOH half of 
the protein. This deletion leads to a temperature-sensitive cryIA(b) 
protein. See M. Geiser, EP 0 440 581, entitled "Temperaturstabiles 
Bacillus thuringiensis-Toxin". Repair of this deletion with the 
corresponding region from the cryIA(a) or cryIA(c) protein improves the 
temperature stability of the repaired protein. Constructs of the 
full-length modified cryIA(b) synthetic gene are designed to insert 
sequences coding for the missing amino acids at the appropriate place in 
the sequence without altering the reading frame and without changing the 
rest of the protein sequence. The full-length synthetic version of the 
gene is assembled by synthesizing a series of double-stranded DNA 
cassettes, each approximately 300 bp in size, using standard techniques of 
DNA synthesis and enzymatic reactions. The repaired gene is said to encode 
a "heat stable" or "temperature-stable" cryIA(b) protein, since it retains 
more biological activity than its native counterpart when exposed to high 
temperatures. Specific sequences of maize optimized, heat stable cryIA(b) 
genes encoding temperature stable proteins are set forth in FIGS. 9 (SEQ 
ID NO:10), 11 (SEQ ID NO:12), 13 (SEQ ID NO:14), and 15 (SEQ ID NO:16), 
and are also described in Example 7, below. 
The present invention encompasses maize optimized coding sequences encoding 
other polypeptides, including those of other Bacillus thuringiensis 
insecticidal polypeptides or insecticidal proteins from other sources. For 
example, cryIB genes can be maize optimized, and then stably introduced 
into plants, particularly maize. The sequence of a maize optimized cryIB 
gene constructed in accordance with the present invention is set forth in 
FIG. 6 (SEQ ID NO:6). 
Optimizing a Bt IP gene for expression in maize using the maize preferred 
codon usage according to the present invention results in a significant 
increase in the expression of the insecticidal gene. It is anticipated 
that other genes can be synthesized using plant codon preferences to 
improve their expression in maize or other plants. Use of maize codon 
preference is a likely method of optimizing and maximizing expression of 
foreign genes in maize. Such genes include genes used as selectable or 
scoreable markers in maize transformation, genes which confer herbicide 
resistance, genes which confer disease resistance, and other genes which 
confer insect resistance. 
The synthetic cryIA(b) gene is also inserted into Agrobacterium vectors 
which are useful for transformation of a large variety of dicotyledenous 
plant species. (Example 44). Plants stably transformed with the synthetic 
cryIA(b) Agrobacterium vectors exhibit insecticidal activity. 
The native Bt cryIA(b) gene is quite A+T rich. The G+C content of the 
full-length native Bt cryIA(b) gene is approximately 39%. The G+C content 
of a truncated native Bt cryIA(b) gene of about 2 Kb in length is 
approximately 37%. In general, maize coding regions tend to be 
predominantly G+C rich. The modifications made to the Bt cryIA(b) gene 
result in a synthetic IP coding region which has greater than 50% G+C 
content, and has about 65% homology at the DNA level with the native 
cryIA(b) gene. The protein encoded by this synthetic CryIA(b) gene is 100% 
homologous with the native protein, and thus retains full function in 
terms of insect activity. The truncated synthetic CryIA(b) IP gene is 
about 2 Kb in length and the gene encodes the active toxin region of the 
native Bt kurstaki CryIA(b) insecticidal protein. The length of the 
protein encoded by the truncated synthetic CryIA(b) gene is 648 amino 
acids. 
The synthetic genes of the present invention are useful for enhanced 
expression in transgenic plants, most preferably in transformed maize. The 
transgenic plants of the present invention may be used to express the 
insecticidal CryIA(b) protein at a high level, resulting in resistance to 
insect pests, preferably coleopteran or lepidopteran insects, and most 
preferably European Corn Borer (ECB) and Sugarcane Borer. 
In the present invention, the DNA coding sequence of the synthetic maize 
optimized gene may be under the control of regulatory elements such as 
promoters which direct expression of the coding sequence. Such regulatory 
elements, for example, include monocot or maize and other monocot 
functional promoters to provide expression of the gene in various parts of 
the maize plant. The regulatory element may be constitutive. That is, it 
may promote continuous and stable expression of the gene. Such promoters 
include but are not limited to the CaMV 35S promoter; the CaMV 19S 
promoter; A. tumefaciens promoters such as octopine synthase promoters, 
mannopine synthase promoters, nopaline synthase promoters, or other opine 
synthase promoters; ubiquitin promoters, actin promoters, histone 
promoters and tubulin promoters. The regulatory element may be a 
tissue-preferential promoter, that is, it may promote higher expression in 
some tissues of a plant than in others. Preferably, the 
tissue-preferential promoter may direct higher expression of the synthetic 
gene in leaves, stems, roots and/or pollen than in seed. The regulatory 
element may also be inducible, such as by heat stress, water stress, 
insect feeding or chemical induction, or may be developmentally regulated. 
Numerous promoters whose expression are known to vary in a tissue specific 
manner are known in the art. One such example is the maize phosphoenol 
pyruvate carboxylase (PEPC), which is green tissue-specific. See, for 
example, Hudspeth, R. L. and Grula, J. W., Plant Molecular Biology 
12:579-589, 1989). Other green tissue-specific promoters include 
chlorophyll a/b binding protein promoters and RubisCO small subunit 
promoters. 
The present invention also provides isolated and purified pith-preferred 
promoters. Preferred pith-preferred promoters are isolated from 
graminaceous monocots such as sugarcane, rice, wheat, sorghum, barley, rye 
and maize; more preferred are those isolated from maize plants. 
In a preferred embodiment, the pith-preferred promoter is isolated from a 
plant TrpA gene; in a most preferred embodiment, it is isolated from a 
maize TrpA gene. That is, the promoter in its native state is operatively 
associated with a maize tryptophan synthase-alpha subunit gene 
(hereinafter "TrpA"). The encoded protein has a molecular mass of about 38 
kD. Together with another alpha subnit and two beta subunits, TrpA forms a 
multimeric enzyme, tryptophan synthase. Each subunit can operate 
separately, but they function more efficiently together. TrpA catalyzes 
the conversion of indole glycerol phosphate to indole. Neither the maize 
TrpA gene nor the encoded protein had been isolated from any plant before 
Applicants' invention. The Arabidopsis thaliana tryptophan synthase beta 
subunit gene has been cloned as described Wright et al., The Plant Cell, 
4:711-719 (1992). The instant maize TrpA gene has no homology to the beta 
subunit encoding gene. 
The present invention also provides purified pollen-specific promoters 
obtainable from a plant calcium-dependent phosphate kinase (CDPK) gene. 
That is, in its native state, the promoter is operably linked to a plant 
CDPK gene. In a preferred embodiment, the promoter is isolated from a 
maize CDPK gene By "pollen-specific," it is meant that the expression of 
an operatively associated structural gene of interest is substantially 
exclusively (i.e. essentially entirely) in the pollen of a plant, and is 
negligible in all other plant parts. By "CDPK," it is meant a plant 
protein kinase which has a high affinity for calcium, but not calmodulin, 
and requires calcium, but not calmodulin, for its catalytic activity. 
To obtain tissue-preferred or tissue specific promoters, genes encoding 
tissue specific messenger RNA (mRNA) can be obtained by differential 
screening of a cDNA library. For example, a pith-preferred cDNA can be 
obtained by subjecting a pith cDNA library to differential screening using 
cDNA probes obtained from pith and seed mRNA. See, Molecular Cloning, A 
Laboratory Manual, Sambrook et al. eds. Cold Spring Harbor Press: New York 
(1989). 
Alternately, tissue specific promoters may be obtained by obtaining tissue 
specific proteins, sequencing the N-terminus, synthesizing oligonucleotide 
probes and using the probes to screen a cDNA library. Such procedures are 
exemplified in the Experimental section for the isolation of a pollen 
specific promoter. 
The scope of the present invention in regard to the pith-preferred and 
pollen-specific promoters encompasses functionally active fragments of a 
full-length promoter that also are able to direct pith-preferred or 
pollen-specific transcription, respectively, of associated structural 
genes. Functionally active fragments of a promoter DNA sequence may be 
derived from a promoter DNA sequence, by several art-recognized 
procedures, such as, for example, by cleaving the promoter DNA sequence 
using restriction enzymes, synthesizing in accordance with the sequence of 
the promoter DNA sequence, or may be obtained through the use of PCR 
technology. See, e.g. Mullis et al., Meth. Enzymol. 155:335-350 (1987); 
Erlich (ed.), PCR Technology, Stockton Press (New York 1989). 
Further included within the scope of the instant invention are 
pith-preferred and pollen-specific promoters "equivalent" to the 
full-length promoters. That is, different nucleotides, or groups of 
nucleotides may be modified, added or deleted in a manner that does not 
abolish promoter activity in accordance with known procedures. 
A pith-preferred promoter obtained from a maize TrpA gene is shown in FIG. 
24 (SEQ ID NO:18). Those skilled in the art, with this sequence 
information in hand, will recognize that pith-preferred promoters included 
within the scope of the present invention can be obtained from other 
plants by probing pith libraries from these plants with probes derived 
from the maize TrpA structural gene. Probes designed from sequences that 
are highly conserved among TrpA subunit genes of various species, as 
discussed generally in Example 17, are preferred. Other pollen-specific 
promoters, which in their native state are linked to plant CDPK genes 
other than maize, can be isolated in similar fashion using probes derived 
from the conserved regions of the maize CDPK gene to probe pollen 
libraries. 
In another embodiment of the present invention, the pith-preferred or 
pollen-specific promoter is operably linked to a DNA sequence, i.e. 
structural gene, encoding a protein of interest, to form a recombinant DNA 
molecule or chimeric gene. The phrase "operably linked to" has an 
art-recognized meaning; it may be used interchangeably with "operatively 
associated with," "linked to," or "fused to". 
The structural gene may be homologous or heterologous with respect to 
origin of the promoter and/or a target plant into which it is transformed. 
Regardless of relative origin, the associated DNA sequence will be 
expressed in the transformed plant in accordance with the expression 
properties of the promoter to which it is linked. Thus, the choice of 
associated DNA sequence should flow from a desire to have the sequence 
expressed in this fashion. Examples of heterologous DNA sequences include 
those which encode insecticidal proteins, e.g. proteins or polypeptides 
toxic or inhibitory to insects or other plant parasitic arthropods, or 
plant pathogens such as fungi, bacteria and nematodes. These heterologous 
DNA sequences encode proteins such as magainins, Zasloff, PNAS USA, 
84:5449-5453 (1987); cecropins, Hultmark et al., Eur. J. Biochem. 
127:207-217 (1982); attacins, Hultmark et al., EMBO J. 2:571-576 (1983); 
melittin, gramicidin S, Katsu et al., Biochem. Biophys. Acta, 939:57-63 
(1988); sodium channel proteins and synthetic fragments, Oiki et al. PNAS 
USA, 85:2395-2397 (1988); the alpha toxin of Staphylococcus aureusm Tobkes 
et al., Biochem., 24:1915-1920 (1985); apolipoproteins and fragments 
thereof, Knott et al., Science 230:37 (1985); Nakagawa et al., J. Am. 
Chem. Soc., 107:7087 (1985); alamethicin and a variety of synthetic 
amphipathic peptides, Kaiser et al., Ann. Rev. Biophys. Biophys. Chem. 
16:561-581 (1987); lectins, Lis et al., Ann. Rev. Biochem., 55:35-68 
(1986); protease and amylase inhibitors; and insecticidal proteins from 
Bacillus thuringiensis, particularly the delta-endotoxins from B. 
thuringiensis; and from other bacteria or fungi. 
In a preferred embodiment of the invention, a pith-preferred promoter 
obtained from a maize TrpA subunit gene or pollen-specific promoter 
obtained from a maize CDPK gene is operably linked to a heterologous DNA 
sequence encoding a Bacillus thuringiensis ("B.t.") insecticidal protein. 
These proteins and the corresponding structural genes are well known in 
the art. See, Hofte and Whiteley, Microbiol. Reviews, 53:242-255 (1989). 
While it is recognized that any promoter capable of directing expression 
can be utilized, it may be preferable to use heterologous promoters rather 
than the native promoter of the protein of interest. In this manner, 
chimeric nucleotide sequences can be constructed which can be determined 
based on the plant to be transformed as well as the insect pest. For 
example, to control insect pests in maize, a monocot or maize promoter can 
be operably linked to a Bt protein. The maize promoter can be selected 
from tissue-preferred and tissue-specific promoters such as pith-preferred 
and pollen-specific promoters, respectively as disclosed herein. 
In some instances, it may be preferred to transform the plant cell with 
more than one chimeric gene construct. Thus, for example, a single plant 
could be transformed with a pith-preferred promoter operably linked to a 
Bt protein as well as a pollen-specific promoter operably linked to a Bt 
protein. The transformed plants would express Bt proteins in the plant 
pith and pollen and to a lesser extent the roots, outer sheath and brace 
roots. 
For various other reasons, particularly management of potential insect 
resistance developing to plant expressed insecticidal proteins, it is 
beneficial to express more than one insecticidal protein (IP) in the same 
plant. One could express two different genes (such as two different 
Bacillus thuringiensis derived delta-endotoxins which bind different 
receptors in the target insect's midgut) in the same tissues, or one can 
selectively express the two toxins in different tissues of the same plant 
using tissue specific promoters. Expressing two Bt genes (or any two 
insecticidal genes) in the same plant using three different tissue 
specific promoters presents a problem for production of a plant expressing 
the desired phenotype. Three different promoters driving two different 
genes yields six different insecticidal genes that need to be introduced 
into the plant at the same time. Also needed for the transformation is a 
selectable marker to aid in identification of transformed plants. This 
means introducing seven different genes into the plant at the same time. 
It is most desired that all genes, especially the insecticidal genes, 
integrate into the plant genome at the same locus so they will behave as a 
single gene trait and not as a multiple gene trait that will be harder to 
track during breeding of commercial hybrids. The total number of genes can 
be reduced by using differential tissue specific expression of the 
different insecticidal proteins. 
For example, by fusing cryIA(b) with the pollen and PEP carboxylase 
promoters, one would obtain expression of this gene in green tissues and 
pollen. Fusing a pith-preferred promoter with the cryIB delta endotoxin 
from Bacillus thuringiensis would produce expression of this insecticidal 
protein most abundantly in the pith of a transformed plant, but not in 
seed tissues. Transformation of a plant with three genes, PEP 
carboxylase/cryIA(b), pollen/cryIA(b), and pith/cryIB produces a plant 
expressing two different Bt insecticidal endotoxins in different tissues 
of the same plant. CryIA(b) would be expressed in the "outside" tissues of 
a plant (particularly maize), that is, in those tissues which European 
corn borer feeds on first after hatching. Should ECB prove resistant to 
cryIA(b) and be able to burrow into the stalk of the plant after feeding 
on leaf tissue and/or pollen, it would then encounter the cryIB 
delta-endotoxin and be exposed to a second insecticidal component. In this 
manner, one can differentially express two different insecticdal 
components in the same plant and decrease the total number of genes 
necessary to introduce as a single genetic unit while at the same time 
providing protection against development of resistance to a single 
insecticidal component. 
Likewise, a plant may be transformed with constructs encoding more than one 
type of insecticidal protein to control various insects. Thus, a number of 
variations may be constructed by one of skill in the art. 
The recombinant DNA molecules of the invention may be prepared by 
manipulating the various elements to place them in proper orientation. 
Thus, adapters or linkers may be employed to join the DNA fragments. Other 
manipulations may be performed to provide for convenient restriction 
sites, removal of restriction sites or superfluous DNA. These 
manipulations can be performed by art-recognized methods. See, Sambrook et 
al., Molecular Cloning:. A Laboratory Manual, Cold Spring Harbor 
Laboratory Press, second edition, 1989. For example, methods such as 
restriction, chewing back or filling in overhangs to provide blunt ends, 
ligation of linkers, complementary ends of the DNA fragments can be 
provided for joining and ligation. See, Sambrook et al., supra. 
Other functional DNA sequences may be included in the recombinant DNA 
molecule, depending upon the way in which the molecule is to be 
incorporated into the target plant genome. For instance, in the case of 
Agrobacterium-mediated transformation, if Ti- or the Ri- plasmid is used 
to transform the plant cells, the right and left borders of the T-DNA of 
the Ti- and Ri- plasmid will be joined as flanking regions to the 
expression cassette. Agrobacterium tumefaciens-mediated transformation of 
plants has been described in Horsch et al., Science, 225:1229 (1985); 
Marton, Cell Culture Somatic Cell Genetics of Plants, 1:514-521 (1984); 
Hoekema, In: The Binary Plant Vector System Offset-Drukkerij Kanters B. 
V., Alblasserdam, 1985, Chapter V Fraley, et al., Crit. Rev. Plant Sci., 
4:1-46; and An et al., EMBO J., 4:277-284 (1985). 
The recombinant DNA molecules of the invention also can include a marker 
gene to facilitate selection in recombinant plant cells. Examples of 
markers include resistance to a biocide such as an antibiotic, e.g. 
kanamycin, hygromycin, chloramphenicol, paramomycin, methotrexate and 
bleomycin, or a herbicide such as imidazolones, sulfonylureas, glyphosate, 
phosphinothricin, or bialaphos. Marker genes are well known in the art. 
In another embodiment of the present invention, plants stably transformed 
with a recombinant DNA molecule or chimeric gene as described hereinabove 
are provided. The resultant transgenic plant contains the transformed gene 
stably incorporated into its genome, and will express the structural gene 
operably associated to the promoter in the respective fashion. 
Transgenic plants encompassed by the instant invention include both 
monocots and dicots. Representative examples include maize, tobacco, 
tomato, cotton, rape seed, soybean, wheat, rice, alfalfa, potato and 
sunflower. [others?]. Preferred plants include maize, particularly inbred 
maize plants. 
All transformed plants encompassed by the instant invention may be prepared 
by several methods known in the art. A. tumefaciens-mediated 
transformation has been disclosed above. Other methods include direct gene 
transfer into protoplasts, Paszkowski et al., EMBO J., 12:2717 (1984); 
Loerz et al., Mol. Gen. & Genet., 1199:178 (1985); Fromm et al., Nature 
319:719 (1986); microprojectile bombardment, Klein et al., Bio/Technology, 
6:559-563 (1988); injection into protoplasts, cultured cells and tissues, 
Reich et al., Bio/Technology, 4:1001-1004 (1986); or injection into 
meristematic tissues or seedlings and plants as described by De La Pena et 
al., Nature, 325:274-276 (1987); Graves et al., Plant Mol. Biol., 7:43-50 
(1986); Hooykaas-Van Slogteren et al., Nature, 311:763-764 (1984); 
Grimsley et al., Bio/Technology, 6:185 (1988); and Grimsley et al., 
Nature, 325:177 (1988); and electroporation, W092/09696. 
The expression pattern of a structural gene operatively associated with an 
instant tissue-preferred or tissue-specific promoter in a transformed 
plant containing the same is critical in the case where the structural 
gene encodes an insecticidal protein. For example, the instantly disclosed 
pith-preferred expression pattern will allow the transgenic plant to 
tolerate and withstand pathogens and herbivores that attack primarily the 
pith, but also the brace roots, outer sheath and leaves of the plant since 
the protein will be expressed to a lesser extent but still in an insect 
controlling amount in these plant parts, but yet in the case of both types 
of promoters, will leave the seed of the plant unaffected. 
EXAMPLES 
The following examples further describe the materials and methods used in 
carrying out the invention. They are offered by way of illustration, and 
not by way of limitation. 
Example 1 
General Methods 
DNA manipulations were done using procedures that are standard in the art. 
These procedures can often be modified and/or substituted without 
substantively changing the result. Except where other references are 
identified, most of these procedures are described in Sambrook et al., 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 
Press, second edition, 1989. 
Synthesis of DNA Oligomers 
DNA oligomers which are from about twenty to about ninety, preferably from 
about sixty to about eighty nucleotides in length, are synthesized using 
an Applied Biosystems model 380B DNA synthesizer and standard procedures. 
The oligomers are made using the updated SSCAF3 cycle on a 0.2 .mu.mole, 
wide pore, small scale ABI column. The end procedure is run trityl off and 
the oligomer is cleaved from the column using the 380B's automatic 
cleavage cycle. The oligomers are then deblocked in excess ammonium 
hydroxide (NH.sub.4 OH) at 55.degree. C. for 8-12 hours. The oligomers are 
then dried in an evaporator using nitrogen gas. After completion, the 
oligomers are resuspended in 0.25-0.5 ml of deionized water. 
Purification of Synthetic Oligomers 
An aliquot of each oligomer is mixed with an equal volume of blue 
dye.backslash.formamide mix with the final solution containing 0.05% 
bromophenol blue, 0.05% xylene cyanol FF, and 25% formamide. This mixture 
is heated at 95.degree. C. for 10 minutes to denature the oligomers. 
Samples are then applied to a 12% polyacrylamide-urea gel containing 7M 
urea (Sambrook et al.). After electrophoresis at 300-400 volts for 3-4 
hours using a Vertical Slab Gel Unit (Hoefer Scientific Instruments, San 
Francisco, Calif.), UV shadowing is used to locate the correct sized 
fragment in the gel which was then excised using a razor blade. The 
purified gel fragment is minced and incubated in 0.4M LiCl, 1 mM EDTA (pH 
8) buffer overnight at 37.degree. C. 
Either of two methods is used to separate the oligomers from the 
polyacrylamide gel remnants: Gene.backslash.X 25 .mu.M porous polyethylene 
filter units or Millipore's ultrafree-MC 0.45 .mu.M filter units. The 
purified oligomers are ethanol precipitated, recovered by centrifuging in 
a microfuge for 20 min at 4.degree. C., and finally resuspended in TE (10 
mM Tris, 1 mM EDTA, pH 8.0). Concentrations are adjusted to 50 
ng.backslash..mu.l based on absorption readings at 260 nm. 
Kinasing Oligomers for Size Determinations 
To check the size of some of the oligomers on a sequencing gel, kinase 
labeling reactions are carried out using purified synthetic oligomers of 
each representative size: 40 mers, 60 mers, 70 mers, 80 mers, and 90 mers. 
In each 20 .mu.l kinasing reaction, one pmole of purified oligomer is used 
in a buffer of 7.0 mM Tris pH 7.5, 10 mM KCl, l mM MgC12), 0.5 mM DTT, 50 
.mu.g/ml BSA, 3000 .mu.Ci (3 pmoles) of 32P-gammaATP, and 8 units of T4 
polynucleotide kinase. The kinase reaction is incubated for 1 hour at 
37.degree. C. followed by a phenol.backslash.chloroform extraction and 
three ethanol precipitations with glycogen as carrier (Tracy, Prep. 
Biochem. 11:251-268 (1981). 
Two gel loadings (one containing 1000 cpm, the other containing 2000 cpm) 
of each reaction are prepared with 25% formamide, 0.05% bromophenol blue, 
and 0.05% xylene cyanol FF. The kinased oligomers are boiled for 5 minutes 
before loading on a 6% polyacrylamide, 7M urea sequencing gel (BRL Gel Mix 
TM6, BRL, Gaithersburg, Md.). A sequencing reaction of plasmid pUC18 is 
run on the same gel to provide size markers. 
After electrophoresis, the gel is dried and exposed to diagnostic X-ray 
film (Kodak, X-OMAT AR). The resulting autoradiograph shows all purified 
oligomers tested to be of the correct size. Oligomers which had not been 
sized directly on the sequencing gel are run on a 6% polyacrylamide, 7M 
urea gel (BRL Gel Mix TM6), using the sized oligomers as size markers. All 
oligomers are denatured first with 25% formamide at 100.degree. C. for 5 
minutes before loading on the gel. Ethidium bromide staining of the 
polyacrylamide gel allows all the oligomers to be visualized for size 
determination. 
Hybridizing Oligomers for Direct Cloning 
Oligomers to be hybridized are pooled together (from 1 .mu.g to 20 .mu.g 
total DNA) and kinased at 37.degree. C. for 1 hour in 1X Promega ligation 
buffer containing 30 mM Tris-HCl pH 7.8, 10 mM MgC12, 10 mM DTT, and 1 mM 
dATP. One to 20 units of T4 polynucleotide kinase is used in the reaction, 
depending on the amount of total DNA present. The kinasing reactions are 
stopped by placing the reaction in a boiling water bath for five minutes. 
Oligomers to form the 5' termini of the hybridized molecules are not 
kinased but are added to the kinased oligomers along with additional 
hybridization buffer after heating. The pooled oligomers are in a volume 
of 50-100 ul with added hybridization buffer used to adjust the final salt 
conditions to 100 mM NaCl, 120 mM Tris pH 7.5, and 10 mM MgC12. The 
kinased and non-kinased oligomers are pooled together and heated in a 
boiling water bath for five minutes and allowed to slowly cool to room 
temperature over a period of about four hours. The hybridized oligomers 
are then phenol.backslash.chloroform extracted, ethanol precipitated, and 
resuspended in 17 .mu.l of TE (10 mM Tris, 1 mM EDTA, pH 8.0). Using this 
17 .mu.l, a ligation reaction with a final volume of 20 .mu.l is assembled 
(final conditions=30 mM Tris-HCl pH 7.8, 10 mM MgC12, 10 mM DTT, 1 mM ATP, 
and 3 units of T4 DNA ligase (Promega, Madison Wis.). The ligation is 
allowed to incubate for about 2 hours at room temperature. The 
hybridized.backslash.ligated fragments are generally purified on 2% 
Nusieve gels before and.backslash.or after cutting with restriction 
enzymes prior to cloning into vectors. A 20 .mu.l volume ligation reaction 
is assembled using 100 ng to 500 ng of each fragment with approximate 
equimolar amounts of DNA in 30 mM Tris-HCl pH 7.8, 10 mM MgC12, 10 mM DTT, 
1 mM ATP, and 3 units of T4 DNA ligase (Promega, Madison, Wis.). Ligations 
are incubated at room temperature for 2 hours. After ligation, DNA is 
transformed into frozen competent E. coli cells using standard procedures 
(Sambrook et al.) and transformants are selected on LB-agar (Sambrook et 
al.) containing 100 .mu.g/ml ampicillin (see below). 
PCR Reactions for Screening Clones in E. coli 
E. coli colonies which contain the correct DNA insert are identified using 
PCR (see generally, Sandhu et al., BioTechniques 7:689-690 (1989)). Using 
a toothpick, colonies are scraped from an overnight plate and added to a 
20 .mu.l to 45 .mu.l PCR reaction mix containing about 50 pmoles of each 
hybridizing primer (see example using primers MK23A28 and MK25A28 to 
select orientation of SacII fragment in pHYB2#6), 200 .mu.m to 400 mM of 
each dNTP, and 1X reaction buffer (Perkin Elmer Cetus, Norwalk, Conn.). 
After boiling the E. coli.backslash.PCR mix in a boiling water bath for 10 
minutes, 5 .mu.l of Taq polymerase (0.5 units)(Perkin Elmer Cetus, 
Norwalk, Conn.) in 1X reaction buffer is added. The PCR reaction 
parameters are generally set with a denaturing step of 94.degree. C. for 
30 seconds, annealing at 55.degree. C. for 45 seconds, and extension at 
72.degree. C. for 45 seconds for 30 to 36 cycles. PCR reaction products 
are run on agarose or Nusieve agarose (FMC) gels to detect the correct 
fragment size amplified. 
Ligations 
Restriction enzyme digested fragments are either purified in 1% LGT (low 
gelling temperature agarose, FMC), 2% Nusieve (FMC), or 0.75% agarose 
using techniques standard in the art. DNA bands are visualized with 
ethidium bromide and bands are recovered from gels by excision with a 
razor blade. Fragments isolated from LGT are ligated directly in the LGT. 
Ten microliters of each recovered DNA fragment is used to assemble the 
ligation reactions, producing final ligation reaction volumes of about 23 
.mu.l. After excision with a razor blade, the recovered gel bands 
containing the desired DNA fragments are melted and brought to 1X ligase 
buffer and 3 units of T4 DNA ligase (Promega) are added as described 
above. Fragments isolated from either regular agarose or Nusieve agarose 
are purified from the agarose using ultrafree-MC 0.45 .mu.M filter units 
(Millipore) and the fragments are ligated as described above. Ligation 
reactions are incubated at room temperature for two hours before 
transforming into frozen competent E. coli cells using standard procedures 
(Sambrook et al.). 
Transformations 
Frozen competent E. coli cells of the strain DH5alpha or HB101 are prepared 
and transformed using standard procedures (Sambrook et al.). E. coli 
"SURE" competent cells are obtained from Stratagene (La Jolla, Calif.). 
For ligations carried out in LGT agarose, after ligation reactions are 
complete, 50 mM CaC12 is added to a final volume of about 150 .mu.l and 
the solution heated at approximately 65.degree. C. for about 10 minutes to 
completely melt the agarose. The solution is then mixed and chilled on ice 
for about 10 minutes before the addition of about 200 .mu.l of competent 
cells which had been thawed on ice. This mixture is allowed to incubate 
for 30 minutes on ice. The mixture is next heat shocked at 42.degree. C. 
for 60 seconds before chilling on ice for two minutes. Next, 800 .mu.l of 
SOC media (20% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 
adjusted to pH 8 with 5N NaOH, 20 mM MgC12:MgSO4 mix, and 20 mM glucose; 
Sambrook et al.) is added and the cells are incubated at 37.degree. C. 
with shaking for about one hour before plating on selective media plates. 
Plates typically are L-agar (Sambrook et al.) containing 100 .mu.g/ml 
ampicillin. 
When ligations are carried out in a solution without agarose, typically 200 
.mu.l of frozen competent E. coli cells (strain DH5alpha (BRL, 
Gaithersburg, Md. or Sure cells, Stratagene, La Jolla, Calif.) are thawed 
on ice and 5 .mu.l of the ligation mixture added. The reaction is 
incubated on ice for about 45 to 60 minutes, the cells are then heat 
shocked at 42.degree. for about 90 seconds. After recovery at room 
temperature for about 10 minutes, 800 .mu.l of SOC medium is added and the 
cells are then incubated 1 hour at 37.degree. C. with shaking and plated 
as above. 
When screening for inserts into the beta-galactosidase gene in some of the 
standard vectors used, 200 .mu.l of the recovered transformation mixture 
is plated on LB-agar plates containing 0.008% X-gal, 80 .mu.M IPTG, and 
100 .mu.g/ml ampicillin (Sambrook et al.). The plates are incubated at 
37.degree. overnight to allow selection and growth of transformants. 
Miniscreening DNA 
Transformants from the selective media plates are grown and their plasmid 
Structure is examined and confirmed using standard plasmid mini-screen 
procedures (Sambrook et al.). Typically, the "boiling" procedure is used 
to produce small amounts of plasmid DNA for analysis (Sambrook et al.). 
Alternatively, an ammonium acetate procedure is used in some cases. This 
procedure is a modification of that reported by Shing-yi Lee et al., 
Biotechniques 9:676-679 (1990). 
1) Inoculate a single bacterial colony from the overnight selection plates 
into 5 ml (can be scaled down to 1 ml) of TB (Sambrook et al.) medium and 
grow in the presence of the appropriate antibiotic. 
2) Incubate on a roller at 37.degree. C. overnight. 
3) Collect 5 ml of bacterial cells in a plastic Oakridge tube and spin for 
5 min. at 5000 rpm in a Sorvall SS-34 rotor at 4.degree. C. 
4) Remove the supernatant. 
5) Resuspend the pellet in 1 ml of lysis buffer (50 mM glucose, 25 mM 
Tris-HCl[pH 8.0], 10 mM EDTA and 5 mg/ml lysozyme), vortex for 5 seconds, 
and incubate at room temperature for 5 min. 
6) Add 2 ml of freshly prepared alkaline solution (0.2N NaOH, 1% sodium 
dodecyl sulfate), tightly secure lid, mix by inverting 5 times and place 
tube in an ice-water bath for 5 min. 
7) Add 1.5 ml of ice-cold 7.5M ammonium acetate (pH 7.6) to the solution, 
mix by inverting the tube gently 5 times and place on an ice-water bath 
for 5 min. 
8) Centrifuge mixture at 9000 rpm for 10 min. at room temperature. 
9) Transfer clear supernatant to a 15 ml Corex tube and add 0.6 volumes of 
isopropanol (approx. 2.5 ml). Let sit at room temperature for 10 min. 
10) Centrifuge the mixture at 9000 rpm for 10 min. at room temperature and 
discard the supernatant. 
11) Resuspend the pellet in 300 ul of TE buffer. Add 6 ul of a stock of 
RNase A & T1 (made as a 200 ul solution by adding 180 ul of RNase A [3254 
Units/mg protein, 5.6 mg protein/ml] and 20 ul of RNase T1[481 Units/ug 
protein, 1.2 mg protein/ml]). These stocks may be purchased from USB(US 
Biochemical). Transfer to a microcentrifuge tube and incubate at 
37.degree. C. for 15 min. 
12) Add 75 ul of distilled water and 100 ul of 7.5M ammonium acetate and 
incubate in an ice-water bath for 10 min. 
13) Centrifuge the mixture at 14,000 rpm for 10 min. in a Beckman microfuge 
at 4.degree. C. 
14) Precipitate by adding 2.5 volumes of 100% EtOH (approx. 1 ml) and 
incubate in an ice-water bath for 10 min. 
15) Spin at 14,000 rpm for 10 min. in a microfuge. 
16) Wash pellet with 70% ethanol (using 0.5 ml-1 ml). Dry the pellet and 
resuspend in 100 .mu.l of 1X New England Biolabs restriction enzyme Buffer 
4 [20 mM Tris-HCl(pH 7.9), 10 mM magnesium acetate, 50 mM potassium 
acetate, 1 mM DTT]. Measure concentration and check purity by 
spectrophotometry at absorbances 260 and 280 nm. 
For a more rapid determination as to whether or not a particular bacterial 
colony harbored a recombinant plasmid, a PCR miniscreen procedure is 
carried out using a modification of the method described by (Sandhu, G. S. 
et al., 1989, BioTechniques, 7: 689-690) . Briefly, the following mixture 
is prepared: 
100 .mu.l primer mix above, 20 .mu.M each primer, 
100 .mu.l dNTP mix (2.5 mM each) 
100 .mu.l 10X AmpliTaq buffer (Perkin-Elmer Cetus, 1X buffer=10 mM Tris-HCl 
pH 8.3, 50 mM KCl, 1.5 mM MgC12, and 0.01% gelatin) 
700 .mu.l deionized water. 
20 .mu.l of the above mixture is put into a a 0.5 ml polyproplyene PCR 
tube. A transformed bacterial colony is picked with a toothpick and 
resuspended in the mixture. The tube is put in a boiling water bath for 10 
minutes and then cooled to room temperature before adding 5 .mu.l of the 
mix described below: 
265 .mu.l deionized water 
30 .mu.l 10X Amplitaq buffer (Perkin-Elmer Cetus, 1X buffer=10 mM Tris-HCl 
pH 8.3, 50 mM KCl, 1.5 mM MgC12, and 0.01% gelatin) 
7.5 .mu.l Taq polymerase 
The samples are overlaid with 50 .mu.l of mineral oil and PCR is carried 
out for 30 cycles using the following parameters: 
denature: 94.degree. for 1 min 
anneal: 55.degree. for 1 min 
extend: 72.degree. for 45 seconds. 
After PCR amplification, 1 .mu.l of loading dye (30% glycerol, 0.25% 
Bromophenol blue, 0.25% xylene cyanol) is added to the whole reaction and 
20 .mu.l of the mixture is loaded on a 2% Nusieve, 1% agarose gel to see 
if there is a PCR product of the expected size. 
This procedure is used as an initial screen. Minipreps are subsequently 
carried out to confirm the structure of the plasmid and its insert prior 
to sequencing. 
Example 2 
AMPLIFICATION AND ASSEMBLY OF EACH QUARTER 
Cloning Fragments of the Synthetic Bt cryIA(b) Gene 
The synthetic gene was designed to be cloned in four pieces, each roughly 
one quarter of the gene. The oligomers for each quarter were pooled to 
either be assembled by PCR, hybridization, or a combination of 
hybridization followed by PCR amplification as described elsewhere. 
Synthetic quarters were pieced together with overlapping restriction sites 
Aat II, NcoI, and Apa I between the 1st and 2nd, 2nd and 3rd, and 3rd and 
4th quarters respectively. 
Each quarter of the gene (representing about 500 bp) was assembled by 
hybridizing the appropriate oligomers and amplifying the desired fragment 
using PCR primers specific for the ends of that quarter. Two different 
sets of PCR reactions employing two sets of slightly different primers 
were used. The PCR products of the two reactions were designed to be 
identical except that in the first reaction there was an additional AATT 
sequence at the 5' end of the coding region and in the second reaction 
there was an AGCT sequence at the 3' end of a given quarter. When the 
products of the two reactions for a particular quarter were mixed (after 
removing the polymerase, primers and incomplete products), denatured, and 
subsequently re-annealed, a certain ratio (theoretically 50%) of the 
annealed product should have non-homologous overhanging ends. These ends 
were designed to correspond to the "sticky ends" formed during restriction 
digestion with EcoRI at the 5' end and Hind III at the 3' end of the 
molecule. The resulting molecules were phosphorylated, ligated into an 
EcoRI/HindIII digested and phosphatased Bluescript vector, and transformed 
into frozen competent E. coli strain DH5alpha. After selection, the E. 
coli colonies containing the desired fragment are identified by 
restriction digest patterns of the DNA. Inserts representing parts of the 
synthetic gene are subsequently purified and sequenced using standard 
procedures. In all cases, clones from multiple PCR reactions are generated 
and sequenced. The quarters are then joined together using the unique 
restriction sites at the junctions to obtain the complete gene. 
Cloned quarters are identified by mini-screen procedures and the gene 
fragment sequenced. It is found that errors are frequently introduced into 
the sequence, most probably during the PCR amplification steps. To correct 
such errors in clones that contain only a few such errors, hybridized 
oligomers are used. Hybridized fragments are digested at restriction 
enzyme recognition sites within the fragment and cloned to replace the 
mutated region in the synthetic gene. Hybridized fragments range from 90 
bp in length (e.g. the region that replaces the fragment between the Sac 
II sites in the 2nd quarter) to the about 350 bp 4th quarter fragment that 
replaces two PCR induced mutations in the 4th quarter of the gene. 
Due to the high error rate of PCR, a plasmid is designed and constructed 
which allows the selection of a cloned gene fragment that contains an open 
reading frame. This plasmid is designed in such a manner that if an open 
reading frame is introduced into the cloning sites, the transformed 
bacteria could grow in the presence of kanamycin. The construction of this 
vector is described in detail below. This selection system greatly 
expedites the progress by allowing one to rapidly identify clones with 
open reading frames without having to sequence a large number of 
independent clones. The synthetic quarters are assembled in various 
plasmids, including BSSK (Stratagene; La Jolla, Calif.), pUC18 (Sambrook 
et al.), and the Km-expression vector. Other suitable plasmids, including 
pUC based plasmids, are known in the art and may also be used. Complete 
sequencing of cloned fragments, western blot analysis of cloned gene 
products, and insect bioassays using European corn borer as the test 
insect verify that fully functional synthetic Bt cryIA(b) genes have been 
obtained. 
Construction of the Km-expression Vector to Select Open Reading Frames 
The Km-expression vector is designed to select for fragments of the 
synthetic gene which contain open-reading frames. PCR oligomers are 
designed which allow the fusion of the NPTII gene from Tn5 starting at 
nucleotide 13 (Reiss et al., EMBO J. 3:3317-3322 (1984)) with pUC18 and 
introduce useful restriction sites between the DNA segments. The 
polylinker region contains restriction sites to allow cloning various 
synthetic Bt IP fragments in-frame with the Km gene. The 88 bp 5' oligomer 
containing the polylinker region is purified on a 6% polyacrylamide gel as 
described above for the oligomer PAGE purification. A PCR reaction is 
assembled with a 1Kb Bgl II.backslash.Sma I template fragment which 
contains the NPT II gene derived from Tn5. The PCR reaction mix contains 
100 ng of template with 100 pmols of oligomers KE72A28 and KE74A28 (see 
sequences below), 200 nM dNTP, and 2.5 Units of Taq polymerase all in a 50 
N1 volume with an equal volume of mineral oil overlaid. Sequences of the 
primers are: 
__________________________________________________________________________ 
KE74A28 
5'-GCAGATCTGG ATCCATGCAC GCCGTGAAGG GCCCTTCTAG AAGGCCTATC 
GATAAAGAGC TCCCCGGGGA TGGATTGCAC GCAGGTTC-3'(SEQ ID NO:29) 
KE72A28 
5'-GCGTTAACAT GTCGACTCAG AAGAACTCGT CAAGAAGGCG-3'(SEQ ID 
__________________________________________________________________________ 
NO:30) 
The PCR parameters used are: 94.degree. C. for 45 seconds (sec), 55.degree. 
C. for 45 sec, and 72.degree. C. for 55 sec with the extension at step 3 
for 3 sec for 20 cycles. All PCR reactions are carried out in a 
Perkin-Elmer Cetus thermocycler. The amplified PCR product is 800 bp and 
contains the polylinker region with a translational start site followed by 
unique restriction sites fused in-frame with the Km gene from base #13 
running through the translational terminator. pUC:KM74 is the 
Km-expression cassette that was assembled from the 800 bp Bgl 
II.backslash.Sal I polylinker/Km fragment cloned in the PUC18 vector. The 
lacZ promoter allows the Km gene to be expressed in E. coli. pUC:KM74 
derivatives has to first be plated on LB-agar plates containing 100 
.mu.g/ml ampicillin to select transformants which can subsequently be 
screened on LB-agar plates containing 25 .mu.g/ml kanamycin/IPTG. 
Synthetic Bt IP gene fragments are assembled from each quarter in the 
Km-cassette to verify cloning of open-reading-frame containing fragments 
pieces. The first ECB active synthetic Bt IP gene fragment, pBt:Km#6, is a 
Bt IP gene that shows Km resistance. This fragment is subsequently 
discovered to contain mutations in the 3rd and 4th quarter which are later 
repaired. 
Example 2A 
SYNTHESIS AND CLONING OF THE FIRST QUARTER OF THE SYNTHETIC GENE [base 
pairs 1 to 550] 
The following procedures are followed in order to clone the first quarter 
of the synthetic DNA sequence encoding a synthetic Bt cryIA(b) gene. The 
same procedures are essentially followed for synthesis and cloning of the 
other quarters, except as noted for primers and restriction sites. 
Template for Quarter 1: Mixture of Equal Amounts of Purified Oligomers 
U1-U7 and L1 to L7 
__________________________________________________________________________ 
PCR Primers: 
__________________________________________________________________________ 
Forward: 
P1 (a): 
5'-GTCGACAAGG ATCCAACAAT GG-3'(SEQ ID NO: 31) 
P1 (b): 
5'-AATTGTCGAC AAGGATCCAA CAATGG-3'(SEQ ID NO: 32) 
Reverse: 
P2 (a): 
5'-ACACGCTGAC GTCGCGCAGC ACG-3'(SEQ ID NO: 33) 
P2 (b): 
5'-AGCTACACGC TGACGTCGCG CAG-3'(SEQ ID NO: 34) 
__________________________________________________________________________ 
Primer pair A1: P1 (b) + P2 (a) 
Primer pair A2: P1 (a) + P2 (b) 
The PCR reaction containing the oligomers comprising the first quarter of 
the synthetic maize-optimized Bt IP gene is set up as follows: 
200 ng oligo mix (all oligos for the quarter mixed in equal amounts based 
on weight) 
10 .mu.l of primer mix (1:1 mix of each at 20 .mu.M; primers are described 
above) 
5 .mu.l of 10X PCR buffer 
PCR buffer used may be either 
(a) 1X concentration=10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.0, 2 
mM MgSO4, and 0.1% Triton X-100), or 
(b) 1X concentration=10mM Tris-HCl pH 8.3, 50 mM KCl 1.5 mM MgCl.sub.2, 
0.01% wt/vol gelatin. 
Components are mixed, heated in a boiling water bath for 5 minutes, and 
incubated at 65.degree. C. for 10 minutes. 
Next, the following reagents are added: 
8 .mu.l of dNTPs mixture (final concentration in the reaction=0.2 mM each) 
5 units polymerase. 
The final reaction volume is 50 microliters. 
Oligomers are then incubated for 3 min at 72.degree. C. and then a PCR 
cycle is run. The PCR reaction is run in a Perkin Elmer thermocycler on a 
step cycle protocol as follows: 
denaturation cycle: 94.degree. for 1 minute 
annealing cycle : 60.degree. for 1 minute 
extension cycle : 72.degree. for 45 seconds (+3 sec per cycle) 
number of cycles: 15 
After the reaction is complete, 10 .mu.l of the PCR reaction is loaded on a 
2% Nusieve-GTG (FMC), 1% agarose analytical gel to monitor the reaction. 
The remaining 40 .mu.l is used to clone the gene fragments as described 
below. 
PCR Products 
The termini of the double stranded PCR product corresponding to the various 
primer pairs are shown (only upper strand): 
__________________________________________________________________________ 
A1 
AATTGTCGAC(SEQ ID NO: 35).sub.-- GCGTGT 
(554 bp) first qtr. 
A2 
GTCGAC.sub.-- GCGTGTAGCT(SEQ ID NO: 36) 
(554 bp) first qtr. 
__________________________________________________________________________ 
Hybridization 
40 .mu.l of each of the PCR reactions described above is purified using a 
chromaspin 400 column (Clonetech, Palo Alto, Calif.) according to 
manufacturers directions. Five .mu.g of carrier DNA was added to the 
reactions before loading on the column. (This is done for most of the 
cloning. However, in some reactions the PCR reactions are 
phenol:chloroform extracted using standard procedures (Sambrook et al.) to 
remove the Taq polymerase and the PCR generated DNA is recovered from the 
aqueous phase using a standard ethanol precipitation procedure.) The 
carrier DNA does not elute with the PCR generated fragments. The A1 and A2 
reaction counterparts for each quarter are mixed, heated in a boiling 
water bath for 10 minutes and then incubated at 65.degree. C. overnight. 
The reactions are then removed from the 65.degree. bath and ethanol 
precipitated with 1 .mu.l (20 .mu.g) of nuclease free glycogen (Tracy, 
Prep. Biochem. 11:251-268 (1981) as carrier. The pellet is resuspended in 
40 .mu.l of deionized water. 
Phosphorylation Reaction 
The phosphorylation reaction is carried out as follows: 
40 .mu.l DNA 
2.5 .mu.l 20 mM ATP 
0.5 .mu.l 10X BSA/DTT (1X=5 mM DTT, 0.5 mg/ml BSA) 
1.0 .mu.l 10X polynucleotide kinase buffer (1X=70 mM Tris.HCl, 
pH 7.6, 0.1M KCl, 10 mM MgC12) 
2.0 .mu.l polynucleotide kinase (New England Biolabs, 20 units). 
Incubation is for 2 hours at 37.degree. C. 
The reaction is then extracted one time with a 1:1 phenol:chloroform 
mixture, then once with chloroform and the aqueous phase ethanol 
precipitated using standard procedures. The pellet is resuspended in 10 
.mu.l of TE. 
Restriction Digests 
20 .mu.g of Bluescript vector (BSSK+, Stratagene, La Jolla, Calif.) 
10 .mu.l 10X restriction buffer (1X=20 mM Tris-HCl pH 8.0, 10 mM MgC12, 100 
mM NaCl) 
5 .mu.l Eco RI (New England Biolabs) 100 units 
5 .mu.l Hind III (New England Biolabs) 100 units 
Final reaction volume is 100 .mu.l. 
Incubation is for 3 hours at 37.degree.. 
When completed, the reaction is extracted with an equal volume of phenol 
saturated with TE (10 mM Tris.HCl pH 8.0 and 10 mM EDTA). After 
centrifugation, the aqueous phase was extracted with an equal volume of 
1:1 mixture of (TE saturated) phenol:chloroform (the "chloroform" is mixed 
in a ratio of 24:1 chloroform:isoamyl alcohol), and finally the aqueous 
phase from this extraction is extracted with an equal volume of 
chloroform. The final aqueous phase is ethanol precipitated (by adding 10 
.mu.l of 3M sodium acetate and 250 .mu.l of absolute ethanol, left at 
4.degree. for 10 min and centrifuged in a microfuge at maximum speed for 
10 minutes. The pellet is rinsed in 70% ethanol and dried at room 
temperature for 5-10 minutes and resuspended in 100 .mu.l of 10 mM 
Tris.HCl (pH 8.3). 
Phosphatase Reaction 
Vector DNA is routinely treated with phosphatase to reduce the number of 
colonies obtained without an insert. Calf intestinal alkaline phosphatase 
is typically used (Sambrook et al.), but other phosphatase enzymes can 
also be used for this step. 
Typical phosphatase reactions are set up as below: 
90 .mu.l of digested DNA described above 
10 .mu.l of 10X Calf intestinal alkaline phosphatase buffer (1X=50 mM 
Tris-HCl (pH 8.3), 10 mM MgC12, 1 mM ZnC12, 10 mM spermidine) 
1 .mu.l (1 unit) of calf intestinal alkaline phosphatase (CIP, Boehringer 
Mannheim, Indianapolis, Ind.) 
Incubation is at 37.degree. C. for 1 hour. 
The DNA is then gel purified (on a 1% low gelling temperature (LGT) agarose 
gel) and the pellet resuspended in 50 .mu.l TE. After electrophoresis, the 
appropriate band is excised from the gel using a razor blade, melted at 
65.degree. for 5 minutes and diluted 1:1 with TE. This solution is 
extracted twice with phenol, once with the above phenol:chloroform 
mixture, and once with chloroform. The final aqueous phase is ethanol 
precipitated and resuspended in TE buffer. 
Ligation 
To ligate fragments of the synthetic gene into vectors, the following 
conditions are typically used. 
5 .mu.l of phosphorylated insert DNA 
2 .mu.l of phosphatased Eco RI/Hind III digested Bluescript vector heated 
at 65.degree. for 5 minutes, then cooled 
1 .mu.l 10X ligase buffer (1X buffer=30 mM Tris.HCl (pH 7.8), 10 mM MgC12, 
10 mM DTT, 1 mM ATP) 
1 .mu.l BSA (1 mg/ml) 
1 .mu.l ligase (3 units, Promega, Madison, Wis.) 
Ligase reactions are typically incubated at 16.degree. overnight or at room 
temperature for two hours. 
Transformation 
Transformation of ligated DNA fragments into E. coli is performed using 
standard procedures (Sambrook et al.) as described above. 
Identification of Recombinants 
White or light blue colonies resulting from overnight incubation of 
transformation plates are selected. Plasmids in the transformants are 
characterized using standard mini-screen procedures (Sambrook et al.) or 
as described above. One of the three procedures listed below are typically 
employed: 
(1) boiling DNA miniprep method 
(2) PCR miniscreen 
(3) Ammonium acetate miniprep. 
The restriction digest of recombinant plasmids believed to contain the 
first quarter is set up as follows: 
(a) Bam HI/Aat II digest: 10 .mu.l DNA+10 .mu.l 1X New England Biolabs 
restriction enzyme Buffer 4 
0.5 .mu.l Bam HI (10 units) 
0.5 .mu.l Aat II (5 units) 
Incubation is for about 2 hours at 37.degree. C. 
Clones identified as having the desired restriction pattern are next 
digested with Pvu II and with Bgl II in separate reactions. Only clones 
with the desired restriction patterns with all three enzyme digestions are 
carried further for sequencing. 
Sequencing of Cloned Gene Fragments 
Sequencing is performed using a modification of Sanger's dideoxy chain 
termination method (Sambrook et al.) using double stranded DNA with the 
Sequenase 2 kit (United States Biochemical Corp., Cleveland, Ohio). In 
all, six first quarter clones are sequenced. Of the clones sequenced, only 
two clones designated pQA1 and pQA5 are found to contain only one deletion 
each. These deletions are of one base pair each located at position 452 in 
pQA1 and position 297 in pQA5. 
Plasmid pQA1 is used with pP1-8 (as described below) to obtain a first 
quarter with the expected sequence. 
Example 2B 
SYNTHESIS AND CLONING OF THE SECOND QUARTER [base pairs 531 to 
__________________________________________________________________________ 
PCR Primers: 
forward: 
P3 (a): 
5'-GCTGCGCGAC GTCAGCGTGT TCGG-3'(SEQ ID NO: 37) 
P3 (b): 
5'-AATTGCTGCG CGACGTCAGC GTG-3'(SEQ ID NO: 38) 
Reverse: 
P4 (a): 
5'-GGCGTTGCCC ATGGTGCCGT ACAGG-3'(SEQ ID NO: 39) 
P4 (b): 
5'-AGCTGGCGT TGCCCATGGT GCCG-3'(SEQ ID NO: 40) 
Primer pair B1: P3 (b) + P4 (a) 
Primer pair B2: P3 (a) + P4 (b) 
PCR Products 
B1 
AATTGCTGCG(SEQ ID NO: 41).sub.-- AACGCC 
(524 bp) second quarter 
B2 
GCTGCG.sub.-- AACGCCAGCT(SEQ ID NO: 42) 
(524 bp) 
__________________________________________________________________________ 
Hybridization, PCR amplification, spin column size fractionation, and 
cloning of this gene fragment in Bluescript digested with Eco RI/Hind III 
are performed as described above for the first quarter (Example 2A). The 
PCR product for this quarter is about 529 bp in size representing the 
second quarter of the gene (nucleotides 531 to 1050). Transformation is 
into frozen competent E. coli cells (DH5alpha) using standard procedures 
described above (Sambrook et al.) 
Miniscreen of pQB Clones 
Miniprep DNA is prepared as described above and digested with (a) Aat 
II/Nco I, (b) Pvu II and (c) with Bgl I to confirm the structure insert in 
the vector before sequencing. 
Sequencing is performed as described above using the dideoxy method of 
Sanger (Sambrook et al.). 
A total of thirteen clones for this quarter are sequenced. The second 
quarter consistently contains one or more deletions between position 884 
and 887. In most cases the G at position 884 is deleted. 
Plasmid pQB5 had only one deletion at position 884. This region lies 
between two Sac II sites (positions 859 and 949). Correction of this 
deletion is described in Example 3. Clones of the first half (1-1050 bp). 
A fragment for cloning the first half (quarters 1 and 2) of the synthetic 
Bt maize gene as a single DNA fragment is obtained by restriction 
digestion of the product of a PCR reaction comprising the first quarter 
and the second quarter. Restriction endonuclease Aat II is used to cut the 
DNA (following phenol extraction and ethanol precipitation) in a 20 .mu.l 
reaction. 15 .mu.l of each of the Aat II digested quarters is mixed and 
ligated (in a 50 .mu.l volume by adding 5 .mu.l of 10X ligase buffer, 
(1X=30 mM Tris-HCl pH 7.8, 10 mM MgC12, 10 mM DTT, 1 mM ATP) 14 .mu.l of 
deionized water and 1 .mu.l of T4 DNA ligase, 3 units, Promega, Madison, 
Wis.) at room temperature for 2 hr. The result is an about 1 kb fragment 
as judged by electrophoresis on a 1% agarose gel run using standard 
conditions (Sambrook et al.) Ten .mu.l of the ligation product is 
amplified by PCR using conditions described previously except that only 5 
cycles were run. 
Primer Pair: HA=P1(a)+P4(b) 
Primer Pair: HB=P1(b)+P4(a) 
The product of these reactions is cloned into Bluescript (Stratagene, La 
Jolla, Calif.) as described for the individual quarters. This procedure is 
only done once i.e., all insert DNA is obtained in a particular region 
from a single PCR reaction. 
Thirty-six colonies are miniscreened with Sal I digests and Pvu II digests. 
All except 4 contain an insert of approximately 1 kb in size of which at 
least 20 contain the correct Pvu II digestion pattern. Eight of these 
clones are selected for sequence analysis. One of the clones, P1-8, has 
the desired sequence between the Eco NI site (396 bp) and the Dra III site 
(640 bp). This clone is used to obtain a plasmid with the desired sequence 
up to the Dra III site (640 bp) in the second quarter with pQA1 (first 
quarter with a deletion at position 452 bp described previously.) 
Example 2C 
CLONING AND SYNTHESIS OF THIRD QUARTER [base pairs 1021 to 1500] 
Template: Oligos U15-U20 and L15-L21 
__________________________________________________________________________ 
PCR primers: 
forward 
P5 (a): 
5'-TTCCCCCTGT ACGGCACCAT GGGCAACGCC GC-3'(SEQ ID NO: 43) 
P5 (b): 
5'-AATTGTACGG CACCATGGGC AAC-3'(SEQ ID NO: 44) 
reverse 
P6 (a): 
5'-GAAGCCGGGG CCCTTCACCA CGCTGG-3'(SEQ ID NO: 45) 
P6 (b): 
5'-AGCTGAAGCC GGGGCCCTTC ACC-3'(SEQ ID NO: 46) 
Primer pair C1: P5 (b) + P6 (a) 
Primer pair C2: P5 (a) + P6 (b) 
PCR Product: 
C1 
AATTGTACGG(SEQ ID NO: 47).sub.-- GGCTTC 
(475 bp) 3d qtr 
C2 
TTCCCCTGTACGG(SEQ ID NO: 48).sub.-- GGCTTCAGCT 
(484 bp) 3d qtr 
__________________________________________________________________________ 
PCR reactions, spin column recovery of the correct sized DNA fragment, and 
ligation into vectors are performed as described above (Example 2A) using 
a Bluescript vector cut with Eco RI and Hind III. The approximately 479 
base pair PCR product represents the third quarter of the synthetic gene 
(NT 1021-1500). 
Transformation into frozen competent E. coli strain DH5alpha cells, 
selection and identification of transformants, characterization of 
transformants by mini-screen procedures, and sequencing of the synthetic 
gene fragment in the vector are all as described above. 
Mini Screen of pQC Clones 
The third quarter is miniscreened using standard procedures (Sambrook et 
al.). Miniprep DNA is cut with (a) Nco I/Apa I and (b) with Pvu II. Clones 
containing the correct restriction digest patterns are sequenced using 
standard procedures. A total of 22 clones of the third quarter are 
sequenced. Three major deletion "hotspots" in the third quarter are 
identified (a) at position 1083 (b) between position 1290-1397 and (c) 
between positions 1356-1362. In all clones except one, pQC8, there is also 
consistently an insertion of a C at position 1365. In addition to these 
mutations, the third quarter clones contain a large number of other 
apparently random deletions. The common factor to the three mutational 
"hotspots" in the third quarter and the one in the second quarter is that 
these regions are all flanked on either side by sequences that are about 
80% C+G. Other regions containing 5 to 9 C-Gs in a row are not affected. 
The oligomers in U15, U16, U18, U19, L15, L16, L18 and L19 are redesigned 
to reduce the C+G content in these regions. Five clones each from PCR 
reaction using the modified oligomers are sequenced. 
Plasmid pQCN103 has the correct sequence for the third quarter except for a 
change at position 1326. This change, which substitutes a G for a C, 
results in the substitution of one amino acid (leucine) for the original 
(phenylalanine). 
Example 2D 
SYNTHESIS AND CLONING OF FOURTH QUARTER [base pairs 1480 to 1960] 
The fourth quarter of the gene is obtained from a clone which is originally 
designed to comprise the third and fourth quarters of the gene. The 
"second half" of the synthetic gene is obtained from PCR reactions to fuse 
the third and fourth quarters. These reactions are run with PCR primers P5 
(a) and P6 (a) described above for the third quarter and primers P7 (a) 
and P8 (a) (described below). The reverse primer is modified to include a 
Sac I site and a termination codon. Separate reactions for each quarter 
are run for 30 cycles using the conditions described above. The two 
quarters are joined together by overlapping PCR and subsequently digested 
with restriction enzymes Nco I and Sac I. The resulting 953 bp fragment is 
cloned directionally into pCIB3054, which has been cut with Nco I/Sac I 
and treated with alkaline phosphatase. 
pCIB3054 is constructed by inserting intron #9 of PEPcarboxylase (PEPC ivs 
#9) in the unique Hpa I site of pCIB246 (described in detail in Example 4) 
pCIB246 is cut with HpaI and phosphatased with CIP using standard 
procedures described in Example 2A. PEPC ivs #9 is obtained by PCR using 
pPEP-10 as the template. pPEP-10 is a genomic subclone containing the 
entire maize PEP carboxylase gene encoding the C.sub.4 photosynthetic 
enzyme, plus about 2.2 Kb of 5'-flanking and 1.8 Kb of 3'-flanking DNA. 
The 10Kb DNA is ligated in the HindIII site of pUC18. (Hudspeth et al., 
Plant Molecular Biology, 12:576-589 (1989). The forward PCR primer used to 
obtain the PEPCivs#9 is GTACAAAAACCAGCAACTC (SEQ ID NO:50) and the reverse 
primer is CTGCACAAAGTGGAGTAGT (SEQ ID NO:51). The PCR product is a 108 bp 
fragment containing only the PEPcarboxylase intron #9 sequences. The PCR 
reaction is extracted with phenol and chloroform, ethanol precipitated 
phosphorylated with polynucleotide kinase and treated with T4 polymerase 
to fill in the 3' nontemplated base addition found in PCR products (Clark, 
J. M., Nucleic Acid Research, 16:9677-9686 (1988)) using standard 
procedures. The kinased fragment is blunt-end cloned into the HpaI site of 
pCIB246, using standard procedures described earlier. 
Amplification and Assembly of the Fourth Quarter 
Template: U21-U26 and L22-L28 
__________________________________________________________________________ 
PCR primers 
FORWARD 
P7 (a): 
5'-TGGTGAAGGG CCCCGGCTTC ACCGG-3'(SEQ ID NO: 52) 
REVERSE 
P8 (a): 
5'-ATCATCGATG AGCTCCTACA CCTGATCGAT GTGGTA-3'(SEQ ID NO: 53) 
PRIMER PAIR 4: P7 (a) + P8 (a) 
PRIMER PAIR 3: P5 (A) + P6 (a) 
Primer pair for overlapping PCR : P7 (a) + P8 (a) 
PCR Product 
fourth quarter: GGTGAA.sub.-- ATCAGGAGCTCATCGATGAT(SEQ ID NO: 54) 
(484 bp) third quarter: TTCCCCCTGTA(SEQ ID NO: 55)-TTCACCGG 
(484 bp) second half: GGTGAA-CATGATGAT (953 bp) 
__________________________________________________________________________ 
Four positive clones are identified by plasmid miniscreen and are 
subsequently sequenced using standard procedures. 
Plasmid Bt.P2 #1 contains approximately the correct fourth quarter sequence 
except for two mutations. These mutations are at position 1523 
(substituting an A for a G, resulting in an amino acid change which 
substitutes a His for an Arg) and at position 1634 (substituting a T for a 
C, resulting in an amino acid substitution of a Ser for a Thr). 
Plasmid Bt.P2#1 is used in the construction of pCIB4414 described below. 
(The mistakes are ultimately corrected by hybridizing all the oligos of 
the fourth quarter, digesting with Apa I/Bst EII and replacing that region 
in pCIB4414. Therefore, only sequences from position 1842-1960 remain of 
Bt.P2#1 in the final construct.) 
Example 3 
ASSEMBLY AND REPAIR OF THE FINAL SYNTHETIC GENE 
The synthetic maize optimized Bt cryIA(b) gene is designed to be cloned in 
quarters. Using the PCR technique, however, results in mutations, which in 
most cases are deletions resulting in frameshift mutations. Plasmids 
containing individual quarters are therefore sequenced and the correct 
parts ligated together using standard procedures. 
After obtaining first and second quarter clones with almost the desired 
sequence, plasmids pEB1Q#4 and pEB1Q#5 are constructed to obtain the 
desired sequence of the synthetic Bt gene up to the Dra III site at the 
base pair position 634 (this mutation destroys the Dra III site). The 
pEB1Q constructs are made by ligating a 3.9 Kb Eco NI.backslash.Bam HI 
fragment from pP1-8 with a 400 bp fragment from pQA1. pEB1Q#5 has the 
desired sequence up to the Dra III site, but pEB1Q#4 has a mutation at 
base pair position 378. 
Plasmids p1H1M4 and p1H1M5 are constructed to repair the Dra III site in 
pEB1Q#4 and pEB1Q#5. Plasmids p1H1M#4 and #5 are made by ligating a 3.5 Kb 
Nco I.backslash.Aat II fragment from pEB1Q#4 and #5 respectively, with a 
500 bp Nco I.backslash.Aat II fragment from pQB5. Plasmid p1H1M5 contains 
a mutation between the Sac II sites at position 884 in the second quarter 
of the synthetic Bt gene. Plasmid p1H1M4 contains the additional mutation 
as described in its precursor construct pEB1Q#4. 
The Sac II site in the Bluescript vector region of p1H1M4 is deleted by 
cutting p1H1M4 with Not I and Sac I and converting these sites to blunt 
ends using T4 DNA polymerase under standard conditions before ligating 
this 3.9 Kb fragment to make p1H1M4 S. Deleting the Sac II site in the 
vector region allows the 90 bp Sac II fragment with the mutation at 
position 884 in the 2nd quarter of p1H1M4 S to be removed prior to 
replacement with a 90 bp Sac II fragment. Oligomers U.backslash.L 12 and 
13 are kinased and hybridized (described above) before cutting with Sac II 
and isolating a 90 bp fragment on a 2% Nusieve gel. The Sac II fragment is 
ligated into the about 3.8 Kb Sac II cut p1H1M4 S vector which has been 
phosphatased with CIP. The repaired Sac II construct is called pHYB2#6. 
The orientation of the Sac II fragment in pHYB2#6 is detected by PCR 
screening as described earlier using the following primers: 
__________________________________________________________________________ 
MK23A28 = 5'-GGGGCTGCGGATGCTGCCCT-3'(SEQ ID NO: 56) 
MK25A28 = 5'-GAGCTGACCCTGACCGTGCT-3'(SEQ ID NO: 57) 
MK26A28 = 5'-CACCTGATGGACATCCTGAA-3'(SEQ ID NO: 58) 
__________________________________________________________________________ 
Running the PCR reactions with 50 pmoles of primers MK23A28 and MK25A28 
produces an approximate 180 bp fragment, indicating the inserted fragment 
bounded by the Sac II sites in pHYB2#6 is in the correct orientation. 
Using primers MK25A28 and MK26A28 in the PCR screening acts as the 
negative control producing an approximate 180 bp fragment only in 
constructs containing the Sac II bounded fragment in the wrong 
orientation. pHYB2#6 sequence is determined using standard procedures. 
pHYB2#6 has one mutation at position 378 which needed to be repaired to 
obtain a first quarter containing the desired sequence. 
Plasmid p1HG#6 contains the desired sequence for the entire first half of 
the synthetic Bt gene. p1HG#6 is made from a 3.4 Kb Aat II.backslash.Nco I 
fragment of p1H1M5#2 ligated to a 500 bp Aat II.backslash.Nco I fragment 
from pHYB2#6. 
To identify clones or partial clones of the synthetic gene which contain 
open reading frames, the kanamycin selection vector (described above) is 
used. The fourth quarter of the synthetic Bt gene is the first put into 
the kanamycin cassette. pKM74-4 contains the approximately 500 bp Apa 
I.backslash.Cla I fragment from plasmid BtP2 (which had been previously 
transformed into a dam- E. coli strain (PO-100) to be able to cut with Cla 
I), ligated to pUC:KM74 cut with Apa I.backslash.Cla I. Plasmid pKM74-4 
displays kanamycin resistance but is later found to contain two 
substitution mutations at positions 1523 and 1634 (mutations are described 
above in the section on cloning the fourth quarter; they are 
substitutions, not deletions or insertions). 
The correct first half of the synthetic Bt gene from plasmid p1HG#6 is 
inserted into plasmid pKM74-4. The resulting plasmid, called pKm124, is 
made from the about 3.9 Kb Apa I.backslash.Bam HI fragment derived from 
pKM74-4 ligated to 1 Kb Apa I.backslash.Bam HI fragment from p1HG#6. 
pKm124 shows kanamycin resistance. This plasmid contains the first, 
second, and fourth quarters of the synthetic gene forming a single open 
reading frame. 
The third quarter of the synthetic gene is next cloned into pKM124. The 
first functional clone, in plasmid pBt:Km#6, is a functional copy of the 
truncated synthetic cryIA(b) gene in the Km-cassette which displays 
kanamycin resistance but which contains deletion mutations between the 
third and fourth quarters. Plasmid pBt:Km#6 is made from the approximately 
5 Kb Apa I.backslash.Nco I pKM124 vector fragment ligated to the 
approximately 500 bp Apa I.backslash.Nco I fragment from pQCN103 (pQCN103 
contains a mismatch mutation at position 1326 which is repaired later). 
Contaminating nuclease activity appears to have deleted the Apa I site 
between the third and fourth quarters in pBt:Km#6. The Bt gene encoded by 
the synthetic gene in plasmid pBT:Km#6 has about 50-60 % of the native 
proteins' activity against ECB. The 2 Kb Sma I.backslash.Bam HI fragment 
from pBt:Km#6 is inserted into a 35S:expression cassette to make a plasmid 
called 35S:Bt6. 
Two functional synthetic Bt clones, each with mutations, are initially 
obtained: plasmids pBT:KM#6 and pCIB4414. pCIB4414, which is 100% active 
in insect bioassays against European corn borer compared with the native 
gene, contains substitution mutations in the third and fourth quarters at 
positions 1323, 1523, and 1634. 
pCIB4414 is constructed from two plasmids, MG3.G4#18 and 1HG which is 
described above. MG3.G4#18 is obtained by cloning the Apa I/Kpn I fragment 
in plasmid Bt.P2#1 into pQCN103 (using those same restriction sites). This 
produces a plasmid containing the third and fourth quarters of the gene. 
The first half of the synthetic gene from plasmid 1HG is cut with Bam HI 
and Nco I and moved into MG3.G4#18 (containing the third and fourth 
quarters of the gene). The resulting plasmid, pCIB4414, contains a 
functional version of the synthetic gene. While being functional, the 
synthetic gene in this plasmid contains three errors; position 1326 (G 
substituted for a C), position 1523 (substitute A for a G), and at 
position 1634 (substitution of a T for a C). 
The fourth quarter in pCIB4414 is replaced with a 354 bp fourth quarter Apa 
I.backslash.Bst EII fragment obtained from hybridizing, ligating, and 
restriction cleaving fourth quarter oligomers as described earlier, and 
isolating the fragment from a 2% Nusieve agarose gel. pCIB4408 is a 
synthetic Bt gene clone obtained by replacing the fourth quarter fragment 
in pCIB4414 with the hybridized fourth quarter fragment. To insert the 
CaMV 35S promoter in front of the synthetic Bt gene, pCIB4406 is made from 
a 4 Kb Eco NI.backslash.Kpn I fragment from plasmid p35SBt6 and 1.8 Kb Eco 
NI.backslash.Kpn I fragment from pCIB4408. 
pCIB4406 is 100% active (as compared with the protein from the native gene) 
against ECB but contains the substitution mutation in the third quarter of 
the synthetic gene at position 1323 resulting in an amino acid 
substitution of a leucine for a phenylalanine. Plasmid pBS123#13 is used 
to repair this mutation. 
The third quarter fragment in plasmid pBS123#13 is made from an 
approximately 479 bp hybridized oligomer generated fragment. Third quarter 
oligomers U15-U20 and L15-L21 are kinased, hybridized, and ligated as 
described above. PCR reactions are carried out as described above with 
primers P5(a) and P6(b) for 15 cycles. The PCR product is treated with 
proteinase K at a final concentration of about 50 .mu.g.backslash.ml in an 
approximate 95 .mu.l volume for 30 minutes at 37.degree. C. followed by 10 
minutes at 65.degree. C. (Crowe et al., Nucleic Acid Research 19:184, 
1991.) Subsequently, the product is phenol.backslash.chloroform extracted 
and ethanol precipitated using standard procedures before cutting with 
restriction enzymes Apa I and Nco I. 
The approximate 450 bp Apa I.backslash.Nco I PCR fragment is ligated to the 
3.8 Kb Apa I.backslash.Nco I vector fragment from p1HG#6 to make 
pBS123#13. Plasmid pBS123#13 contains the desired sequence for the third 
quarter of the maize optimized cryIA(b) gene from position 1319 at the Nsp 
I site through the Apa I site at position 1493. This 170 bp Nsp 
I.backslash.Apa I fragment from pBS123#13 is used in the fully active 
synthetic cryIA(b) gene in plasmid pCIB4418. 
Western Blot Analysis 
Western blot analyses of various transformants are performed using crude 
extracts obtained from E. coli grown on selective plates. Using a 
toothpick, cultures are scraped from fresh plates containing the 
transformants of interest which have been grown overnight at 37.degree. C. 
The positive control for expression of the Bt gene in E. coli was a 
construct called pCIB3069 which contains the native Bt-k gene fused with 
the plant expressible CaMV 35S promoter. pCIB3069 also contains the 35S 
promoter operably linked to the hygromycin resistance gene, 35S promoter, 
with Adh intron #1 operably linked to the GUS gene, and 35S promoter 
operably linked to a gene coding for the production of the native Bt 
cryIA(b) IP. A negative control of E. coli which does not contain a Bt 
gene is also included in the analyses. Cultures are resuspended in 100 
.mu.l of loading buffer containing 62 mM Tris-HCl pH 6.8, 1% SDS, 0.0025% 
bromophenol blue, 10% glycerol and 7.5% mercaptoethanol. After heating the 
mixtures at 95.degree. C. for 10 minutes, the preparations are sonicated 
for 1-3 seconds. The debris is centrifuged in a microfuge at room 
temperature for about 5 minutes and 10 to 15 .mu.l of each sample is 
loaded onto an acrylamide gel with a 10% running gel below a 6% stacking 
gel (Laemmli, Nature 227;680-685(1970)). After electrophoresis overnight 
at 10 mAmps, proteins are transferred from the gel to an Immobilon 
membrane (Millipore). The transfer is done using an electrophoretic 
Blotting Unit (American BioNuclear, Emeryville, Calif.) in transfer buffer 
(20 mM Tris, 150 mM glycine, and 20% methanol) for 1.5 hours at 450 mAmps. 
Buffers for western blotting included: 
Blocking buffer: 
2% Tween-20 
30 mM Tris-HCl pH 10.2 
150 mM NaCl 
Wash buffer: 
0.05% Tween-20 
30 mM Tris-HCl pH 10.2 
150 mM NaCl 
Developing buffer: 
100 mM Tris-HCl pH 9.6 
100 mM NaCl 
10 mM MgC12 
After transfer is complete, the membrane is incubated for about ten minutes 
in the blocking buffer. Three 15 minute washes with wash buffer are done 
before the first antibody treatment. The first antibody is an 
immunoaffinity purified rabbit or goat antibody prepared using the 
CryIA(b) protein as the antigen (Ciba-Geigy, RTP, N.C.; Rockland Inc., 
Gilbertsville, Pa.; and Berkeley Antibody CO., Richmond, Calif.). The 
cryIA(b) specific antibody is treated immediately before use with E. coli 
lysate from Bio-Rad in a 1 ml volume with 5 .mu.g of antibody, 50 .mu.l of 
E. coli lysate in the wash buffer solution. This mixture is incubated for 
1 hour at room temperature before diluting it 1 to 30 for a final dilution 
of 1:6000 with wash buffer. Incubation of the membrane with the first 
antibody is at room temperature for 1.5 hours. 
Three 10 minute washes are done between the 1st and 2nd antibody 
treatments. The second antibody is either rabbit anti-goat or goat 
anti-rabbit/alkaline phosphatase conjugate (Sigma, St. Louis, Mo.). 
Incubation with the alkaline phosphatase conjugate is carried out at room 
temperature for one hour using a 1 to 6000 dilution in wash buffer. Six 10 
minute washes are done between the second antibody treatment and 
developing the western blot. The western blot is developed in 100 ml of 
developing buffer with 440 .mu.l of nitroblue tetrazolium in 70% dimethyl 
formamide (75 mg.backslash.ml), and 330 .mu.l of 
5-bromo-4-chloro-indolyl-phosphate in 100% dimethyl formamide (50 
mg.backslash.ml). After developing for 15 to 30 minutes, the membrane is 
washed in water and air dried. 
Example 4 
CONSTRUCTION OF TRANSFORMATION VECTORS 
Construction of pCIB710 and Derivatives 
CaMV 35S Promoter Cassette Plasmids pCIB709 and pCIB710 are constructed as 
shown in Rothstein et al., Gene 53:153-161 (1987). pCIB710 contains CaMV 
promoter and transcription termination sequences for the 35S RNA 
transcript [Covey et al., Nucl. Acids. Res., 9:6735-6747 (1981)]. A 1149 
bp BglII restriction fragment of CaMV DNA [bp 6494-7643 in Hohn et al., 
Current Topics in Microbiology and Immunology, 96:194-220 and Appendices A 
to G (1982)] is isolated from CaMV DNA by preparative agarose gel 
electrophoresis as described earlier The fragment is mixed with 
BamHI-cleaved plasmid pUC19 DNA, treated with T4 DNA ligase, and 
transformed into E. coli. (Note the BamHI restriction site in the 
resulting plasmid is destroyed by ligation of the BglII cohesive ends to 
the BamHI cohesive ends.) 
The resulting plasmid, called pUC19/35S, is then used in 
oligonucleotide-directed in-vitro mutagenesis to insert the BamHI 
recognition sequence GGATCC immediately following CaMV nucleotide 7483 in 
the Hohn reference. The resulting plasmid, pCIB710, contains the CaMV 35S 
promoter region and transcription termination region separated by a BamHI 
restriction site. DNA sequences inserted into this BamHI site will be 
expressed in plants by these CaMV transcription regulation sequences. 
(Also note that pCIB710 does not contain any ATG translation initiation 
codons between the start of transcription and the BamHI site). 
pCIB710 is modified to produce pCIB709 by inserting a Bam HI fragment 
containing the coding sequence for hygromycin phosphotransferase from 
pLG90 [Rothstein et al., Gene, 53:153-161 (1987)] in the Bam HI site. 
pCIB709 is modified to produce pCIB996 by removing the ATG just upstream 
from the initiation codon of the hygromycin phosphotranserase gene using 
standard mutagenesis techniques while inserting a Bgl II restriction site 
at this location. The resulting plasmid, pCIB996, is further modified to 
remove the Bam HI, Sma I and Bgl II sites in the 5' untranslated leader 
region located 5' of the initiation codon for the initiation codon. The 
result is a change of DNA base sequence from --TATAAGGATC CCGGGGGCA 
AGATCTGAGA TATG(SEQ ID NO:59)-Hyg to --TATAAGGATC TGAGATATG (SEQ ID NO:59 
with nucleotides 11-24 deleted)-Hyg. The resulting plasmid is known as 
pCIB3073. 
Alternatively, pCIB710 is modified to produce pCIB900, by inserting the Bam 
HI-Bcl I fragment of pCIB10/35SBt, which contains the 645 amino acid Bt 
coding sequence, described in Part C4 below, into the Bam HI site of 
pCIB710 to create pCIB710/35SBt. To introduce an antibiotic resistance 
marker, pCIB709 is cut with Sal I, a Kpn I/Sal I adaptor is ligated and 
the resulting ligation product is cut with Kpn I. The Kpn fragment of 
pCIB709 containing the 35S/hygromycin resistance gene is inserted into the 
Kpn I site of pCIB710/35SBt to produce pCIB900. 
Genes useful as the selectable marker gene include the hygromycin 
resistance gene described in Rothstein et al., Gene 53:153-161 (1987). The 
hygromycin gene described in this reference is moved into a pUC plasmid 
such as pCIB710 or pCIB709 and the "extra" ATG upstream from the 
hygromycin phosphotransferase coding sequence is removed to create 
pCIB996. This modified pCIB996 gene is further modified to remove a BglII, 
BamHI and SmaI sites from the 5' region of the gene using standard 
techniques of molecular biology to make pCIB3073. 
pCIB932 is a pUC19-based plasmid containing the chimeric gene 
Pep-C:promoter.backslash.Bt.backslash.Pep-C:terminator. It is composed of 
fragments derived from pPEP-10, a HindIII subclone of a genomic clone, 
Hi-lambda-14, PNAS USA, 83:2884-2888 (1986), of the maize gene encoding 
the PEP carboxylase enzyme active in photosynthesis, and from pCIB930, 
which is a BamHI fragment containing the 645 amino acid truncated form of 
the the cryIAb endotoxin gene in the BamHI site of pUC18. 
The 2.6 kb EcoRI-XhoI fragment from pPEP-10, containing the polyA addition 
site from the PEP carboxylase gene, is isolated and digested with PstI and 
HincII. The restriction digest is ligated with PstI/HincII digested pUC18, 
transformed into E. coli and transformants screened for those containing a 
412 bp PstI-HincII insert in pUC18 and the insert verified by sequencing. 
The resulting plasmid is called pCIB931. 
The nuclear gene encoding the phosphoenolpyruvate carboxylase isozyme 
("Pep-C") is described in Hudspeth et al., Plant Molecular Biology, 
12:579-589 (1989). pCIB932 is constructed by the ligation of three 
fragments. The first fragment, containing the PEP-C transcription 
terminator, is produced by digesting pCIB931 to completion with HindIII, 
partially with SphI and the 3098 bp fragment isolated. The second 
fragment, containing the Bt endotoxin coding sequence, is produced by 
digesting pCIB930 with NcoI and SphI and isolating the 1950 bp fragment. 
The third fragment, containing the PEP-C promoter, is produced by 
digesting pPEP-10 to completion with HindIII, partially with NcoI and 
isolating the 2.3 kb fragment. The ligation mix is transformed into E. 
coli, transformants with the correct insertion identified and the insert 
verified by sequencing. 
pCIB932 is cut with PvuII to generate a 4.9 Kb fragment containing the 
maize Pep-C:promoter.backslash.Bt.backslash.Pep-C:terminator and purified 
on a 1% LGT agarose gel in 1X TAE. The linearized pCIB3079 vector and the 
4.9 Kb insert from pCIB932 are ligated using T4 DNA ligase in LGT to make 
pCIB4401. pCIB4401 is a maize transformation vector containing the 
chimeric genes: 35S:promoter.backslash.PAT.backslash.35S:terminator, 
Pep-C:promoter.backslash.Bt.backslash.Pep-C: terminator, and 
35S:promoter.backslash.AdhI #1 intron.backslash.GUS.backslash.35S: 
terminator. 
Construction of pCIB246 (35S-GUS-35S) 
A CaMV 35S promoter cassette, pCIB246, is constructed as follows. 
The DdeI restriction site at nucleotide position 7482 of the CaMV genome 
[Franck et al., Cell, 21:285-294 (1980)] is modified by insertion of a 48 
bp oligonucleotide containing several restriction enzyme sites including 
an NcoI (CCATGG) site, a SalI (GTCGAC) site, and an SstI (GAGCTC) site. 
This altered CaMV 35S promoter is inserted into a pUC19 vector that had 
been modified to destroy the vector's SstI and SalI sites. Thus, the CaMV 
35S promoter of pCIB1500 contains unique SstI and SalI sites for cloning. 
pCIB1500 is digested with SstI/NcoI and ligated with the GUS gene obtained 
from pBI221 (Clontech Laboratories, Inc., Palo Alto, Calif.). The NcoI 
site is fused to the GUS gene such that the ATG of the NcoI site functions 
as the start codon for the translation of the GUS gene. The CaMV 35S 
polyadenylation and termination signals are used for the 3' end of the 
chimeric gene. 
Construction of pCIB3069 (35S-Adh1-GUS-35S) 
pCIB246 is modified by adding the maize alcohol dehydrogenase gene Adh1 
intron number 1 (Adh1) (Dennis et al., Nucleic Acids Research, 
12:3983-4000 (1984)) into the Sal I site of pCIB246 to produce plasmid 
pCIB3007. The Adh1 intron is excised from the maize Adh1 gene as a Bal 
I/Pst I fragment and subcloned into pUC18 that was cut with Sma I/Pst I to 
make a plasmid called Adh 1026. Adh 1026 is cut with Pvu II/Sac II, the 
fragments are made blunt ended with T4 DNA polymerase, Sal I linkers are 
added using standard procedures and a fragment of about 560 bp is 
recovered from a 3% NuSeive gel and ligated into Sal I cut/phosphatase 
treated pUC18. The Sal I linkered Adh intron #1 in the resulting plasmid 
is cut out with Sal I, gel purified, and ligated into Sal I 
cut/phosphatase treated pCIB246 to make plasmid pCIB3007. 
pCIB3007 is cut with PstI and the ends made blunt by using T4 DNA 
polymerase (NEW England Biolabs) according to the suppliers' 
specifications. The resulting blunt ended molecules are cut with Sph I and 
the approximately 5.8 Kb fragment with one blunt end and one Sph I end is 
purified on a low gelling temperature (LGT) agarose gel using standard 
procedures. pCIB900 is cut with Sma I/Sph I and the fragment containing 
the 35S/Bt gene is purified on a LGT agarose gel. The two gel purified 
fragments are ligated in LGT agarose using T4 DNA ligase according to 
standard conditions. The resulting ligated fragments are transformed into 
E. coli using standard procedures and the resulting plasmid is called 
pCIB3062. There are two versions of pCIB3062. pCIB3062#1 has a Sma I site 
regenerated where the Sma I site and the T4 polymerase blunted ends are 
ligated. This most likely results from the T4 polymerase nibbling a few 
base pairs from the Pst I site during the blunting reaction. pCIB3062#3 
does not have this SmaI site. 
pCIB3062#3 is cut with KpnI and made blunt-ended using T4 DNA polymerase, 
and subsequently cut with Pvu II to yield a 6.4 Kb fragment with blunt 
ends containing the 35S/GUS and 35S/Bt genes. This blunt-end fragment is 
ligated into Sma I cut pCIB3073 to produce pCIB3063 or pCIB3069. pCIB3069 
contains the same fragment used to make pCIB3063, but the chimeric genes 
in pCIB3069 are all in the same relative orientation, unlike those in 
pCIB3063. These plasmids contain a) a 35S promoter operably linked to the 
hygromycin resistance gene; b) a 35S promoter, with Adh intron #1, 
operably linked to the GUS gene; and c) a 35S promoter operably linked to 
a gene coding for the production of the synthetic cryIA(b) insecticidal 
protein from Bacillus thuringiensis, as described above. 
GUS Assays 
GUS assays are done essentially as described in Jefferson, Plant Mol. Bio. 
Reporter, 5:387-405 (1987). As shown above, plasmid pCIB246 contains a 
CaMV 35S promoter fused with the GUS gene. The 5' untranslated leader of 
this chimeric gene contains a copy of the maize Adh1 intron #1. It is used 
here as a transformation control. Although the same amount of pCIB246 is 
added to each transformation, the calculated activity varied among Bt 
constructs tested. The values reported below are averages of 3 replicates. 
pCIB4407 was tested twice. 
______________________________________ 
pCIB3069 28 nM MU/ug/min 
pCIB4407 0.7 nM MU/ug/min, 2.3 nM MU/ug/min 
______________________________________ 
Example 5A 
ASSAY OF SYNTHETIC cryIA(b) gene FOR INSECTICIDAL ACTIVITY AGAINST EUROPEAN 
CORN BORER 
The synthetic cryIA(b) gene in pCIB4414 in E. coli is assayed for 
insecticidal activity against European corn borer according to the 
following protocol. 
Molten artifical insect diet is poured into a 60 mm Gellman snap-cap petri 
dish. After solidification, E. coli cells, suspended in 0.1% Triton X-100, 
are spread over the surface at a concentration of 3.times.107 cells/cm2. 
The plates are air dried. Ten first instar European corn borer, Ostrinia 
nubilalis, which are less than 12 hours old are then placed onto the diet 
surface. The test is incubated at 30 C. in complete darkness for 2-5 days. 
At the end of the test percent mortality is recorded. A positive clone has 
been defined as one giving 50% or higher mortality when control E. coli 
cells give 0-10% background mortality. 
For comparison, the native cryIA(b) gene in pCIB3069 is tested at the same 
concentration. Clones are tested at 3.times.10.sup.7 cells/cm.sup.2 diet; 
20 insects per clone. 
The following results are observed: 
______________________________________ 
Clone Percent Mortality 
______________________________________ 
Control 0 
pCIB3069 100 
pCIB4414 100 
______________________________________ 
These results indicate that the insecticidal crystal protein produced by 
the synthetic cryIA(b) gene demonstrates activity against European corn 
borer comparable to that of the IP produced by the native cryIA(b). Other 
plasmids containing a synthetic cryIA(b) gene were assayed in a similar 
manner. 
Example 5AB 
ASSAY OF CRYIA(b) PROTEIN FOR INSECTICIDAL ACTIVITY AGAINST SUGARCANE 
BORER. 
CryIA(b) was expressed in E. coli and assayed for insecticidal activity 
against Sugarcane borer (Diatrea saccharalis) according to the same 
protocol used for European corn borer, described immediately above. The 
results are summarized in the Table. 
TABLE 
______________________________________ 
SUGARCANE BORER ASSAY WITH Bt PROTEIN 
FROM E. COLI 
Protein Percent 
Concentration Mortality 
(ng/g) CryIA(b) 
______________________________________ 
10 0 
25 0 
50 7 
100 13 
250 40 
500 53 
1000 80 
LC50 380 
95% Cl 249-646 
______________________________________ 
The results indicate that the insecticidal protein produced by a maize 
optimized Bt gene is effective against Sugarcane borer. The upper 
concentrations of CryIA(b) protein, 250 ng/g1000 ng/g, are achievable in 
transgenic maize plants produced in accordance with the instant invention 
 
Example 6 
MAIZE PROTOPLAST ISOLATION AND TRANSFORMATION WITH THE SYNTHETIC BT GENE 
Expression of the synthetic Bt gene is assayed in transiently transformed 
maize protoplasts. 
Protoplast Isolation Procedure 
1. The contents of 10 two day old maize 2717 Line 6 suspension cultures are 
pipetted into 50 ml sterile tubes and allowed to settle. All culture media 
is then removed and discarded. 
2. Cells (3-5 ml Packed Cell Volume) are resuspended in 30 ml protoplast 
enzyme solution. Recipe follows: 
3% Cellulase RS 
1% Macerozyme R10 in KMC Buffer 
______________________________________ 
KMC Buffer (recipe for 1 liter) 
______________________________________ 
KCl 8.65 g 
MgCl.sub.2 --6H.sub.2 O 
16.47 g 
CaCl.sub.2 --2H.sub.2 O 
12.50 g 
MES 5.0 g 
pH 5.6, filter sterilize 
______________________________________ 
3. Mix cells well and aliquot into 100.times.25 mm petri dishes, about 15 
ml per plate. Shake on a gyratory shaker for 4 hours to digest. 
4. Pipette 10 ml KMC through each 100 micron sieve to be used. Filter 
contents of dishes through sieve. Wash sieve with an equal volume KMC. 
5. Pipette sieved protoplasts carefully into 50 ml tubes and spin in a 
Beckman TJ-6 centrifuge for 10 minutes at 1000 rpm (500.times.g). 
6. Remove supernatant and resuspend pellet carefully in 10 ml KMC. Combine 
contents of 3 tubes into one and bring volume to 50 ml with KMC. 
7. Spin and wash again by repeating the above step. 
8. Resuspend all washed protoplasts in 50 ml KMC. Count in a hemocytometer. 
Spin protoplasts and resuspend at 8.times.10.sup.6 /ml in resuspending 
buffer (RS Buffer). 
______________________________________ 
RS Buffer (recipe for 500 ml) 
______________________________________ 
mannitol 27.33 g 
CaCl.sub.2 (0.1 M stock) 
75 ml 
MES 0.5 g 
pH 5.8, filter sterilize 
______________________________________ 
Protoplast Transformation Procedure 
1. Aliquot 50 .mu.g plasmid DNA (Bt IP constructs, both synthetic 
(pCIB4407) and native (pCIB3069)) to 15 ml polystyrene culture tubes. Also 
aliquot 25 .mu.g GUS-containing plasmid DNA (which does not contain Bt IP 
(pCIB246) to all tubes. 3 replications are used per construct to be 
tested, with 1 rep containing no DNA as a control. 
______________________________________ 
Bt constructs: GUS construct: 
______________________________________ 
pCIB3069 pCIB246 
pCIB4407 
______________________________________ 
2. Gently mix protoplasts well and aliquot 0.5 ml per tube. 
3. Add 0.5 ml PEG-40 to each tube. 
PEG-40 
0.4M mannitol 
0.1M Ca(NO.sub.3).sub.2 --4H.sub.2 O 
pH 8.0, filter sterilize 
4. Mix gently to combine protoplasts with PEG. Wait 30 minutes. 
5. Sequentially add 1 ml, 2 ml, and 5 ml W5 solution at 5 minute intervals. 
W5 Solution 
154 mM NaCl 
125 mM CaCl.sub.2 --H.sub.2 O 
5 mM KCl 
5 mM glucose 
pH 7.0, filter sterilize 
6. Spin for 10 minutes in a Beckman TJ-6 centrifuge at about 1000 rpm 
(500g). Remove supernatant. 
7. Gently resuspend pellet in 1.5 ml FW media and plate carefully in 
35.times.10 mm petri dishes. 
FW Media (Recipe for 1 liter) 
______________________________________ 
MS salts 4.3 g 
200X B5 vits. 5 ml 
sucrose 30 g 
proline 1.5 g 
mannitol 54 g 
2,4 D 3 mg 
pH 5.7, filter sterilize 
______________________________________ 
8. Incubate overnight in the dark at room temperature. 
9. Perform GUS assays, insect bioassays, and ELISA's on protoplast extracts 
as described below. 
Example 7 
CONSTRUCTION OF A FULL-LENGTH SYNTHETIC MAIZE OPTIMIZED CRYIA(b) GENE 
SEQ ID NO:4 shows the synthetic maize optimized sequence encoding the 
full-length cryIA(b) insecticidal protein from B. thuringiensis. The 
truncated version described above represents the first approximately 2 Kb 
of this gene. The remainder of the full-length gene is cloned using the 
procedures described above. Briefly, this procedure entails synthesizing 
DNA oligomers of 40 to 90 NT in length, typically using 80 mers as an 
average size. The oligomers are purified using standard procedures of HPLC 
or recovery from a polyacrylamide gel. Purified oligomers are kinased and 
hybridized to form fragments of about 500 bp. The hybridized oligomers can 
be amplified using PCR under standard conditions. The 500 bp fragments, 
either directly from hybridizations, from PCR amplification, or recovered 
from agarose gels after either hybridization or PCR amplification, are 
then cloned into a plasmid and transformed into E. coli using standard 
procedures. 
Recombinant plasmids containing the desired inserts are identified, as 
described above, using PCR and/or standard miniscreen procedures. Inserts 
that appear correct based upon their PCR and/or restriction enzyme profile 
are then sequenced to identify those clones containing the desired open 
reading frame. The fragments are then ligated together with the 
approximately 2 Kb synthetic sequence described in Example 2 to produce a 
full-length maize optimized synthetic cryIA(b) gene useful for expression 
of high levels of CryIA(b) protein in maize. 
______________________________________ 
G + C Content of native and synthetic Bt genes: 
______________________________________ 
Full-length native 38.8% 
Truncated native 37.2% 
Full-length synthetic 64.8% 
Truncated synthetic 64.6% 
______________________________________ 
% homology of the final truncated version of the Bt gene relative to a 
"pure" maize codon usage gene: 98.25% Example 8 
Construction of a plant expressible, full-length, hybrid partially maize 
optimized cryIA(b) gene 
pCIB4434 contains a full length CryIA(b) gene (SEQ ID NO:8) comprised of 
about 2 Kb of the synthetic maize optimized cryIA(b) gene with the 
remainder (COOH terminal encoding portion) of the gene derived from the 
native gene. Thus, the coding region is a chimera between the synthetic 
gene and the native gene, but the resulting protein is identical to the 
native cryIA(b) protein. The synthetic region is from nucleotide 1-1938 
(amino acids 1 to 646) and the native coding sequence is from nucleotide 
1939-3468 (amino acids 647 to 1155). The sequence of this gene is set 
forth in FIG. 7. A map of pCIB4434 is shown in FIG. 8. 
The following oligos were designed to make pCIB4434: 
__________________________________________________________________________ 
KE134A28 = 5'-CGTGACCGAC TACCACATCG ATCAAGTATC CAATTTAGTT 
GAGT-3'(SEQ ID NO: 60) 
KE135A28 = 5'-ACTCAACTAA ATTGGATACT TGATCGATGT GGTAGTCGGTC 
ACG-3'(SEQ ID NO: 61) 
KE136A28 = 5'-GCAGATCTGA GCTCTTAGGT ACCCAATAGC GTAACGT-3'(SEQ ID NO: 62) 
KE137A28 = 5'-GCTGATTATG CATCAGCCTAT-3'(SEQ ID NO: 63) 
KE138A28 = 5'-GCAGATCTGA GCTCTTATTC CTCCATAAGA AGTAATTC-3'(SEQ ID NO: 
64) 
MK05A28 = 5'-CAAAGGTACC CAATAGCGTA ACG-3'(SEQ ID NO: 65) 
MK35A28 = 5'-AACGAGGTGT ACATCGACCG-3'(SEQ ID NO: 66) 
__________________________________________________________________________ 
pCIB4434 is made using a four-way ligation with a 5.7 kb fragment from 
pCIB4418, a 346 bp Bst E II.backslash.Kpn I PCR-generated synthetic:native 
fusion fragment, a 108 bp Kpn I.backslash.Nsi I native CryIA(b) fragment 
from pCIB1315, and a 224 bp Nsi I.backslash.Bgl II PCR-generated fragment. 
Standard conditions for ligation and transformation are as described 
previously. 
A synthetic:native gene fusion fragment is made in two steps using PCR. The 
first 253 bp of the PCR fusion fragment is made using 100 pmols of oligos 
KE134A28 and MK04A28 with approximately 200 ng of native cryIA(b) template 
in a 100 ul volume with 200 nm of each dNTP, 1X PCR buffer (Perkin Elmer 
Cetus), 20% glycerol, and 5 units of Taq polymerase (Perkin Elmer Cetus). 
The PCR reaction is run with the following parameters: 1 minute at 
94.degree. C., 1 minute at 55.degree. C., 45 seconds at 72.degree. C., 
with extension 3 for 3 seconds for 25 cycles. A fraction (1%) of this 
first PCR reaction is used as a template along with 200 ng of the 
synthetic cryIA(b) DNA to make the complete 351 bp synthetic:native fusion 
fragment. Oligos used as PCR primers in this second PCR reaction are 50 
pmols of MK35A28, 50 pmols of MK04A28, and 25 pmols of KE135A28. The PCR 
reaction mix and parameters are the same as those listed above. The 
resultant 351 bp synthetic:native fusion fragment is treated with 
Proteinase K at 50 ug.backslash.ml total concentration and 
phenol.backslash.chloroform extraction followed by ethanol precipitation 
before cutting with Bst E II.backslash.Kpn I using standard conditions. 
The 224 bp Nsi I.backslash.Bgl II PCR fragment used in making pCIB4434 is 
made using 100 pmols of oligos KE137A28 and KE138A28 and 200 ng of the 
native cryIA(b) gene as template in 100 ul volume with the same PCR 
reaction mix and parameters as listed above. The 230 bp PCR native 
cryIA(b) fragment is treated with Proteinase K, 
phenol.backslash.chloroform extracted, and ethanol precipitated as 
described above, before cutting with Nsi I.backslash.Bgl II. 
pCIB4434 was transformed into maize protoplasts as described above. Line 6 
2717 protoplasts were used with pCIB4434 and pCIB4419 as a control for 
comparison. The results are shown below: 
______________________________________ 
ng Bt/mg protein 
______________________________________ 
4419(35S) 14,400 .+-. 2,100 
4434(full-length) 
2,200 .+-. 900 
______________________________________ 
Background = 13 ng Bt/mg protein for untransformed protoplasts 
The results indicate that pCIB4434 expresses at a level of about 15% of 
pCIB4419. 
Western blot analysis shows at least one-third of the cryIA(b) protein 
produced by pCIB4434 in this system is about 130 kD in size. Therefore, a 
significant amount of full-length cryIA(b) protein is produced in maize 
cells from the expression of pCIB4434. 
Example 7 
Construction of a full-length, cryIA(b) genes encoding a temperature-stable 
cryIA(b) protein. 
Constructs pCIB5511-5515, each containing a full-length, cryIA(b) gene are 
described below. In these sequences, the 26 amino acid deletion between 
amino acids 793 and 794, KCGEPNRCAPHLEWNPDLDCSCRDGE, (see SEQ ID NOS:8, 
10, 12, 14, 16) present in cryIA(a) and cryIA(c) but not in cryIA(b), has 
been repaired. The gene in pCIB5513 is synthetic; the other four genes are 
hybrids, and thus are partially maize optimized. 
Construction of pCIB5511 
This plasmid is a derivative of pCIB4434. A map of pCIB5511 is shown in 
FIG. 10. A 435 bp segment of DNA between bp 2165 and 2590 was constructed 
by hybridization of synthetic oligomers designed to represent the upper 
and lower strand as described above for the construction of the truncated 
cryIA(b) gene. This segment of synthetic DNA is synthesized using standard 
techniques known in the art and includes the 26 amino acid deletion found 
to occur naturally in the cryIA(b) protein in Bacillus thuringiensis 
kurstaki HD-1. The entire inserted segment of DNA uses maize optimized 
codon preferences to encode amino acids. The 26 amino acids used to repair 
the naturally occurring deletion are contained within this fragment. They 
are inserted starting at position 2387 between the KpnI site at nt 2170 
and the XbaI site at nt 2508 (2586 in pCIB5511) of pCIB4434. pCIB5511 is 
constructed via a three way ligation using a 3.2 Kb fragment obtained by 
restriction digestion of pCIB4434 with SphI and KpnI, a 3.8 Kb fragment 
obtained by digestion of pCIB4434 with SphI and XbaI, and a 416 bp 
fragment obtained by digestion of the synthetic DNA described above, with 
KpnI and XbaI. Enzymatic reactions are carried out under standard 
conditions. After ligation, the DNA mixture is transformed into competent 
E. coli cells using standard procedures. Transformants are selected on 
L-agar containing 100 .mu.g/ml ampicillin. Plasmids in transformants are 
characterized using standard mini-screen procedures. The sequence of the 
repaired cryIA(b) gene encoding the cryIA(b) temperature (heat) stable 
protein is set forth in FIG. 9 (SEQ ID NO:10). 
Construction of pCIB5512 
This plasmid construct is a derivative of pCIB4434. A map of pCIB5512 is 
shown in FIG. 12. DNA to repair the 26 amino acid deletion is prepared 
using standard techniques of DNA synthesis and enzymatic reaction. Three 
double stranded DNA cassettes, pGFcas1, pGFcas2 and pGFcas3, each about 
300 bp in size, are prepared. These cassettes are designed to contain the 
maize optimized codons while maintaining 100% amino acid identity with the 
insecticidal protein. These cassettes are used to replace the region 
between restriction site BstEII at position 1824 and XbaI at position 2508 
and include the insertion of the additional 78 bp which encode the missing 
26 amino acids (described above for pCIB5511 in pCIB4434). Each of these 
cassettes is cloned into the EcoRV site of the vector Bluescript 
(Stratagene) by standard techniques. The three cassettes are designed to 
contain overlapping restriction sites. Cassette 1 has restriction sites 
BstEII at the 5' end and EcoRV at the 3' end: cassette 2 has EcoRV at the 
5' end and ClaI at the 3' end and cassette 3 has ClaI at the 5' end and 
Xba I at the 3' end. They are cloned individually in Bluescript and the 
the complete 762 bp fragment is subsequently assembled by ligation using 
standard techniques. pCIB5512 is assembled using this 762 bp fragment and 
ligating it with a 6.65 Kb fragment obtained by a complete digestion of 
pCIB4434 with BstEII and a partial digestion with XbaI. Alternatively, a 
four way ligation using the same vector and the three cassettes digested 
with the specific enzymes can be employed. Enzymatic reactions are carried 
out under standard conditions. After ligation, the DNA mixture is 
transformed into competent E. coli cells using standard procedures. 
Transformants are selected on L-agar containing 100 .mu.g/ml ampicillin. 
Plasmids in transformants are characterized using standard mini-screen 
procedures. The resulting plasmid is pCIB5512. The sequence of the 
repaired cryIA(b) gene is illustrated in FIG. 11 (SEQ ID NO:12). This 
repaired cryIA(b) differs from that carried in pCIB5511 in that a larger 
region of the cryIA(b) coding region is optimized for maize expression by 
using maize preferred codons. 
Construction of pCIB5513 
This plasmid contains a repaired cryIA(b) gene derived from pCIB5512. A map 
of pCIB5513 is shown in FIG. 14. The region 3' from the XbaI site at 
position 2586 to the end of the gene (BglII site at position 3572) is 
replaced entirely with maize optimized codons. This region is synthesized, 
using standard techniques of DNA synthesis and enzymatic reaction, well 
known in the art, as four double stranded DNA cassettes (cassettes #4, 5, 
6, 7). Adjacent cassettes have overlapping restriction sites to facilitate 
assembly between cassettes. These are XbaI and XhoI at the 5' and 3' ends 
of cassette 4; XhoI and SacI at the 5' and 3' ends, respectively, of 
cassette 5; SacI and BstXI at the 5' and 3' ends, respectively, of 
cassette 6; and BstXI and BglII at the 5' and 3' ends, respectively, of 
cassette 7. As described for pCIB5512, the cassettes are cloned into the 
blunt-end EcoRV site of the Bluescript vector (Stratagene) and the 
full-length "repaired" cryIA(b) gene cloned either by sequential assembly 
of the above cassettes in Bluescript followed by ligation of the complete 
967 bp synthetic region with a 6448 bp fragment obtained by a complete 
digestion of pCIB5512 with BglII and a partial digestion with XbaI. 
Alternately, the plasmid containing the full-length genes is obtained by a 
5-way ligation of each of the four cassettes (after cleavage with the 
appropriate enzymes) and the same vector as above. The sequence of the 
full-length, "repaired" cryIA(b) gene is set forth in FIG. 13 (SEQ ID 
NO:14). The protein encoded by the various synthetic and synthetic/native 
coding region chimeras encode the same protein. This protein is the 
heat-stable version of cryIA(b) produced by repairing the naturally 
occurring 26 amino acid deletion found in the cryIA(b) gene from Bacillus 
thuringiensis kurstaki HD-1 when the homologous region is compared with 
either cryIA(a) or cryIA(c) Bacillus thuringiensis delta-endotoxins. 
Construction of pCIB5514 
This plasmid is a derivative of pCIB4434. A map of pCIB5514 is shown in 
FIG. 16. It is made using synthetic DNA cassette #3 (see above) which 
contains a maize optimized sequence of the region between the ClaI site 
(position 2396) found in the 26 amino acid thermostable region and the 
XbaI site at position 2508 in pCIB4434 (2586 in pCIB5511). The region 
between nt 2113 of pCIB4434 and the junction of the thermostable region is 
PCR amplified by using pCIB4434 as template with the following primers: 
__________________________________________________________________________ 
forward: 5'GCACCGATATCACCATCCAAGGAGGCGATGACGTATTCAAAG-3'(SEQ ID NO: 67) 
reverse: 
5'-AGCGCATCGATTCGGCTCCCCGCACTTGCCGATTGGACTTGGGGCTGAAAG-3'(SEQ ID NO: 
__________________________________________________________________________ 
68) 
The PCR product is then digested with restriction enzymes KpnI and ClaI and 
ligated in a four part reaction with a 189 bp fragment obtained by 
digestion of cassette 3 with ClaI and XbaI, a 3.2 Kb fragment of pCIB4434 
digested with SphI and KpnI, and a 3.8 Kb fragment of pCIB4434 obtained by 
digestion with SphI and Xba. Enzymatic reactions are carried out under 
standard conditions. The ligation product is transformed into competent E. 
coli cells, selected with ampicillin and screened using standard 
procedures described above. The sequence of the repaired cryIA(b) gene 
contained in pCIB5514 is shown in FIG. 15 (SEQ ID NO:16). 
Construction of pCIB5515 
pCIB4434 was modified by adding the 78bp Geiser thermostable element 
(Geiser TSE), described above, between the Kpn I site (2170 bp) and the 
Xba I site (2508 bp) in the native Btk region. The exact insertion site 
starts at the nucleotide #2379. The region containing the Geiser TSE was 
amplified by two sets of PCR reactions, i.e. the Kpn I--Geiser TSE 
fragment and the Geiser TSE--Xba I fragment. 
__________________________________________________________________________ 
PCR primer#1: (Kpn I site) 
ATTACGTTAC GCTATTGGGT ACCTTTGATG - 3' (SEQ ID NO: 69) 
PCR primer#2: (Geiser TSE bottom) 
TCCCCGTCCC TGCAGCTGCA GTCTAGGTCC GGGTTCCACT 
CCAGGTGCGG AGCGCATCGA TTCGGCTCCC CGCACTTGCC 
GATTGGACTT GGGGCTGA - 3' (SEQ ID NO: 70) 
PCR primer#3: (Geiser TSE top) 
CAAGTGCGGG GAGCCGAATC GATGCGCTCC GCACCTGGAG 
TGGAACCCGG ACCTAGACTG CAGCTGCAGG GACGGGGAAA 
AATGTGCCCA TCATTCCC - 3' (SEQ ID NO: 71) 
PCR primer#4: (Xba I site) 
TGGTTTCTCT TCGAGAAATT CTAGATTTCC - 3' (SEQ ID NO: 72) 
__________________________________________________________________________ 
After the amplification, the PCR fragments were digested with (Kpn I+Cla I) 
and (Cla I+Xba I), respectively. These two fragments were ligated to the 
Kpn I and Xba I digested pCIB4434. The resulting construct pCIB5515 is 
pCIB4434 with a Geiser TSE and an extra Cla I site flanked by Kpn I and 
Xba I. A map of pCIB5515 is illustrated in FIG. 38. The cryIA(b) gene 
contained herein, which encodes a temperature stable cryIA(b) protein, is 
shown in FIG. 37 (SEQ ID NO:27). 
Examples 9-20 set forth below are directed to the isolation and 
characterization of a pith-preferred promoter. 
Example 9 
RNA Isolation and Northern Blots 
All RNA was isolated from plants grown under greenhouse conditions. Total 
RNA was isolated as described in Kramer et al., Plant Physiol., 
90:1214-1220 (1990) from the following tissues of Funk maize line 5N984: 
8, 11, 15, 25, 35, 40, and 60 day old green leaves; 8, 11, 15, 25, 35, 39, 
46, 60 and 70 day old pith; 60 and 70 day old brace roots from Funk maize 
line 5N984; 60 and 70 day 5N984 sheath and ear stock. RNA was also 
isolated from 14 day 211D roots and from developing seed at weekly 
intervals for weeks one through five post-pollenation. Poly A+ RNA was 
isolated using oligo-dT as described by Sambrook et al., Molecular 
Cloning: A Laboratory Manual (2nd ed.), 1989, and Northern blots were 
carried out, also as per Sambrook et al. using either total RNA (30 .mu.g) 
or poly A+ RNA (2-10 .mu.g). After electrophoresis, RNA was blotted onto 
Nitroplus 2000 membranes (Micron Separations Inc). The RNA was linked to 
the filter using the Stratalinker (Stratagene) at 0.2 mJoules. The 
northerns were probed with the 1200 bp EcoRI pith (TRpA) 8-2 cDNA 
fragment, isolated by using 0.8% low melting temperature agarose in a TBE 
buffer system. Northerns were hybridized and washed and the filters 
exposed to film as described in Isolation of cDNA clones. 
Example 10 
Isolation of cDNA Clones 
First strand cDNA synthesis was carried out using the BRL AMV reverse 
transcriptase system I using conditions specified by the supplier (Life 
Technologies, Inc., Gaithersburg, Md.). Specifically, 25 .mu.l reactions 
containing 50 mM Tris-HCl pH 8.3, 20 mM KCl, 1 mM DTT, 6 mM MgCl2, 1 mM 
each of each dNTP, 0.1 mM oligo (dT) 12-18, 2 .mu.g pith poly(A+) RNA, 100 
.mu.g/ml BSA, 50 .mu.g/ml actinomycin D, 8 units placental RNase 
inhibitor, 1 .mu.l (10 mM Ci/ml) 32P dCTP &gt;3000 mCi/mM as tracer, and 30 
units AMV reverse transcriptase were incubated at 42.degree. C. for 30 
min. Additional KCl was added to a concentration of 50 mM and incubation 
continued a further 30 min. at 42.degree. C. KCl was added again to yield 
a final concentration of 100 mM. Additional AMV reverse transcriptase 
reaction buffer was added to maintain starting concentrations of the other 
components plus an additional 10 units, and the incubation continued at 
42.degree. C. for another 30 min. Second strand synthesis was completed 
using the Riboclone cDNA synthesis system with Eco RI linkers (Promega, 
Madison, Wis.). Double stranded cDNA was sized on an 1% agarose gel using 
Tris-borate-EDTA buffer as disclosed in Sambrook et al., and showed an 
average size of about 1.2 Kb. The cDNA was size fractionated using NA45 
DEAE membrane so as to retain those molecules of about 1000 bp or larger 
using conditions specified by the supplier (Schleicher and Schuell). Size 
fractionated cDNA was ligated into the Lambda ZapII vector (Stratagene, La 
Jolla, Calif.) and packaged into lambda particles using Gigapack II Plus 
(Stratagene, La Jolla, Calif.). The unamplified library had a titer of 
315,000 pfu while the amplified library had a titer of 3.5 billion/ml 
using PLK-F' cells. 
Recombinant phage were plated at a density of 5000 pfu on 150.times.15mm 
L-agar plates. A total of 50,000 phage were screened using duplicate lifts 
from each plate and probes of first strand cDNA generated from either pith 
derived mRNA or seed derived mRNA. The lifts were done as described in 
Sambrook et al. using nitrocellulose filters. DNA was fixed to the filters 
by UV crosslinking using a Stratalinker (Stratagene, La Jolla, Calif.) at 
0.2 mJoule. Prehybridization and hybridization of the filter were carried 
out in a solution of 10.times. Denhardts solution, 150 .mu.g/ml sheared 
salmon sperm DNA, 1% SDS, 50 mM sodium phosphate pH 7, 5 mM EDTA, 6.times. 
SSC, 0.05% sodium pyrophosphate. Prehybridization was at 62.degree. C. for 
4 hours and hybridization was at 62.degree. C. for 18 hours (overnight) 
with 1 million cpm/ml in a volume of 40 ml. Filters were washed in 500 ml 
of 2.times. SSC, 0.5% SDS at room temperature for 15 min. then at 
63.degree. C. in 0.1.times. SSC, 0.5% SDS for 30 min. for each wash. 
Radiolabeled DNA probes were made using a BRL random prime labeling system 
and unincorporated counts removed using Nick Columns (Pharmacia). Filters 
were exposed overnight to Kodak X-Omat AR X-ray film with (DuPont) Cronex 
Lightning Plus intensifying screens at -80.degree. C. Plaques showing 
hybridization with the pith-derived probe and not the seed-derived probe 
were plaque purified for further characterization. 
Example 11 
Isolation of Genomic Clones 
Genomic DNA from Funk inbred maize line 211D was isolated as described by 
Shure et al., Cell, 35:225-233 (1988). The DNA was partially digested with 
Sau 3A and subsequently size fractionated on 10-40% sucrose gradients 
centrifuged in a Beckman SW40 rotor at 22,000 rpm for 20 hours at 
20.degree. C. Fractions in the range of 9-23 Kb were pooled and ethanol 
precipitated. Lambda Dash II (Stratagene) cut with Bam HI was used as 
described by the supplier. The library was screened unamplified and a 
total of 300,000 pfu were screened using the conditions described above. 
The library was probed using pith-specific (TrpA) cDNA clone 8-2, pCIB5600 
which was identified in the differential screen of the cDNA library. 
Isolated clones were plaque purified and a large scale phage preparation 
was made using Lambdasorb (Promega) as described by the supplier. Isolated 
genomic clones were digested with Eco RI and the 4.8 kb EcoRI fragment was 
subcloned into Bluescript vector (Stratagene). 
Example 12 
DNA Sequence and Computer Analysis 
Nucleotide sequencing was performed using the dideoxy chain-termination 
method disclosed in Sanger et al., PNAS, 74:5463-5467 (1977). Sequencing 
primers were synthesized on an Applied Biosystems model 380B DNA 
synthesizer using standard conditions. Sequencing reactions were carried 
out using the Sequenase system (US Biochemical Corp.). Gel analysis was 
performed on 40 cm gels of 6% polyacrylamide with 7M urea in 
Tris-Borate-EDTA buffer (BRL Gel-Mix 6). Analysis of sequences and 
comparison with sequences in GenBank were done using the U. of Wisconsin 
Genetic Computer Group Sequence Analysis Software (UWGCG). 
Example 13 
Mapping the Transcriptional Start Site 
Primer extension was carried according to the procedure of Metraux et al., 
PNAS, 86:896-900 (1988). Briefly, 30 .mu.g of maize pith total RNA were 
annealed with the primer in 50 mM Tris pH 7.5, 40 mM KCl, 3 mM MgCl2 (RT 
buffer) by heating to 80.degree. C. for 10 minutes and slow cooling to 
42.degree. C. The RNA/primer mix was allowed to hybridize overnight. 
Additional RT buffer, DTT to 6 mM, BSA to 0.1 mg/ml, RNAsin at 4 U/ml and 
dNTP's at 1 mM each were added. Then 8 units AMV reverse transcriptase 
were added and reaction placed at 37.degree. C. for one hour. The primer 
used was 5'-CCGTTCGTTC CTCCTTCGTC GAGG-3' (SEQ ID NO:73), which starts at 
+90 bp relative to the transcription start. See. FIG. 29A. A sequencing 
ladder using the same primer as in the primer extension reaction was 
generated using the 4.8 Kb genomic clone to allow determination of the 
transcriptional start site. The sequencing reaction was carried out as 
described in Example 12. 
RNase protection was used to determine if the the 371 bp sequence from +2 
bp to +373 bp (start of cDNA) was contiguous or if it contained one or 
more introns. A 385 bp SphI-NcoI fragment spanning +2 bp to +387 bp 
relative to transcriptional start see FIG. 29B was cloned into pGEM-5Zf(+) 
(Promega) and transcribed using the Riboprobe Gemini system (Promega) from 
the SP6 promoter to generate radioactive antisense RNA probes as described 
by the supplier. RNase protection was carried out as described in Sambrook 
et al. pBR322 (cut with HpaII and end labelled with 32P-dCTP) and Klenow 
fragment were used molecular weight markers. Gels were 6% acrylamide/7M 
urea (BRL Gel-Mix 6) and were run at 60 watts constant power. 
Example 14 
Genomic Southern Blots 
Genomic DNA was isolated from maize line 211D using the procedure of Shure 
et al., supra. 8 .mu.g of genomic DNA were used for each restriction 
enzyme digest. The following enzymes were used in the buffer suggested by 
the supplier: BamHI, EcoRI, EcoRV, HindIII, and SacI. Pith cDNA clone 
number 8-2 was used for estimating gene copy number. The digested DNA was 
run on a 0.7% agarose gel using Tris-Borate-EDTA buffer system. The gel 
was pretreated with 250 mM HCl for 15 min. to facilitate transfer of high 
molecular weight DNA. The DNA was transferred to Nitroplus 2000 membrane 
and subsequently probed with the pith cDNA 8-2. The blot was washed as 
described in Example 10. 
Example 15 
PCR Material and Methods 
PCR reactions were preformed using the GeneAmp DNA Amplication reagent kit 
and AmpliTaq recombinant Taq DNA polmerase (Perkin Elmer Cetus). Reaction 
condition were as follows: 0.1 to 0.5 uM of each of the two primers used 
per reaction, 25 ng of the pith 4.8 Kb EcoRI fragment in Bluescript, plus 
the PCR reaction mix described by the supplier for a total volume of 50 uL 
in 0.5 mL GeneAmp reaction tube (Perkin Elmer Cetus). The DNA Thermal 
Cycler (Perkin Elmer Cetus) using the Step-Cycle program set to denature 
at 94.degree. C. for 60 s, anneal at 55.degree. C. for 60 s, and extend at 
72.degree. C. for 45 s followed by a 3-s-per-cycle extension for a total 
of 30 cycles. The following primer sets were used: I. 83.times.84, -429 bp 
to -2 bp; II. 49.times.73, -69 bp to +91 bp; III. 38.times.41, +136 bp to 
+258 bp; and IV. 40.times.75, +239 bp to +372 bp. These are marked on FIG. 
24. 
Example 16 
Isolation of a Pith-Preferred Gene 
A cDNA library derived from pith mRNA cloned into Lambda Zap and screened 
using first strand cDNA derived from either pith or seed mRNA. Clones 
which hybridized with only the pith probe were plaque purified and again 
screened. Clones passing the second screen were used as probes in northern 
blots containing RNA from various maize tissues. 
Example 17 
Gene Structure and Sequence Analysis 
The 1.2 Kb insert of the cDNA clone 8-2 was sequenced using the dideoxy 
method of Sanger et al., supra. Likewise, the genomic equivalent contained 
on a 4.8 Kb EcoRI fragment in Bluescript denoted as pCIB5601, was 
sequenced. This information revealed that the genomic copy of the coding 
region spans 1.7 Kb and contains five introns. The mRNA transcript 
represents six exons. This is shown in FIG. 24. The exons range in size 
from 43 bp to 313 bp and the introns vary in size from 76 bp to 130 bp. 
The entire sequence of the gene and its corresponding deduced amino acid 
sequence are shown in FIG. 24 (SEQ ID NOS: 18 and 19). 
This gene encodes a protein of 346 amino acids with a molecular mass of 
about 38 kD. As illustrated in Table 1, the predicted protein shows 62% 
similarity and 41% identity with the subunit protein of Pseudomonas 
aeruginosa and has high homology with trpA proteins from other organisms. 
TABLE 1 
______________________________________ 
Conservation of TrpA sequences between a maize TrpA gene and 
other organisms. 
Organisms % amino acid 
% amino acid 
compared Similarity Identity 
______________________________________ 
Haloferax volancii 
56.4 36.1 
Methanococcus voltae 
58.1 35.1 
Pseudomonas aeruginosa 
62.5 41.8 
Neurospora crassa 
61.4 39.3 
Saccharomyces cerevisiae 
56.7 36.1 
______________________________________ 
Similarity groupings, I = L = M = V, D = E, F = Y, K = R, N = Q, S = T 
Similarities and indentities were done using the GAP program from UWGCG. 
Crawford et al., Ann. Rev. Microbiol., 43:567-600 (1989), incorporated 
herein by reference, found regions of conserved amino acids in bacterial 
trpA genes. These are amino acids 49 to 58, amino acids 181 to 184, and 
amino acids 213 to 216, with the rest of the gene showing greater 
variability than is seen in the TrpB sequence. An alignment of known trpA 
proteins with the maize TrpA protein (not shown) illustrates that the 
homology between the maize gene and other trpA proteins is considerable. 
Also, it is comparable to the level of homology observed when other TrpA 
proteins are compared to each other as described in Crawford et al., 
supra. 
To determine the location of the transcription start site and whether or 
not there were introns present in this region, four polymerase chain 
reaction (PCR) generated fragments of about 122 bp to 427 bp from the 
region -429 bp to 372 bp were used for northern analysis. The results of 
the northerns showed that PCR probes II, III, IV hybridized to pith total 
RNA and PCR probe I did not hybridize. This indicated that the 
transcription start was in the -69 bp to +90 bp region. To more precisely 
locate the transcriptional start site, primer extension was employed. FIG. 
28A shows that when a primer (#73) located at +90 bp relative to the 
transcriptional start is used for primer extension, the transcriptional 
start site is located at +1, 1726 bp on the genomic sequence. 
The first ATG from the transcriptional start site is at +114 bp. This is 
the ATG that would be expected to serve as the site for translational 
initiation. This ATG begins an open reading that runs into the open 
reading frame found in the cDNA clone. The first 60 amino acids of this 
predicted open reading frame strongly resemble a chloroplast transit 
peptide. See Berlyn et al. PNAS, 86:4604-4608 (1989) and Neumann-Karlin et 
al., EMBO J., 5:9-13 (1986). This result suggests that this protein is 
targeted to a plastid and is likely processed to yield the active protein. 
Transient expression assays in a maize mesophyll protoplast system using a 
maize optimized B.t. gene driven by the trpA promoter showed that when the 
ATG at 114 bp is used as the fusion point, the highest levels of 
expression are obtained. Using either of the next two ATGs in the sequence 
substantially reduces the level of expression of the reporter gene. The 
ATG at +390 bp gave some activity, but at a much lower level than the +114 
ATG, and the ATG at +201 bp gave no activity. 
Athough a number of TATA like boxes are located upstream of the upstream of 
the transcriptional start site at 1 bp, the TATAAT at -132 bp is most like 
the plant consensus of TATAAA. See Joshi, Nuc. Acids Res., 15:6643-6653 
(1987). The presumptive CCAAT like box was found at -231 bp . The 
nucleotide sequence surrounding the ATG start (GCGACATGGC see SEQ ID 
NO:18) has homology to other maize translation starts as described in 
Messing et al., Genetic Engineering of Plants: An Agricultural 
Perspective, Plenum Press, pp. 211-227 (1983), but differs from that 
considered a consensus sequence in plants (ANNATGGC). See, Joshi, above. 
The presumptive poly(A) addition signal is located at 3719 bp (AATAAA) on 
the genomic sequence, 52 bp from the end of the cDNA. The sequence matches 
known sequences for maize as described in Dean et al., Nuc. Acids Res., 
14:2229-2240 (1986), and is located 346 bp downstream from the end of 
protein translation. See Dean et al., Nuc. Acids Res., 14:2229-2240 
(1986). The 3' untranslated sequence of the cDNA ends at 3775 bp on the 
genomic sequence. 
FIG. 27 shows a Southern blot of maize 211D genomic DNA with the 
approximate gene copy number as reconstructed using pith gene 8-2 cDNA. 
From the restriction digests and reconstruction there appear to be 1-2 
copies of the gene present per haploid genome. There do not appear to be 
other genes with lower levels of homology with this gene. Therefore, this 
represents a unique or small member gene family in maize. 
Example 18 
RNase Protection 
The structure of the 5' end of the mRNA was determined using RNase 
protection. The RNase protection was carried out using a probe 
representing 385 nt from +2 bp to +387 bp. This region from the genomic 
clone was placed in the RNA transcription vector pGEM-5Zf(+) and a 32P 
labelled RNA probe generated using SP6 polymerase. The probe and the extra 
bases from the multiple cloning site produce a transcript of 461 nt. The 
probe was hybridized with total pith RNA and subsequently digested with a 
mixture of RNase A and T1 and the protected fragments analyzed on 
denaturing polyacrylamide gels. Analysis of the gels shows a protected 
fragment of about 355 nt and another fragment of about 160 nt. See FIG. 
28B. 
The fact that primer extension using a primer (#73) at +80 bp produces a 
product of 90 NT in length argues that the 5' end of the transcript is 
located at position +1 bp. Primer extension from a primer in this region 
produces a product, so one would expect this also to be detected by the 
RNase protection assay. This primer is located in the 5' region of the 
RNase protection probe. The cDNA clone contains sequences present in the 
3' end of the RNase protection probe and hence were expected to be 
protected in this assay. Since only one band is present on the gel which 
could account for both of these sequences, we are confident that the 
protected fragment is indeed the larger band and that the smaller single 
band is an artifact. If there were an intron in this region, fragments 
from each end would be present in the probe, and hence would be detectable 
on the gel. Of the two bands seen, one of them appears to represent the 
entire 5' region, therefore we do not believe that there is an intron 
located in this region. 
Example 19 
Complementation of E. coli TrpA Mutant with the Pith cDNA 8-2 
E. coli strain CGSC strain 5531 from the E. coli Genetic Stock Center, Yale 
University (O. H. Smith lab strain designation, #M5004) with chromosomal 
markers glnA3, TrpA9825, l-, IN(rrnD-rrnE), thi-1 as described in Mayer et 
al., Mol. Gen. Gentet., 137:131-142 (1975), was transformed with either 
the pith (TRpA) cDNA 8-2 or Bluescript plasmid (Stratagene) as described 
in Sambrook et al., supra. The transformants containing the TrpA cDNA 8-2 
had the ability to grow without the presence of tryptophan on minimal 
medium whereas the transformants with the Bluescript (Stratagene) plasmid 
or untransformed control were not able to grow without tryptophan. The 
cells transformed with the maize TrpA gene grew very slowly with colonies 
visible after seven days growth at room temperature. All strains were 
grown on M9 minimal medium supplemented with 200 ug/ml glutamine, 0.01 
ug/ml thiamine and with or without 20 ug/ml tryptophan. All transformants 
were checked for the presence of the appropriate plasmid by restriction 
enzyme analysis. Colonies growing in the absence of tryptophan all 
contained clone 8-2 containing the cDNA for the putative maize TrpA gene, 
as confirmed by Southern hybridization (data not shown). These results 
support the conclusion that this is the maize tryptophan synthase subunit 
A protein. 
Example 20 
Gene Expression 
The expression pattern of the pith-preferential gene throughout the plant 
was examined. Different maize genotypes were also examined for patterns of 
expression of this gene. The following tissues were used as the source of 
RNA for these studies: upper, middle, and lower pith, brace roots, ear 
shank, cob in genotype 5N984; upper, middle, lower pith, 10 day old 
leaves, 14 day old roots and pith from the entire plant in genotype 211D, 
and seed from genotype 211D which had been harvested at weekly intervals 
one to five weeks post-pollination. Lower pith is derived from, i.e. 
constitutes the two internodes above brace roots; middle pith is derived 
from the next three internodes; upper pith represents the last two 
internodes before the tassel in 60 and 70 day plants. Only two internodes 
were present in 39 day old plants and three internodes for 46 day old 
plants. Northern blot analysis shows that transcripts hybridizing with a 
probe derived from the pith cDNA accumulate rapidly in young pith and 
young leaf. As the age of the plant increases and one moves up the stalk, 
there is a significant decrease in the amount of transcript detected. See 
FIGS. 25A-D. At no time is message from this gene detected in seed derived 
RNA, either total RNA or poly A+ RNA. See FIG. 26. Transcript is also 
detected in root, earshank, and sheath but not at the high levels detected 
in the pith and young leaf tissues. See FIGS. 25B, 25C. Some message is 
detected in brace roots, but only at a very low level. See FIG. 25D. Six 
maize undifferentiated callus lines were analyzed by northern blot 
analysis and no expression was found for this gene (data not shown) in any 
callus sample. The level of expression of this gene is extremely high 
since a very strong signal to a probe from TrpA gene 8-2 can be detected 
in pith and leaf as little as two hours after exposure of the blot to film 
(FIG. 25A). The amount of mRNA made is comparable to that derived from the 
maize phosphoenolpyruvate carboxylase gene disclosed in Hudspeth et al., 
Plant Mol. Biology, 12:579-589 (1989), another highly expressed maize 
gene. Hudspeth is incorporated herein by reference. 
The expression pattern of this gene is not temporally constant. Expression 
is very high in the lower and middle pith of plants less than 60 days old 
and decreases rapidly near the top of the plant. As the plant reaches 
maturity, e.g. over 70 days old, the expression drops to nearly 
undetectable levels except in the lower pith and earshank. The 
accumulation of transcript in young leaf is nearly as high as that seen in 
lower pith but expression decreases rapidly and is undetectable in leaves 
over 40 days of age. Expression in leaf was found to be variable depending 
on the season when it is grown. 
Examples 21-39 set forth below are directed to the isolation, 
characterization and expression analysis of a pollen-specific promoter 
according to the present invention. 
Identification of Pollen-specific Proteins 
Example 21 
Maize Plant Growth 
Maize plants (Zea mays Funk inbred 211D) were grown from seed in a 
vermiculite/sand mixture in a greenhouse under a 16 hour light/8 hour dark 
regime. 
Example 22 
Total Pollen Protein Isolation 
Mature pollen was isolated from maize plants at the time of maximum pollen 
shed. It was sieved to remove debris, frozen in liquid nitrogen, and a 3-4 
ml volume of frozen pollen was ground in a mortar and pestle with an equal 
volume of 75-150 .mu.m glass beads. 40 ml of grinding buffer (2 mM EDTA, 5 
mM DTT, 0.1% SDS, 100 mM Hepes pH 8) was added and the mixture was ground 
again. The glass beads and intact pollen grains were pelleted by low speed 
centrifugation, and mixture was clarified by centrifugation at 10,000 g 
for 15 minutes. Protein was precipitated from the supernatant by addition 
of acetone to 90%. 
Example 23 
Pollen Exine Protein Isolation 
Exine Protein was isolated from maize 211D shed pollen as described in 
Matousek and Tupy, J., Plant Physiology 119:169-178 (1985). 
Example 24 
Leaf Protein Isolation 
Young leaves (about 60% expanded) were cut from the maize plant the midrib 
removed. Total protein was isolated as for pollen, except that the 
material was not frozen and grinding was in a Waring blender without glass 
beads. 
Example 25 
Kernel Protein Isolation 
Ears with fully developed, but still moist kernels were removed from the 
plant and the kernels cut off with a scalpel. Total protein was isolated 
as for leaves. 
Example 26 
Gel Electrophoresis of Maize Proteins 
Pollen, leaf and kernel proteins were separated on SDS polyacrylamide gels 
as described in Sambrook et al, Molecular Cloning, A Laboratory Manual, 
Cold Spring Harbor Laboratory Press: New York (1989). Following staining 
by Coomasie blue, protein bands from pollen, leaf and kernel were compared 
and abundant proteins of approximately 10 kD, 13 kD, 20 kD, 45 kD, 55 kD 
and 57 kD were determined to be pollen specific. 
Identification of Pollen-Specific cDNA clones 
Example 27 
Partial Sequence Determination of Pollen-Specific Proteins 
Protein bands determined to be pollen-specific were purified by 
electroblotting from the polyacrylamide gel onto PVDF membrane 
(Matsudaira, P., J. Biol. Chem. 261:10035-10038 (1987)) or by reverse 
phase HPLC. N-terminal sequence of the purified proteins was determined by 
automated Edman egradation with an Applied Biosystems 470A gas-phase 
sequencer. Phenylthiohydantoin (PTH) amino acids were identified using an 
Applied Biosystems 120A PTH analyzer. To obtain internal sequence, 
proteins were digested with endoproteinase Lys-C (Boehringer Mannheim) in 
0.1M Tris-HCl, pH 8.5, for 24 hours at room temperature using an 
enzyme:substrate ratio of 1:10. Resulting peptides were isolated by HPLC 
using an Aquapore C-8 column eluted with a linear acetonitrile/isopropanol 
(1:1 ratio) gradient (0 to 60%) in 0.1% TFA. Sequence of isolated Lys-C 
peptides was determined as above. The following sequences were determined 
for the 13 kD pollen-specific protein: 
______________________________________ 
N-terminus: 
TTPLTFQVGKGSKPGHLILTPNVATI 
(SEQ ID NO: 74) 
LysC 61: 
KPGHLILTPNVATISDVVIK (SEQ ID NO: 75) 
LysC 54: 
SGGTRIADDVIPADFK (SEQ ID NO: 76) 
LysC 49: 
EHGGDDFSFTLK (SEQ ID NO: 77) 
LysC 43: 
EGPTGTWTLDTK (SEQ ID NO: 78) 
______________________________________ 
Example 28 
Synthesis of Oligonucleotide Probes for Pollen-Specific cDNAs 
Regions of peptide sequence in the 13 kD protein with low codon redundancy 
were selected, and suitable oligonucleotide probes for the gene encoding 
these regions were synthesized on an Applied Biosystems 380A synthesizer. 
The following oligonucleotides were synthesized: 
______________________________________ 
Oligo #51 5'-AA RTC RTC ABC ACC RTG YTC-3' 
(SEQ ID NO: 79) 
Oligo #58 5'-CC YTT NCC CAC YTG RAA-3' 
(SEQ ID NO: 80) 
______________________________________ 
where the columns of nucleotides represent bases that were incorporated 
randomly in equal proportions at the indicated position in the oligo. 
Oligo #51 encodes the amino acid sequence EHGGDDF (amino acids 1 to 7 of 
SEQ ID NO:77) found in peptide LysC 49, and Oligo #58 encodes the amino 
acid sequence FQVGKG (amino acids 6 to 11 of SEQ ID NO:74) found in 
peptide N-terminus. Use of these mixed oligonucleotides to screen a cDNA 
library for the pollen-specific gene will be described below. 
Example 29 
Construction of a Maize Pollen cDNA Library 
Total maize RNA from maize 211D shed pollen was isolated as described in 
Glisen et al, Biochemistry 13:2633-2637 (1974). Poly A+ mRNA was purified 
from total RNA as described in Sambrook et al. Using this mRNA, cDNA was 
prepared using a cDNA synthesis kit purchased from Promega, following 
protocols supplied with the kit. The EcoRI linkers were added to the cDNA 
and it was ligated into arms of the cloning vector lambda Zap, purchased 
from Stratagene and using the protocol supplied by the manufacturer. The 
ligation product was packaged in a lambda packaging extract also purchased 
from Stratagene, and used to infect E. coli BB4 cells. 
Example 30 
Isolation of Pollen-specific cDNA Clones 
The maize pollen cDNA library was probed using the synthetic 
oligonucleotides probes specific for the 13 kD protein gene, as described 
in Sambrook et al. Briefly, about 100,000 phage plaques of the pollen cDNA 
library were plated and lifted to nitrocellulose filters. The filters were 
probed using oligonucleotides #51 and #58 which had been 32P end-labeled 
using polynucleotide kinase. The probes were hybridized to the filters at 
low stringency (50 degrees C. in 1M NaCl, 10% dextran sulfate, 0.5% SDS), 
washed 30 minutes at room temperature and then 30 minutes at 45 degrees C. 
in 6.times. SSC, 0.1% SDS, and exposed to X-ray film to identify positive 
clones. Putative clones were purified through four rounds of plaque 
hybridization. Three classes of cDNA clones were isolated. Type I 
contained EcoRI fragments of 0.2 kb and 1.8 kb. Type II contained EcoRI 
fragments of 0.6 kb, 0.5 kb and 1.0 kb, and Type III contained an EcoRI 
fragment of 2.3 kb. 
Example 31 
Characterization of Pollen-specific cDNA Clones 
The EcoRI fragments of the Type II cDNA clone were subcloned into the 
plasmid vector pBluescript SK+, purchased from Stratagene. See FIG. 29. 
The 0.6 kb fragment in pBluescript was named II-.6, the 0.5 kb fragment in 
pBluescript was named II-.5 (later renamed pCIB3169) and the 1.0 kb 
fragment in pBluescript was named II-1.0 (later renamed pCIB3168). As will 
be described below, the 0.5 kb and 1.0 kb fragments encode the maize 
pollen-specific CDPK gene. RNA from anthers, pollen, leaf, root and silk 
was denatured with glyoxal, electrophoresed on a 1% agarose gel, 
transferred to nitrocellulose, and probed separately with the three EcoRI 
fragments that had been labeled with 32P by random primer extension as 
described in Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Laboratory Press: New York (1989). The blots were exposed to 
X-ray film, and an mRNA band of approximately 1.5 kb was identified with 
the 0.6 kb fragment probe, while the 0.5 and 1.0 kb fragments hybridized 
to an approximately 2.0 kb mRNA. In all cases hybridization was only seen 
in the pollen RNA lane, with the exception that the 0.6 kb fragment showed 
a slight signal in anther mRNA. The conclusion from these data was that 
the original cDNA clone was a fusion cDNA molecules derived from two 
different mRNAs. The 0.6 kb fragment was a partial cDNA of a 1.5 kb 
pollen-specific mRNA, and this mRNA encodes the peptides LysC 49 and 
N-terminus. The 1.0 and 0.5 kb fragments comprise a partial cDNA of a 2.0 
kb pollen-specific mRNA unrelated to the peptides and oligonucleotide 
probes used for probes. This conclusion was verified when the fragments 
were sequenced using the dideoxy chain termination method as described in 
Sambrook et al. The cDNA sequence is shown in FIG. 30 (SEQ ID NO:20). 
Example 32 
Determination of Specificity of mRNA Expression 
To determine if the 2.0 kb RNA represented by cDNA clones pCIB3169 and 
pCIB3168 were present only in pollen, total RNA was isolated from maize 
211D roots, leaves, pollen, anthers or silks. The RNAs were denatured with 
glyoxal, electrophoresed on a 1% agarose gel, transferred to 
nitrocellulose, and probed with 32P-labeled EcoRI insert from plasmid 
pCIB3168 or pCIB3169, all using standard techniques as described in 
Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor 
Laboratory Press: New York (1989). Exposure of this blot to photographic 
film demonstrates that the gene represented by these two clones is only 
transcriptionally active in the pollen (FIG. 31). 
Identification of a Pollen-Specific Promoter 
Example 33 
Construction of a Maize Genomic DNA Library 
Genomic DNA from maize line 211D young shoots was isolated as described in 
Shure et l, Cell 35:225-233 (1983). The DNA was provided to Stratagene, 
where a genomic DNA library was constructed by cloning Sau3AI partially 
digested DNA into Stratagene's Lambda Dash cloning vector. 
Example 34 
Genomic DNA Blot Hybridization to Determine Gene Copy Number 
Genomic DNA from maize line 211D was digested with a number of restriction 
enzymes, the individual digests electrophoresed on an agarose gel, 
transferred to nitrocellulose, and probed with 32P-labeled EcoRI insert 
from plasmid pCIB3168 (1.0 kb fragment), pCIB3169 (0.5 kb fragment) or 
clone II-.6 using standard techniques described in Sambrook et al. More 
than 10 bands were detected by the II-.6 probe on most digests, indicating 
that this cDNA is derived from a large, multigene family. Probing with the 
1.0 kb fragment detected from 3 to 6 bands, and probing with the 0.5 kb 
fragment detected only from 1 to 3 bands which were a subset of those 
detected by the 1.0 kb fragment. Due to the smaller gene family size 
detected by the 1.0 kb and 0.5 kb fragments, it was decided to attempt to 
isolate the genomic clone corresponding to them. 
Example 35 
Isolation of a Pollen-specific Genomic Clone 
The Stratagene maize 211D genomic library was screened by probing plaque 
lifts with 32P labeled inserts from plasmid pCIB3168 (1.0 kb fragment) and 
pCIB3169 (0.5 kb fragment) using standard procedures as described in the 
Stratagene manual accompanying the library. Using this strategy, Lambda 
clone MG14 was isolated, and it hybridized to both probes. The 9.0 kb 
BamHI fragment of MG14, which also hybridized to both probes, was 
subcloned into the BamHI site of pBluescript SK+ to create plasmid 
pCIB379. 1800 bp of pCIB379, in the region corresponding to the cDNA 
sequence, was sequenced as described above. Comparison of the cDNA and 
genomic sequences showed only 91% identity. pCIB379 insert represents a 
related pollen-specific gene. 
A second maize 211D genomic library was constructed in the vector lambda 
GEM-11, purchased from Promega, using the procedures described in the 
Promega manual. Screening this un-amplified library as above yielded clone 
GEM11-1, which hybridized to both 0.5 and 1.0 kb probes. The 20 kb HindIII 
fragment of GEM11-1, which also hybridized to both probes, was subcloned 
into the HindIII site of pBluescript SK+ to yield pCIB3166. The DNA 
sequence of 4.1 kb of pCIB3166 was determined (FIG. 35; SEQ ID NO:26) and 
after accounting for six introns in the genomic clone, was 100% identical 
to the cDNA sequence of pCIB3168 and pCIB3169. Comparison of the pCIB3166 
sequence to the Genbank/EMBL database revealed that the 5' portion, 
through the 3 exon, was 34.6% identical to rat calmodulin-dependent 
protein kinase II at the amino acid level (FIG. 32), while the fourth 
through seventh exons were 39.4% identical to human calmodulin. See FIG. 
33. No other pollen-specific kinase has been described, and at the time 
this a protein combining kinase and calmodulin domains was unknown. 
Subsequently, Harper et al., Science 252:951-954 (1991) have disclosed the 
cDNA sequence of a similar protein from soybean, although this gene is not 
pollen-specific in expression. Comparison of the soybean calcium-Dependent 
Protein Kinase (CDPK) and the maize pollen CDPK reveals 38% identity at 
the amino acid level. See FIG. 34. 
Example 36 
Identification of the Promoter's Transcriptional Start Site by Primer 
Extension 
Oligonucleotide PE51, with the following sequence was synthesized as a 
primer. 
______________________________________ 
5'-TGGCCCATGGCTGCGGCGGGGAACGAGTGCGGC-3' 
(SEQ ID NO: 81) 
______________________________________ 
Primer extension analysis was carried out on polyA+ pollen mRNA as 
described in Metraux et al., PNAS USA 86:896-890 (1989). The transcription 
initiation site was determined to be between bases 1415 and 1425 on the 
partial sequence of pCIB3166 shown in FIG. 35. 
Testing Promoter Function in Transgenic Plants 
Example 37 
Construction of Promoter Vectors for Plant Transformation 
To demonstrate that the pollen CDPK promoter can drive expression of a 
linked gene in transgenic plants, a gene fusion of the pollen CDPK 
promoter to the Beta-glucuronidase gene of E. coli was constructed as 
follows. The 10 kb BamHI fragment from lambda GEM11-1 containing the first 
exon and part of the first intron of the pollen CDPK gene plus 9 kb 
upstream of the gene was subcloned into the BamHI site of pBluescript SK+ 
to create plasmid pCIB3167. The 2.3 kb BamHI-HindIII fragment from 
pCIB3167 was subcloned into the BamHI and HindIII sites of pBluescript SK+ 
to create plasmid pSK105. The pSK105 was digested with AvaI and HindIII, 
and the 1.75 kb HindIII-AvaI fragment was isolated on an agarose gel. A 
PCR reaction was run under standard conditions as described in Sambrook et 
al. using intact pSK105 as a template and the following primers: 
__________________________________________________________________________ 
#42: 5'-AGCGGTCGACCTGCAGGCATGCGATCTGCACCTCCCGCCG-3' (SEQ ID NO: 82) 
#43: 5'-ATGGGCAAGGAGCTCGGG-3 (SEQ ID NO: 83) 
__________________________________________________________________________ 
The PCR reaction products were digested with AvaI and SalI and the 
resulting fragment isolated on an agarose gel. pBluescript SK+ was 
digested with HindIII and SalI. The 1.75 kb HindIII-AvaI fragment, PCR 
derived AvaI-SalI fragment, and pBluescript vector with HindIII and SalI 
ends were ligated in a three way ligation to create plasmid pSK110. 
A fusion of the promoter fragment in pSK110 to the Beta-glucuronidase (GUS) 
gene was created by digesting pSK110 with HindIII and SalI, isolating the 
1.9 kb fragment on an agarose gel and ligating it into HindIII and SalI 
sites of pCIB3054, to create plasmid pKL2, a plasmid derived from pUC19 
containing the GUS gene followed by plant intron from the maize PEPC gene 
and a polyA signal from cauliflower mosaic virus. This promoter fusion was 
inactive in plants, probably due to the presence of out of frame ATG 
codons in the leader sequence preceding the GUS gene ATG. 
A function fusion of the promoter was created by digesting pKL2 with XbaI 
and SalI to remove the previous fusion junction. A new fusion junction was 
produced in a PCR reaction using pSK105 as a template and the following 
primers: 
______________________________________ 
#SK50: 5'-CCCTTCAAAATCTAGAAACCT-3' 
(SEQ ID NO: 84) 
#SK49: 5'-TAATGTCGACGAACGGCGAGAGATGGA-3' 
(SEQ ID NO: 85) 
______________________________________ 
The PCR product was digested with XbaI and SalI and purified on an agarose 
gel. The purified fragment was ligated into the XbaI and SalI sites of 
pKL2 to created plasmid pCIB3171. This plasmid contains a functional 
fusion of pollen CDPK promoter and GUS which directs expression the GUS 
gene exclusively in pollen. 
To create a vector containing the pollen CDPK promoter-GUS fusion suitable 
for use in Agrobacterium tumefaciens-mediated plant transformation, the 
fusion gene was isolated from pCIB3171 by digestion with HindIII and SalI. 
The resulting fragment was ligated into the HindIII and SalI sites of 
pBI101 (purchased from Clontech) to create plasmid pCIB3175. 
Example 38 
Production of Transgenic Plants 
pCIB3175 was transformed into Agrobacterium tumefaciens containing the 
helper plasmid pCIB542, and the resulting culture used to transform leaf 
disks from tobacco shoot tip cultures as described by Horsch et al., 
Science 227:1229-1231 (1985) except that nurse cultures were omitted and 
selection was on 100 mg/l kanamycin. Transgenic plants were regenerated 
and verified for presence of the transgene by PCR. 
Example 39 
GUS Gene Expression Analysis 
Pollen from primary transformants and their progeny were analyzed 
histochemically for expression of the GUS gene as described by Guerrero et 
al., Mol. Gen. Genet. 224:161-168 (1990). The percentage of pollen grains 
expressing the GUS gene, as demonstrated by blue staining in the X-gluc 
buffer, is shown in the table below. 
______________________________________ 
Plant Number % Blue Pollen 
______________________________________ 
PP1-51 28% 
PP1-54 54% 
PP1-55 none 
PP1-61 very few 
PP1-63 51% 
PP1-67 15% 
PP1-80 10% 
PP1-83 12% 
______________________________________ 
Primary transformants in which a single pollen CDPK promoter-GUS gene was 
integrated would produce a maximum 50% GUS positive pollen due to 
segregation of the single gene. 
Flouometric GUS assays were done on pollen, stem, root, leaf and pistil 
tissue of selected plants to demonstrate the specificity of pollen CDPK 
promoter expression. Assays were performed as described in Jefferson, 
Plant Mol. Biol. 14:995-1006 (1990), and GUS activity values are expressed 
as nmoles MU/ug protein/minute. 
______________________________________ 
Untransformed 
Plant Plant GUS Net GUS 
number Tissue GUS Activity 
Activity Activity 
______________________________________ 
PP1-51 stem 0.01 0.02 0 
leaf 0 0 0 
root 0.15 0.10 0.05 
pistil 0.02 0.01 0.01 
pollen 0.24 0.02 0.22 
PP1-54 stem 0.01 0.02 0 
leaf 0 0 0 
root 0.13 0.1 0.03 
pistil 0.01 0.01 0 
pollen 0.60 0.02 0.58 
PP1-63 stem 0.01 0.02 0 
leaf 0 0 0 
root 0.07 0.1 0 
pistil 0.01 0.01 0 
pollen 0.57 0.02 0.55 
______________________________________ 
Examples 40-50 are directed primarily to the preparation of chimeric 
constructs, i.e. recombinant DNA molecules, containing constitutive, 
tissue-preferred, or tissue-specific promoters operably linked to an 
instant B.t. gene, insertion of same into vectors, production of 
transgenic platns containing the vectors, and analysis of expression 
levels of B.t. proteins of the transgenic plants. 
Example 40 
Construction of Maize Optimized Bt Transformation Vectors 
To demonstrate the effectiveness of the synthetic Bt cryIA(b) gene in 
maize, the PepC and pith specific promoters are fused to the synthetic Bt 
cryIA(b) gene using PCR. Oligomers designed for the PCR fusions were: 
__________________________________________________________________________ 
(PEPC) 
KE99A28 = 5'-TGCGGTTACC GCCGATCACATG-3' (SEQ ID NO: 86) 
KE97A28 = 5'-GCGGTACCGC GTCGACGCGG ATCCCGCGGC GGGAAGCTAAG-3' (SEQ ID NO: 
87) 
(PITH) 
KE100A28 = 5'-GTCGTCGACC GCAACA-3' (SEQ ID NO: 88) 
KE98A28 = 5'-GCGGTACCGC GTTAACGCGG ATCCTGTCCG ACACCGGAC-3' (SEQ ID NO: 
89) 
KE104A28 = 5'-GATGTCGTCG ACCGCAACAC-3' (SEQ ID NO: 90) 
KE103A28 = 5'-GCGGTACCGC GGATCCTGTC CGACACCGGA CGGCT-3' (SEQ ID NO: 
__________________________________________________________________________ 
91) 
PCR primers are designed to replace the Nco I sites in the 5' untranslated 
leader region of each of these tissue specific genes (containing ATG 
translational start sites) with Bam HI sites to facilitate cloning of the 
synthetic cryIA(b) gene into this Bam HI site. Subsequent construction of 
vectors containing the tissue specific promoters fused to the synthetic 
cryIA(b) gene and also containing the 35S:PAT:35S marker gene involves 
several intermediate constructs. 
1. pCIB4406 (35S:synthetic-cryIA(b):pepC ivs#9:35S) 
pCIB4406 contains the 2 Kb Bam HI.backslash.Cla I synthetic cryIA(b) gene 
fused with the CaMV 35S promoter (Rothstein et al., Gene 53:153-161 
(1987)). The gene also contains intron #6 derived from the maize PEP 
carboxylase gene (ivs#9) in the 3' untranslated region of the gene, which 
uses the CaMV 3' end. (PNAS USA, 83:2884-2888 (1986), Hudspeth et al., 
Plant Molecular Biology, 12:579-589 (1989)). pCIB4406 is ligated and 
transformed into the "SURE" strain of E. coli cells (Stratagene, La Jolla, 
Calif.) as described above. One mutation is found in pCIB4406's cryIA(b) 
gene at amino acid #436 which resulted in the desired Phe being changed to 
a Leu. pCIB4406 is fully active against European corn borer when tested in 
insect bioassays and produces a CryIA(b) protein of the expected size as 
determined by western blot analysis. 
2. pCIB4407 (35S:synthetic-cryIA(b):pepC ivs#9:35S+35S:PAT:35S) 
pCIB4407 is made from an approximately 4 Kb Hind III.backslash.Eco RI 
fragment containing the 35S:PAT:35S gene, and the 3.1 Kb.backslash.Hind 
III.backslash.Eco RI 35S:synthetic-cryIA(b):35S gene from pCIB4406. 
pCIB4407 is ligated and transformed into "SURE", DH5alpha, and HB101 
strains of E. coli using standard procedures (Sambrook et al.). The 
synthetic cryIA(b) gene has the same properties as its precursor pCIB4406. 
3. pCIB4416 (35S:synthetic-cryIA(b):pepC ivs#9:35S+35S:PAT:35S+35S:Adh 
intron:GUS:35S.) 
pCIB4407 is cut with Eco RI and treated with calf intestinal alkaline 
phosphatase (CIP) under standard conditions (Sambrook et al.) to produce 
an about 7.2 Kb fragment that is ligated with a 3.4 Kb Eco RI 
35S:Adh.backslash.GUS:35S fragment to produce pCIB4416. Ligations and 
transformations into "SURE" cells is as described above. The synthetic 
cryIA(b) gene in pCIB4416 has the same properties as the gene in pCIB4406. 
4. pCIB4418 (35S:synthetic-cryIA(b):pepC ivs#9:35S) 
pCIB4406 is digested with Apa I and Bam HI and treated with CIP. pCIB4406 
is digested with Bam HI and Nsp I. pBS123#13 is digested with Nsp I and 
Apa I. A three-way ligation is made consisting of a 4.3 Kb Apa 
I.backslash.Bam HI fragment from pCIB4406, a 1.3 Kb Bam HI.backslash.Nsp I 
fragment from pCIB4406, and a 170 bp Nsp I.backslash.Apa I fragment from 
pBS123#13 to form pCIB4418. The host E. coli strain for pCIB4418 is HB101. 
5. pCIB4419 (35S:synthetic-cryIA(b):pepC ivs#9:35S+35S:PAT:35S+35S:Adh 
intron:GUS:35S.) 
pCIB4416 and pCIB4418 are digested with Bst E II and Eco NI and fragments 
of pCIB4416 are treated with CIP. A 9.1 Kb fragment from pCIB4416 ligated 
to a 1.4 Kb fragment from pCIB4418 to form pCIB4419. pCIB4419 transformed 
in HB101 competent E. coli cells demonstrates full activity in insect 
bioassays against European corn borer. 
6. pCIB4420 (Pith:synthetic-cryIA(b):PEPC ivs#9:35S+35S:PAT:35S) 
Intermediate constructs in making pCIB4420 are pBTin1, pBtin2, p4420A and 
pBtin3. pBtin1 (pith promoter:second half of the synthetic Bt 
gene+35S:PAT:35S) is made by ligating the 2.1 Kb Xba I.backslash.Nco I 
pith promoter fragment from plasmid pith(3-1) with a 5.2 Kb Xba 
I.backslash.Nco I fragment from pCIB4407. pBtin2 is an intermediate 
construct containing the pith promoter modified with a 210 bp PCR fragment 
made using primers KE100A28 and KE98A28 listed above. The PCR reaction mix 
contains approximately 100 ng of a 2.1 Kb Bam HI.backslash.Nco I pith 
promoter fragment with 100 pmol of each oligomer, 200 nM of each dNTP, 
1.times. buffer (Cetus) and 2.5 units of thermal stable polymerase. Since 
the Tm is relatively low (between 40.degree. and 50.degree. C.), PCR 
reactions are run with the following parameters: 
denaturation cycle: 94.degree. C. for 1 minute 
annealing cycle: 37.degree. C. for 1 minute 
extension cycle: 72.degree. C. for 45 seconds (+3 seconds per cycle) 
number of cycles: 25 
PCR reactions are treated with proteinase K as described above prior to 
cutting with Sal I.backslash.Kpn I followed by phenol.backslash.chloroform 
extraction and ethanol precipitation as described above. The 210 bp 
fragment is purified on a 2% Nusieve gel and extracted from the gel using 
Millipore's filter units. The 210 bp Sal I.backslash.Kpn I fragment is 
ligated to the 4.9 Kb Sal I.backslash.Kpn I fragment from pith(3-1) to 
make pBtin2. p4420A (pith:synthetic-Bt:Pep intron:35S+35S:PAT:35S) is made 
with a three-way ligation consisting of a 700 bp Nsi I.backslash.Bam HI 
fragment from pBtin2, a 1.8 Kb Bam HI.backslash.Bst E II fragment from 
pCIB4418, and a 5.9 Kb Bst E II.backslash.Nsi I fragment from pBtin1. 
After p4420A is made three mutations are discovered in pBtin2. A second 
PCR fragment is made to modify the Nco I site in the pith leader using 
primers KE104A28 and KE103A28 with Tm values around 65.degree. C. The PCR 
reaction mix is identical to that listed above with the addition of 
glycerol to 20% to reduce mutations in G+C rich areas (Henry et al., Plant 
Molecular Biology Reporter 9(2):139-144, 1991). PCR parameters are as 
follows: 
File I: 
94.degree. C.: 3 minutes , 1 cycle 
File II: 
60.degree. C.: 1 minute 
94.degree. C.: 1 minute 
25 cycles 
File III: 
72.degree. C.: 5 minutes, 1 cycle 
PCR reactions are treated as above and cut with restriction endonucleases 
Sal I and Kpn I. The 210 bp Sal I.backslash.Kpn I PCR (glycerol in the 
reaction) fragment is ligated to the 4.9 Kb Sal I.backslash.Kpn I fragment 
from plasmid pith(3-1) to make pBtin3. Sequence data on pBtin3-G#1 shows 
this PCR generated fragment to be correct. 
pBtin3-G#1 is used to make pCIB4420 (also called p4420B "G#6"). pCIB4420 is 
constructed with a three-way ligation using the 700 bp Nsi I.backslash.Bam 
HI fragment from pBtin3-G#1, a 1.8 Kb Bam HI.backslash.Bst E II fragment 
from pCIB4418, and a 5.9 Kb Bst E II.backslash.Nsi I fragment from pBtin1. 
pCIB4420 is used in mesophyll protoplast experiments and demonstrates full 
activity of the synthetic cryIA(b) gene against European corn borer. 
7. pCIB4413 (PEPC:synthetic-Bt (Phe mutation):PEPC intron:35S.) 
A fusion fragment is generated by PCR using primers KE99A28 and KE97A28 
with a 2.3 KB Hind III.backslash.Sal I template from pGUS4.5. The PCR mix 
contains the same concentration of primers, template, dNTPs, salts, and 
thermal stable polymerase as described above. PCR reaction parameters are: 
denaturation cycle: 94.degree. C. for 1 minute 
annealing cycle: 55.degree. C. for 1 minute 
extension cycle: 72.degree. C. for 45 seconds (+3 seconds per cycle) 
number of cycles: 30 
After completion, PCR reactions are treated with proteinase K followed by 
phenol.backslash.chloroform extraction and ethanol precipitation as 
described above prior to cutting with restriction endonucleases Bam HI and 
Bst E II. 
pCIB4413 is made with a three-way ligation using the 210 bp Bam 
HI.backslash.Bst E II PCR fragment, a 4.7 Kb Bam HI.backslash.Hind III 
fragment from pCIB4406, and a 2.2 Kb Hind III.backslash.Bst E II fragment 
from pGUS4.5. 
8. pCIB4421 (PEPC:synthetic-cryIA(b):PEPC intron:35S.) 
pCIB4421 is made to replace the synthetic cryIA(b) gene containing the Phe 
mutation in pCIB4413 with the synthetic cryIA(b) gene from pCIB4419. 
pCIB4421 is made by ligating a 5.2 Kb Bam HI.backslash.Sac I fragment from 
pCIB4413 with a 1.9 Kb Bam HI.backslash.Sac I fragment from pCIB4419. 
9. pCIB4423 (PEPC:synthetic-cryIA(b):PepC intron:35S+35S:PAT:35S) 
The 2.4 Kb Bam HI.backslash.Hind III PEPC promoter fragment from pCIB4421 
is ligated to the 6.2 Kb Bam HI.backslash.Hind III fragment in pCIB4420 to 
make pCIB4423. The Hind III site is deleted by exonucleases in the cloning 
of pCIB4423. pCIB4423 contains the synthetic cryIA(b) gene under the 
control of the PEPC promoter, and the PAT gene under the control of the 
35S promoter. 
10. Synthetic cryIA(b) gene in Agrobacterium strains: 
Agrobacterium strains made with the synthetic cryIA(b) gene allow transfer 
of this gene in a range of dicotyledenous plants. Agrobacterium vector 
pCIB4417 contains the 3.3 Kb Hind III.backslash.Eco RI 
35S:synthetic-CryIA(b):PepC:ivs#9:35S fragment from pCIB4406 (Phe 
mutation) ligated to the 14 Kb Hind III.backslash.Eco RI fragment from 
pBI101 (Clontech). Using electroporation, pCIB4417 is transferred into the 
A. tumefaciens strain LBA4404 (Diethard et al., Nucleic Acids Research, 
Vol17: #16:6747, 1989.). 
200 ng of pCIB4417 and 40 ul of thawed on ice LBA4404 competent cell are 
electroporated in a pre-cooled 0.2 cm electroporation cuvette (Bio-Rad 
Laboratories Ltd.). Using Gene Pulser-.TM. with the Pulse Controller unit 
(Bio-Rad), an electric pulse is applied immediately with the voltage set 
at 2.5 kV, and the capacity set at 25 uF. After the pulse, cells are 
immediately transferred to 1 ml of YEB medium and shaken at 27 C. for 3 
hours before plating 10 ul on ABmin:Km50 plates. After incubating at 28 C. 
for approximately 60 hours colonies are selected for miniscreen 
preparation to do restriction enzyme analysis. The final Agrobacterium 
strain is called pCIB4417:LBA4404. 
Example 41 
ELISA Analysis of Transformed Maize Protoplasts 
The presence of the cryIA(b) toxin protein is detected by utilizing 
enzyme-linked immunosorbent assay (ELISA). ELISAS are very sensitive, 
specific assays for antigenic material. ELISA assays are useful to 
determine the expression of polypeptide gene products. Antiserum for these 
assays is produced in response to immunizing rabbits with 
gradient-purified Bt crystals [Ang et al., Applied Environ. Microbiol., 
36:625-626 (1978)] solubilized with sodium dodecyl sulfate. ELISA analysis 
of extracts from transiently transformed maize cells is carried out using 
standard procedures (see for example Harlow, E., and Lane, D. in 
"Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 
1988). ELISA techniques are further described in Clark et al., Methods in 
Enzymology, 118:742-766 (1986); and Bradford, Anal. Biochem., 72:248 
(1976). Thus, these procedures are well-known to those skilled in the art. 
The disclosure of these references is hereby incorporated herein by 
reference. 
ELISA assays are performed to detect the production of CryIA(b) protein in 
maize protoplasts. Protein produced is reported below as ng of Bt per mg 
total protein (ng Bt/mg). Each construct was tested twice. 
pCIB3069 
No detectable Bt (both tests) 
pCIB4407 
21,900 ng Bt/mg total protein, 
21,000 ng Bt/mg total protein 
The transformed maize cells produce high levels, on the order of 
approximately 20,000 ng of Bt CryIA(b) protein per mg total soluble 
protein, of the Bt IP when transformed with the maize optimized Bt gene. 
The level of detection of these ELISA based assays is about 1 to 5 ng 
CryIA(b) protein per mg protein. Therefore, the maize optimized Bt gene 
produces as much as approximately a 20,000 fold increase in expression of 
this protein in maize cells. 
Example 42 
Assay of Extract from Transformed Protoplasts for Insecticidal Activity 
against European Corn Borer 
Western blot analysis is also performed using extracts obtained from maize 
cells which had been transiently transformed with DNA to express the maize 
optimized gene. When examined by western blots, this protein appears 
identical with the protein produced in E. coli. In contrast, as 
demonstrated in Example 6 above, no detectable Bt cryIA(b) insecticidal 
protein is produced by maize cells transformed with comparable vectors 
attempting to express the native Bt derived coding region. 
Qualitative insect toxicity testing can be carried out using harvested 
protoplasts. Suspensions are prepared for each replicate tested in all 
bioassays. A replicate is considered positive if it causes significantly 
higher mortality than the controls. For example, replicates are tested for 
their activity against insects in the order Lepidoptera by using the 
European corn borer, Ostrinia nubilalis. One-hundred .mu.l of a protoplast 
suspension in 0.1% Triton X-100 is pipetted onto the surface of artificial 
Black cutworm diet, (Bioserv, Inc., Frenchtown, N.J.; F9240) in 50 
mm.times.10 mm snap-cap petri dishes. After air drying 10 neonatal larvae 
are added to each plate. Mortality is recorded after about 4 days. When 
this protein is fed to European corn borers, it produces 100% mortality. 
Example 43 
Expression of Synthetic Bt in Maize Mesophyll Protoplasts 
The general procedure for the isolation of corn mesophyll protoplasts is 
adapted from Sheen et al., The Plant Cell, 2:1027-1038 (1990). The 
protoplast transformation system used in Sheen et al. is modified by using 
PEG mediated transformation, rather than electroporation. That procedure, 
as well as changes made in the isolation procedure, is described below. 
Maize Mesophyll Protoplast Isolation/Transformation 
1. Sterilize and germinate corn seeds for leaf material. Seedlings are 
grown in the light at 25C. 
2. Surface sterilize leaf pieces of 10-12 day old seedlings with 5% Clorox 
for 5 minutes followed by several washes with sterile distilled water. 
3. Aliquot enzyme solution (see recipe below); 25 ml/dish (100.times.25 mm 
petri dish). 
4. Remove any excess water from leaves and place 6-8 2 inch pieces in each 
dish of enzyme. 14 plates are usually set up with the leaf material from 
about 100 seedlings. 
5. Cut leaves in longitudinal strips as thin as possible (2-5 mm). 
6. Shake slowly at 25 C. for 6.5 to 7 hours. Cover plates so that 
incubation takes place in the dark. 
7. Before filtering protoplasts, wash 100 um sieves with 10 ml 0.6M 
mannitol. Pipet protoplasts slowly through sieves. Wash plates with 0.6M 
mannitol to gather any protoplasts left in the dishes. 
8. Pipet filtered liquid carefully into 50 ml sterile tubes. Add equal 
volumes of 0.6M mannitol to dilute. 
9. Spin for 10 minutes at 1000 rpm/500 g in table-top centrifuge (Beckman 
Model TJ-6). 
10. Remove enzyme solution and discard. Resuspend pellets carefully in 5 ml 
mannitol. Pool several pellets. Bring volume to 50 ml with 0.6M mannitol 
and spin. 
11. Resuspend to a known volume (50 ml) and count. 
12. After counting and pelleting, resuspend protoplasts at 2 million/ml in 
resuspending buffer (recipe below). Allow ppts to incubate in the 
resuspending buffer for at least 30 min before transformation. 
Transformation 
1. Aliquot plasmids to tubes (Fisherbrand polystyrene 17.times.100 mm Snap 
Cap culture tubes); at least three replicates per treatment; use equimolar 
amounts of plasmids so that equal gene copy numbers are compared. 
2. Add 0.5 ml protoplasts and 0.5 ml 40% PEG made with 0.6M mannitol. 
3. Shake gently to mix and incubate at 25 C. for 30 min. 
4. Add protoplast culture media at 5 min intervals: 1,2,5 ml 
5. Spin for 10 min at 1000 rpm/500 g. 
6. Remove liquid from pellet and resuspend in 1 ml culture media (BMV 
media) 
7. Incubate overnight at 25 C. in the dark. 
Recipes 
Enzyme Solution 
0.6M mannitol 
10 mM MES, pH 5.7 
1 mM CaCL.sub.2 
1 mM MgCl.sub.2 
0.1% BSA 
filter-sterilize 
To this solution, add the following enzymes: 
1% Cellulase RS, and 0.1% Macerozyme R10 
Wash Buffer: 0.6M mannitol, filter-sterilize 
Resuspending Buffer: 0.6M mannitol, 20 mM KCl, filter-sterilize 
Culture Media: BMV media recipe from: 
Okuno et al., Phytopathology 67:610-615 (1977). 
0.6M mannitol 
4 mM MES, pH 5.7 
0.2 mM KH.sub.2 PO.sub.4 
1 mM KNO.sub.3 
1 mM MgSO.sub.4 
10 mM CaCl.sub.2 
1.times. K3 micronutrients 
filter-sterilize 
ELISA analysis of transformed protoplasts is done one day after 
transformation. ELISA's are done as previously described. The following 
three experiments are done with maize inbred line 211D. Of course, other 
lines of maize may be used. 50 ug of plasmid pCIB4419 and equimolar 
amounts of other plasmids are used. Total soluble protein is determined 
using the BioRad protein assay. (Bradford, Anal. Biochem, 72:248 (1976). 
Transformation Experiment 
Constructs tested: 
1. pCIB4419 (Construct contains synthetic Bt under control of CaMV 35S 
promoter and 35S/PAT and 35S/GUS marker genes) 
2. pCIB4420 (Construct contains synthetic Bt under control of Pith promoter 
and PAT marker gene) 
3. pCIB4421 (Construct contains synthetic Bt under control of PEPC 
promoter) 
4. pCIB4423 (Construct contains synthetic Bt under control of PEPC promoter 
and PAT marker gene) 
(PEPC:synthetic-cryIA(b):PepC intron:35S+35S:PAT:35S) 
In the following experiments, 10 or 11 day old 211D seedlings are analyzed 
for production of the Bt CryIA(b) protein in the Biorad protein assay: 
______________________________________ 
Experiment 1 (11 day seedlings): 
pCIB4419 15,000 .+-. 3,000 
ng Bt/mg protein 
pCIB4420 280 .+-. 65 ng Bt/mg protein 
pCIB4421 9,000 .+-. 800 
ng Bt/mg protein 
Experiment 2 (10 day seedlings): 
pCIB4419 5,000 .+-. 270 
ng Bt/mg protein 
pCIB4420 80 .+-. 14 ng Bt/mg protein 
pCIB4421 1,600 .+-. 220 
ng Bt/mg protein 
Experiment 3 (11 day seedlings): 
pCIB4419 21,500 .+-. 1,800 
ng Bt/mg protein 
pCIB4420 260 .+-. 50 ng Bt/mg protein 
pCIB4421 11,900 .+-. 4,000 
ng Bt/mg protein 
pCIB4423 7,200 .+-. 3,400 
ng Bt/mg protein 
______________________________________ 
The above experiments confirm that both the CaMV 35S and PEPC promoters 
express the synthetic Bt CryIA(b) protein at very high levels. The pith 
promoter, while less efficient, is also effective for the expression of 
synthetic CryIA(b) protein. 
Example 44 
Stable Expression of Synthetic Bt in Lettuce 
The synthetic Bt gene in the Agrobacterium vector pCIB4417 is transformed 
into Lactuca sativa cv. Redprize (lettuce). The transformation procedure 
used is described in Enomoto et al., Plant Cell Reports, 9:6-9 (1990). 
Transformation Procedure 
Lettuce seeds are suface sterilized in 5% Clorox for 5 minutes followed by 
several washes in sterile distilled water. Surface-sterilized seeds are 
plated on half strength MS media (Murashige and Skoog, Physiol. Plant. 
15:473-497 (1962)). Cotyledons of 6-day-old Redprize seedlings, grown 
under illumination of 3,000 1.times. 16 hr at 25 C., are used as the 
explants for Agrobacterium infection. The base and tip of each cotyledon 
are removed with a scalpel. The explants are soaked for 10 minutes in the 
bacterial solution which have been cultured for 48 hours in AB minimal 
media with the apropriate antibiotics at 28 C. After blotting excess 
bacterial solution on sterile filter paper, the explants are plated on MS 
media (0.1 mg/l BA and 0.1 mg/l NAA) for 2 days. Explants are then 
transferred to selective media containing 500 mg/l carbenicillin and 50 
mg/l kanamycin. The explants are subcultured to fresh media weekly. The 
growth chamber conditions are 16 hour 2,000 1.times. light at 25 C. After 
approximately 4 weeks, an ELISA is done on healthy looking callus from 
each of four plates being subcultured. The ELISA procedure is the same as 
described above for protoplasts; soluble protein is again determined by 
the Biorad assay described above. 
______________________________________ 
Results: 
______________________________________ 
pCIB3021 (kan control) 
0 
pCIB4417 (plate 1) 0 
pCIB4417 (plate 2) 505 ng Bt/mg protein 
pCIB4417 (plate 3) 45 ng Bt/mg protein 
pCIB4417 (plate 4) 1,200 ng Bt/mg protein 
______________________________________ 
This example demonstrates that dicot plants can also show increased 
expression of the optimized insecticidal gene. 
Example 45 
Construction of pCIB4429 
pCIB4429 contains a preferred maize pollen-specific promoter fused with the 
maize optimized cryIA(b) gene. The pollen-specific maize promoter used in 
this construct was obtained from the plasmid pKL2, described in Example 
37. The maize optimized cryIA(b) gene was obtained from plasmid pCIB4418, 
also described in Example 37. 
pKL2 is a plasmid that contains a preferred maize pollen-specific promoter 
fused with the E. coli beta-glucuronidase gene. It was constructed from 
plasmids pSK110 and pCIB3054. pSK110 contains the pollen specific maize 
promoter. pCIB3054, a pUC19 derivative, contains the E. coli 
beta-glucuronidase (GUS) gene fused with the cauliflower mosaic virus 
(CaMV) 35S promoter. It's construction is described elsewhere in this 
application. This promoter can be removed from this plasmid by cutting 
with SalI/HindIII to yield a fragment containing the GUS gene, a bacterial 
ampicillin resistance gene and a ColEI origin of replication. A second 
fragment contains the CaMV 35S promoter. 
pCIB3054 was cut with the restriction enzymes SalI and HindIII, using 
standard conditions, for 2 hours at room temperature. The reaction was 
then extracted with phenol/chloroform using standard conditions and the 
DNA recovered by ethanol precipitation using standard conditions. The 
recovered DNA was resuspended in buffer appropriate for reaction with calf 
intestinal alkaline phosphatase (CIP) and reacted with 2.5 units of CIP at 
37.degree. C. overnight. After the CIP reaction, the DNA was purified on 
an agarose gel using standard conditions described elsewhere in this 
application. pSK110 was cut with SalI/HindIII under standard conditions 
for 2 hours at room temperature and the DNA subsequently purified on an 
agarose gel using standard conditions. The recovered DNA fragments were 
ligated using standard conditions for two hours at room temperature and 
subsequently transformed into competent E. coli strain HB101 cells using 
standard conditions. Transformants were selected on L-agar containing 100 
.mu.g ampicillin/ml. Transformants were characterized for the desired 
plasmid construct using standard plasmid mini-screen procedures. The 
correct construct was named pKL2. 
To make pCIB4429, a three way ligation was performed using standard 
conditions known to those in the art. The three fragments ligated were: 
1) a HindIII/BamHI fragment from pCIB4418, of about 4.7 kb in size, 
containing the cryIA(b) gene, the bacterial ampicillin resistance gene, 
and the ColEI origin of replication 
2) a HindIII/XbaI fragment from pKL2 of about 1.3 kb in size and containing 
the pollen specific promoter from maize 
3) a PCR generated fragment derived from the pollen promoter with a BamHI 
site introduced downstream from the start of transcription. This fragment 
is approximately 120 bp and has ends cut with the restriction enzymes 
XbaI/BamHI. 
The PCR fragment was generated using a 100 .mu.l reaction volume and 
standard conditions described above. The primers used were: 
__________________________________________________________________________ 
SK50: 
5'-CCC TTC AAA ATC TAG AAA CCT-3' (SEQ ID NO: 84) 
KE127: 
5'-GCG GAT CCG GCT GCG GCG GGG AAC GA-3' (SEQ ID NO: 
__________________________________________________________________________ 
92) 
The above primers were mixed in a PCR reaction with plasmid pSK105, a 
plasmid that contains the pollen specific promoter from maize. 
After the PCR reaction was complete, 10 .mu.l of the reaction was run on an 
agarose gel, using standard condition, to make sure the reaction produced 
the expected size product. The remaining 90 .mu.l was treated with 
proteinase K at a final concentration of 50 .mu.g/ml for 30 min. at 
37.degree. C. The reaction was then heated at 65.degree. C. for 10 min., 
then phenol/chloroform extracted using standard procedures. The DNA was 
recovered from the supernatant by precipitating with two volumes of 
ethanol using standard conditions. After precipitation, the DNA was 
recovered by centrifuging in a microfuge. The pellet was rinsed one time 
with 70% ethanol (as is standard in the art), briefly dried to remove all 
ethanol, and the pellet resuspended in 17 .mu.l TE buffer. 2 .mu.l of 
10.times. restriction enzyme buffer was added as were 0.5 .mu.l BamHI and 
0.5 .mu.l XbaI. The DNA was digested for 1 hour at 37.degree. C. to 
produce a DNA fragment cut with XbaI/BamHI. After digestion with the 
restriction enzymes, this fragment was purified on an agarose gel composed 
of 2% NuSieve (FMC)/1% agarose gel. Millipore filter units were used to 
elute the DNA from the agarose using the manufacturer's specifications. 
After elution, the DNA was used in the three-way ligation described above. 
After ligation, the DNA was transformed into competent E. coli strain HB101 
cells using standard techniques. Transformants were selected on L-agar 
plates containing ampicillin at 100 .mu.g/ml. Colonies that grew under 
selective conditions were characterized for plasmid inserts using 
techniques standard in the art. 
Example 46 
Construction of pCIB4431, a Vector for Tissue Specific Expression of the 
Synthetic CryIA(b) Gene in Plants 
pCIB4431 is a vector designed to transform maize. It contains two chimeric 
Bt endotoxin genes expressible in maize. These genes are the PEP 
carboxylase promoter/synthetic-cryIA(b) and a pollen 
promoter/synthetic-cryIA(b). The PEP carboxylase/cryIA(b) gene in this 
vector is derived from pCIB4421 described above. The pollen promoter is 
also described above. FIG. 20 is a map of plasmid pCIB4431. pCIB4431 was 
constructed via a three part ligation using the about 3.5 Kb Kpn I/Hind 
III fragment (containing pollen/synthetic-cryIA(b) from pCIB4429, the 
about 4.5 Kb Hind III/Eco RI (PEPC/synthetic-cryIA(b) and the about 2.6 Kb 
Kpn I/Eco RI fragment from the vector Bluescript. 
Other vectors including the pollen promoter/synthetic CryIA(b) chimeric 
gene include pCIB4428 and pCIB4430. See FIGS. 21 and 22. pCIB4430 also 
contains the PEPC/synthetic-Bt gene described above. 
Example 47 
Production of Transgenic Maize Plants Containing the Synthetic Maize 
Optimized CryIA(b) Gene 
The example below utilizes Biolistics to introduce DNA coated particles 
into maize cells, from which transformed plants are generated. 
Experiment KC-65 
Production of Transgenic Maize Plants Expressing the Synthetic cryIA(b) 
Gene Using a Tissue-specific Promoter 
Tissue 
Immature maize embryos, approximately 1.5-2.5 mm in length, were excised 
from an ear of genotype 6N615 14-15 days after pollination. The mother 
plant was grown in the greenhouse. Before excision, the ear was surface 
sterilized with 20% Clorox for 20 minutes and rinse 3 times with sterile 
water. Individual embryos were plated scutellum side in a 2 cm square 
area, 36 embryos to a plate, on the callus initiation medium, 2DG4+5 
chloramben medium (N6 major salts, B5 minor salts, MS iron, 2% sucrose, 
with 5 mg/l chloramben, 20 mg/l glucose, and ml G4 additions (Table 1) 
added after autoclaving. 
TABLE 1 
______________________________________ 
G4 Additions 
Ingredient per liter medium 
______________________________________ 
Casein hydrolysate 0.5 gm 
Proline 1.38 gm 
Nicotinic acid .2 mg 
Pyridoxine-HCl .2 mg 
Thiamine-HCl .5 mg 
Choline-HCl .1 mg 
Riboflavin .05 mg 
Biotin .1 mg 
Folic acid .05 mg 
Ca pantothenate .1 mg 
p-aminobenzoic acid .05 mg 
B12 .136 .mu.g 
______________________________________ 
Bombardment Tissue was bombarded using the PDS-1000He Biolistics device. 
The tissue was placed on the shelf 8 cm below the stopping screen shelf. 
The tissue was shot one time with the DNA/gold microcarrier solution, 10 
.mu.l dried onto the macrocarrier. The stopping screen used was hand 
punched at ABRU using 10.times.10 stainless steel mesh screen. Rupture 
discs of psi value were used. After bombardment, the embryos were cultured 
in the dark at 25.degree. C. 
Preparation of DNA for Delivery 
The microcarrier was prepared essentially according to the instructions 
supplied with the Biolistic device. While vortexing 50 .mu.l 1.0.mu. gold 
microcarrier, added 5 .mu.l pCIB4431 (1.23 .mu.g/.mu.l) [#898]+2 .mu.l 
pCIB3064 0.895 .mu.g/.mu.l) [#456] followed by 50 .mu.l 2.5M CaCl.sub.2, 
then 20 .mu.l 0.1M spermidine (free base, TC grade). The resulting mixture 
was vortexed 3 minutes and microfuged for 10 sec. The supernatant was 
removed and the icrocarriers washed 2 times with 250 .mu.l of 100% EtOH 
(HPLC grade) by vortexing briefly, centrifuging and removing the 
supernatant. The microcarriers are resuspended in 65 .mu.l 100% EtOH. 
Callus Formation 
Embryos were transferred to callus initiation medium with 3 mg/l PPT 1 day 
after bombardment. Embryos were scored for callus initiation at 2 and 3 
weeks after bombardment. Any responses were transferred to callus 
maintenance medium, 2DG4+0.5 2,4-D medium with 3 mg/L PPT. Callus 
maintenance medium is N6 major salts, B5 minor salts, MS iron, 2% sucrose, 
with 0.5 mg/l 2,4-D, 20 mg/l glucose, and 10 ml G4 additions added after 
autoclaving. Embryogenic callus was subcultured every 2 weeks to fresh 
maintenance medium containing 3 mg/L PPT. All callus was incubated in the 
dark at 25.degree. C. 
The Type I callus formation response was 15%. Every embryo which produced 
callus was cultured as an individual event giving rise to an individual 
line. 
Regeneration 
After 12 weeks on selection, the tissue was removed from callus maintenance 
edium with PPT and was placed on regeneration medium. Regeneration medium 
is 0.25MS3S5BA (0.25 mg/l 2,4 D, 5 mg/l BAP, MS salts, 3% sucrose) for 2 
weeks followed by subculture to MS3S medium for regeneration of plants. 
After 4 to 10 weeks, plants were removed and put into GA 7's. Our line 
KC65 0-6, which became the #176 BT event, produced a total of 38 plants. 
Assays 
All plants, as they became established in the GA7's, were tested by the 
chlorophenol red (CR) test for resistance to PPT as described in U.S. 
patent application Ser. No. 07/759,243, filed Sep. 13, 1991, the relevant 
portions of which are hereby incorporated herein by reference. This assay 
utilizes a pH sensitive indicator dye to show which cells are growing in 
the presence of PPT. Cells which grow produce a pH change in the media and 
turn the indicator yellow (from red). Plants expressing the resistance 
gene to PPT are easily seen in this test. (#176=8 positive/30 negative) 
Plants positive by the CR test were assayed by PCR for the presence of the 
synthetic BT gene. (#176=5 positive/2 negative/1 dead) 
Plants positive by PCR for the syn-BT gene were sent to the phytotron. Once 
established in the phytotron, they were characterized using insect 
bioassays and ELISA analysis. Plants were insect bioassayed using a 
standard European Corn Borer assay (described in Example 5A) in which 
small pieces of leaf of clipped from a plant and placed in a small petri 
dish with a number of ECB neonate larvae. Plants are typically assayed at 
a height of about 6 inches. Plants showing 100% mortality to ECB in this 
assay are characterized further. ELISA data are shown below. Positive 
plants are moved to the greenhouse. 
Greenhouse/Fertility 
Plant number #176-11 was pollinated with wild-type 6N615 pollen. One tassel 
ear and one ear shoot were produced. All of the embryos from the tassel 
ear (11) and 56 kernels from Ear 1 were rescued. 294 kernels remained on 
the ear and dried down naturally. 
Pollen from #176-11 was outcrossed to various maize genotypes 5N984, 5NA89, 
and 3N961. Embryos have been rescued from all 3 outcrosses (5N984=45; 
5NA89=30; 3N961=8). Most of the kernels remained on the ears on the plants 
in the greenhouse and were dried down naturally. DNA was isolated from 
plant #176-11 using standard techniques and analysed by Southern blot 
analysis. It was found to contain sequences which hybridize with probes 
generated from the synthetic cryIA(b) gene and with a probe generated from 
the PAT gene. These results showed integration of these genes into the 
genome of maize. 
Experiment KC-64 
Production of Transgenic Maize Plants Expressing the Synthetic cryIA(b) 
Gene Using a Constitutive Promoter 
Tissue 
Immature maize embryos, approximately 1.5-2.5 mm in length, were excised 
from an ear of genotype 6N615 14-15 days after pollination. The mother 
plant was grown in the greenhouse. Before excision, the ear was surface 
sterilized with 20% Clorox for 20 minutes and rinse 3 times with sterile 
water. Individual embryos were plated scutellum side in a 2 cm square 
area, 36 embryos to a plate, on the callus initiation medium, 2DG4+5 
chloramben medium (N6 major salts, B5 minor salts, MS iron, 2% sucrose, 
with 5 mg/l chloramben, 20 mg/l glucose, and 10 ml G4 additions Table 1) 
added after autoclaving. 
TABLE 1 
______________________________________ 
G4 Additions 
Ingredient per liter medium 
______________________________________ 
Casein hydrolysate 0.5 gm 
Proline 1.38 gm 
Nicotinic acid .2 mg 
Pyridoxine-HCl .2 mg 
Thiamine-HCl .5 mg 
Choline-HCl .1 mg 
Riboflavin .05 mg 
Biotin .1 mg 
Folic acid .05 mg 
Ca pantothenate .1 mg 
p-aminobenzoic acid .05 mg 
B12 .136 .mu.g 
______________________________________ 
Bombardment 
Tissue was bombarded using the PDS-1000He Biolistics device. The tissue was 
placed on the shelf 8 cm below the stopping screen shelf. The tissue was 
shot one time with the DNA/gold microcarrier solution, 10 .mu.l dried onto 
the macrocarrier. The stopping screen used was hand punched at ABRU using 
10.times.10 stainless steel mesh screen. Rupture discs of 1550 psi value 
were used. After bombardment, the embryos were cultured in the dark at 
25.degree. C. 
Preparation of DNA for Delivery 
The microcarrier was prepared essentially according to the instructions 
supplied with the Biolistic device. While vortexing 50 .mu.l 1.0.mu. gold 
microcarrier, added 3.2 .mu.l pCIB4418 (0.85 .mu.g/.mu.l) [#905]+2 .mu.l 
pCIB3064 0.895 .mu.g/.mu.l) [#456]+1.6 .mu.l pCIB3007A (1.7 .mu.g/.mu.l) 
[#152] followed by 50 .mu.l 2.5M CaCl.sub.2, then 20 .mu.l 0.1M spermidine 
(free base, TC grade). The resulting mixture was vortexed 3 minutes and 
microfuged for 10 sec. The supernatant was removed and the microcarriers 
washed 2 times with 250 .mu.l of 100% EtOH (HPLC grade) by vortexing 
briefly, centrifuging and removing the supernatant. The microcarriers are 
resuspended in 65 .mu.l 100% EtOH. 
Callus Formation 
Embryos were transferred to callus initiation medium with 3 mg/l PPT 1 day 
after bombardment. Embryos were scored for callus initiation at 2 and 3 
weeks after bombardment. Any responses were transferred to callus 
maintenance medium, 2DG4+0.5 2,4-D medium with 3 mg/L PPT. Callus 
maintenance medium is N6 major salts, B5 minor salts, MS iron, 2% sucrose, 
with 0.5 mg/l 2,4-D, 20 mg/l glucose, and 10 ml G4 additions added after 
utoclaving. Embryogenic callus was subcultured every 2 weeks to fresh 
maintenance medium containing 3 mg/L PPT. All callus was incubated in the 
dark at 25.degree. C. 
The Type I callus formation response was 18%. Every embryo which produced 
callus was cultured as an individual event giving rise to an individual 
line. 
Regeneration 
After 12 weeks on selection, the tissue was removed from callus maintenance 
medium with PPT and was placed on regeneration medium and incubated at 
25.degree. C. using a 16 hour light (50 .mu.E .m-2 . s-1)/8 hour dark 
photoperiod. Regeneration medium is 0.25MS3S5BA (0.25 mg/l 2,4 D, 5 mg/l 
BAP, MS salts, 3% sucrose) for 2 weeks followed by subculture to MS3S 
medium for regeneration of plants. After 4 to 10 weeks, plants were 
removed and put into GA 7's. Our line KC64 0-1, which became the #170 BT 
event, produced 55 plants. Our line KC64 0-7, which became the #171BT 
event, produced a total of 33 plants. 
Assays 
Eleven plants, as they became established in the GA7's, were tested by the 
chlorophenol red (CR) test for resistance to PPT as per Shillito, et al, 
above. This assay utilizes a pH sensitive indicator dye to show which 
cells are growing in the presence of PPT. Cells which grow produce a pH 
change in the media and turn the indicator yellow (from red). Plants 
expressing the resistance gene to PPT are easily seen in this test. Plants 
positive by the CR test were assayed by PCR for the presence of the 
synthetic BT gene. (Event 170=37 positive/18 negative; #171=25 positive/8 
negative). 
Plants positive by PCR for the syn-Bt gene were sent to the phytotron. Once 
established in the phytotron, they were characterized using insect 
bioassays and ELISA analysis. Plants were insect bioassayed using a 
standard European corn borer assay (see below) in which small pieces of 
leaf of clipped from a lant and placed in a small petri dish with a number 
of ECB neonate larvae. Plants are typically assayed at a height of about 6 
inches. Plants showing 100% mortality to ECB in this assay are 
characterized further. ELISA data are shown below. Positive plants are 
moved to the greenhouse. 
Basta screening 
Eight of the mature plants from the #170 event were selected for evaluation 
of Basta [Hoechst] resistance. On one middle leaf per plant, an area 
approximately 10-14 cm long.times.the leaf width was painted with 0, 0.4, 
1.0 or 2.0% (10 ml of 200 g/L diluted to 100 ml with deionized water) 
aqueous Basta containing 2 drops of Tween 20/100 ml. Two plants were 
tested per level. Eight wild-type 6N615 plants of the same approximate age 
were treated as controls. All plants were observed at 4 and 7 days. All of 
the control plants eventually died. Throughout the study, none of the #170 
plants displayed any damage due to the herbicide. 
Pollination 
All tassel ears, first ear and, if available, the second ear on the #170 
and #171 plants were pollinated with wild-type 6N615 pollen. At least 90% 
of the plants were female fertile. 
Pollen from #171 plants was outcrossed to genotypes 6N615, 5N984, 5NA89, 
6F010, 5NA56, 2N217AF, 2NDO1 and 3N961. At least 90% of the plants were 
shown to be male fertile. 
Embryo Rescue 
Embryos from the #171 event have been "rescued." Fourteen to 16 days after 
pollination, the ear tip with 25-50 kernels was cut from the ear with a 
coping saw. Prior to cutting, the husks were gently peeled away to expose 
the upper portion of the ear. The cut end of the ear on the plant was 
painted with Captan fungicide and the husks replaced. The seed remaining 
on the plant was allowed to dry naturally. 
The excised ear piece was surface sterilized with 20% Clorox for 20 minutes 
and rinsed 3 times with sterile water. Individual embryos were excised and 
plated scutellum side up on B5 medium [Gamborg] containing 2% sucrose. B5 
vitamins are added to the medium after autoclaving. Four embryos were 
plated per GA7 container and the containers incubated in the dark. When 
germination occurred, the containers were moved to a light culture room 
and incubated at 25.degree. C. using a 16 hour light (50 .mu.E .m-2 . 
s-1)/8 hour dark photoperiod. The germination frequency is 94%. 
Progeny from 15 plants of the #171 event and 2 of the #176 event were 
rescued using standard embryo rescue techniques and evaluated. All plants 
were evaluated by insect assay. Plants from the #171 event were also 
tested in the histochemical GUS assay. In both the insect assay and the 
GUS assay, the ratio of segregation of the transgenes was 1:1, as expected 
for a single locus insertion event. 
Example 48 
Analysis of Transgenic Maize Plants 
ELISA ASSAY 
Detection of cryIA(b) gene expression in transgenic maize is monitored 
using European corn borer(ECB) insect bioassays and ELISA analysis for a 
quantitative determination of the level of cryIA(b) protein obtained. 
Quantitative determination of cryIA(b) IP in the leaves of transgenic 
plants was performed using enzyme-linked immunosorbant assays (ELISA) as 
disclosed in Clark M. F., Lister R. M., Bar-Joseph M.: ELISA Techniques. 
In: Weissbach A., Weissbach H. (eds) Methods in Enzymology 118:742-766, 
Academic Press, Florida (1986). Immunoaffinity purified polyclonal rabbit 
and goat antibodies specific for the B. thuringiensis subsp. kurstaki IP 
were used to determine ng IP per mg soluble protein from crude extracts of 
leaf samples. The sensitivity of the double sandwich ELISA is 1-5 ng IP 
per mg soluble protein using 50 ug of total protein per ELISA microtiter 
dish well. 
Corn extracts were made by grinding leaf tissue in gauze lined plastic bags 
using a hand held ball-bearing homogenizer (AGDIA, Elkart Ind.) in the 
presence of extraction buffer (50 mM Na.sub.2 CO.sub.3 pH 9.5, 100 mM 
NaCl, 0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 .mu.M leupeptin). Protein 
determination was performed using the Bio-Rad (Richmond, Calif.) protein 
assay. 
Using the above procedure, the primary maize transformants described above 
were analyzed for the presence of cryIA(b) protein using ELISA. These 
plants varied in height from 6 inches to about three feet at the time of 
analysis. 
______________________________________ 
Plant Bt ng/mg soluble protein 
5/27/91 
______________________________________ 
176-8 0 0 
176-10 700 1585 
176-11 760 2195 
171-4A 59 
171-6 50 
171-8 60 
171-9 280 
171-13 77 
171-14A 43 
171-14B 60 
171-15 55 
171-16A 13 
171-16B 19 
171-18 19 
176-30 1160 
171-32 980 
171-31 166 
171-30 370 
71-14 
#10 leaf 26 
1 leaf 17 
plant 171-16 
#9 leaf 40 
#1 leaf 120 
______________________________________ 
EUROPEAN CORN BORER ASSAY 
1. One to four 4 cm sections are cut from an extended leaf of a corn plant. 
2. Each leaf piece is placed on a moistened filter disc in a 50.times.9 mm 
petri dish. 
3. Five neonate European corn borer larvae are placed on each leaf piece. 
(Making a total of 5-20 larvae per plant.) 
4. The petri dishes are incubated at 29.5.degree. C. 
5. Leaf feeding damage and mortality data are scored at 24, 48, and 72 
hours. 
Example 49 
Expression of Bt Endotoxin in Progeny of Transformed Maize Plants 
The transformed maize plants were fully fertile and were crossed with 
several genotypes of maize. Progeny from these crosses were analyzed for 
their ability to kill European corn borer (ECB) in a standard ECB bioassay 
(described immediately above) as well as for the presence of the cryIA(b) 
protein using ELISA as described above. The ability to kill ECB and the 
production of cryIA(b) protein correlated. These traits segregated to the 
progeny with a 1:1 ratio, indicating a single site of insertion for the 
active copy of the synthetic gene. This 1:1 ratio was true for both the 
constitutive Promoter/synthetic-cryIA(b) plants and the tissue specific 
Promoter/synthetic-cryIA(b) plants (data not shown). 
FIG. 23A is a table containing a small subset of the total number of 
progeny analyzed. This table is representative of a number of different 
crosses. 
Insect assays were done with Diatrea saccharalis and Ostrinia nubilalis 
using leaf material (as described above) of transgenic progeny containing 
a maize optimized CryIA(b) gene. The results of these assays are shown in 
FIG. 23B. They demonstrate that the maize optimized CryIA(b) gene 
functions in transformed maize to provide resistance to Sugarcane borer 
and Ostrinia nubilalis. 
Example 50 
Expression of the cryIA(b) Gene in Maize Pollen 
Progeny of the transformed maize plants containing the chimeric pollen 
promoter/synthetic cryIA(b) gene derived from pCIB4431 were grown in the 
field to maturity. Pollen was collected and analyzed for the presence of 
the cryIA(b) protein using standard ELISA techniquesd as described 
elsewhere. High levels of cryIA(b) protein were detected in the pollen. 
Progeny from the 35S promoter/synthetic cryIA(b) transformed plant were 
grown in the greenhouse. Pollen from these plants was analyzed using 
ELISA, and cryIA(b) protein was detected. 
Results are shown below in FIG. 23C. 
It is recognized that factors including selection of plant lines, plant 
genotypes, synthetic sequences and the like, may also affect expression. 
Example 51 
Expression of the CryIA(b) Gene Fused to a Pith-preferred Promoter 
pCIB4433 (FIG. 36) is a plasmid containing the maize optimized CryIA(b) 
gene fused with the pith-preferred promoter isolated from maize. This 
plasmid was constructed using a three-way ligation consisting of: 
1) pCIB4418, cut with BstEII and BamHI; 1.8 Kb fragment 
2) pBtin1, cut with NsiI and BstEII; 5.9 Kb fragment; pBtin1 is described 
elsewhere in this application 
3) PCR fragment VI-151 was generated in a PCR reaction using standard 
conditions as described elsewhere in this application. 
PCR primers utilized were: 
__________________________________________________________________________ 
KE150A28: 
ATT CGC ATG CAT GTT TCA TTA TC-3' (SEQ ID NO: 93) 
KE151A28: 
GCT GGT ACC ACG GAT CCG TCG CTT CTG TGC AAC AAC 
C-3' (SEQ ID NO: 94) 
__________________________________________________________________________ 
After the PCR reaction, the DNA was checked on an agarose gel to make sure 
the reaction had proceeded properly. DNA was recovered from the PCR 
reaction using standard conditions described elsewhere and subsequently 
cut with the restriction enzymes NsiI and BamHI using standard condition. 
After cutting, the fragment was run on a 2% NuSieve gel and the desired 
band recovered as described elsewhere. The DNA was used in the ligation 
described above. 
After ligation (under standard condition), the DNA was transformed into 
competent E. coli cell. 
Transformation was carried out using microprojectile bombardment 
essentially as described elsewhere in this application. Embryos were 
transferred to medium containing 10.differential. .mu.g/ml PPT 24 hours 
after microprojectile bombardment. Resulting callus was transferred to 
medium containing 40 .mu.g/ml PPT after four weeks. Plants were 
regenerated without selection. 
A small sample of plants (3-5) was assayed by PCR for each event. Further 
codes were added to indicate different positions and distances of embryos 
with respect to the microprojectile bombardment device. Plants were sent 
to the greenhouse having the following codes: 
______________________________________ 
JS21A TOP Plants B.t. PCR Positive 
JS21A MID Plants B.t. PCR Positive 
JS21C BOT Plants B.t. PCR Positive 
JS22D MID Plants B.t. PCR Positive 
JS23B MID Plants B.t. PCR Negative (for control) 
______________________________________ 
Leaf samples from the regenerated plants were bioassayed for insecticidal 
activity against European corn borer as described in Example 48 with the 
results shown in FIG. 23D. 
ELISA analysis of leaf samples to quantify the level of CryIA(b) protein 
expressed in the leaves was carried out as described in Example 48 with 
the results shown in FIG. 23E. 
DEPOSITS 
The following plasmids have been deposited with the Agricultural Research 
Culture Collection (NRRL)(1818 N. University St., Peoria, Ill. 61604) 
under the provisions of the Budapest Treaty: pCIB4418, pCIB4420, pCIB4429, 
pCIB4431, pCIB4433, pCIB5601, pCIB3166 and pCIB3171. 
The present invention has been described with reference to specific 
embodiments thereof; however it will be appreciated that numerous 
variations, modifications, and embodiments are possible. Accordingly, all 
such variations, modifications and embodiments are to be regarded as being 
within the spirit and scope of the present invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 94 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3468 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Bacillus thuringiensis kurstaki 
(B) STRAIN: HD-1 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..3468 
(D) OTHER INFORMATION: /product="Full-length native 
cryIA(b)" 
/note= "Appears in Figures 1 and 4 as BTHKURHD." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGGATAACAATCCGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAA60 
GTAGAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTG120 
TCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTA180 
GTTGATATAATATGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCTTGTACAAATT240 
GAACAGTTAATTAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTA300 
GAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGAT360 
CCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCC420 
CTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTA480 
TATGTTCAAGCTGCAAATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAA540 
AGGTGGGGATTTGATGCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATT600 
GGCAACTATACAGATCATGCTGTACGCTGGTACAATACGGGATTAGAGCGTGTATGGGGA660 
CCGGATTCTAGAGATTGGATAAGATATAATCAATTTAGAAGAGAATTAACACTAACTGTA720 
TTAGATATCGTTTCTCTATTTCCGAACTATGATAGTAGAACGTATCCAATTCGAACAGTT780 
TCCCAATTAACAAGAGAAATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTT840 
CGAGGCTCGGCTCAGGGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGATATACTT900 
AACAGTATAACCATCTATACGGATGCTCATAGAGGAGAATATTATTGGTCAGGGCATCAA960 
ATAATGGCTTCTCCTGTAGGGTTTTCGGGGCCAGAATTCACTTTTCCGCTATATGGAACT1020 
ATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGA1080 
ACATTATCGTCCACTTTATATAGAAGACCTTTTAATATAGGGATAAATAATCAACAACTA1140 
TCTGTTCTTGACGGGACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTA1200 
TACAGAAAAAGCGGAACGGTAGATTCGCTGGATGAAATACCGCCACAGAATAACAACGTG1260 
CCACCTAGGCAAGGATTTAGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTT1320 
AGTAATAGTAGTGTAAGTATAATAAGAGCTCCTATGTTCTCTTGGATACATCGTAGTGCT1380 
GAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACT1440 
AATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTT1500 
CGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGAGTAAATATTACTGCACCATTATCA1560 
CAAAGATATCGGGTAAGAATTCGCTACGCTTCTACCACAAATTTACAATTCCATACATCA1620 
ATTGACGGAAGACCTATTAATCAGGGGAATTTTTCAGCAACTATGAGTAGTGGGAGTAAT1680 
TTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGA1740 
TCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTATATAGAT1800 
CGAATTGAATTTGTTCCGGCAGAAGTAACCTTTGAGGCAGAATATGATTTAGAAAGAGCA1860 
CAAAAGGCGGTGAATGAGCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACAGATGTG1920 
ACGGATTATCATATTGATCAAGTATCCAATTTAGTTGAGTGTTTATCTGATGAATTTTGT1980 
CTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTTAGTGATGAG2040 
CGGAATTTACTTCAAGATCCAAACTTTAGAGGGATCAATAGACAACTAGACCGTGGCTGG2100 
AGAGGAAGTACGGATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTT2160 
ACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAAAAAATAGATGAG2220 
TCGAAATTAAAAGCCTATACCCGTTACCAATTAAGAGGGTATATCGAAGATAGTCAAGAC2280 
TTAGAAATCTATTTAATTCGCTACAATGCCAAACACGAAACAGTAAATGTGCCAGGTACG2340 
GGTTCCTTATGGCCGCTTTCAGCCCCAAGTCCAATCGGAAAATGTGCCCATCATTCCCAT2400 
CATTTCTCCTTGGACATTGATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGG2460 
GTGATATTCAAGATTAAGACGCAAGATGGCCATGCAAGACTAGGAAATCTAGAATTTCTC2520 
GAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAAAGAGCGGAGAAAAAATGG2580 
AGAGACAAACGTGAAAAATTGGAATGGGAAACAAATATTGTTTATAAAGAGGCAAAAGAA2640 
TCTGTAGATGCTTTATTTGTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATC2700 
GCGATGATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTGCCTGAG2760 
CTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAATTAGAAGGGCGTATTTTC2820 
ACTGCATTCTCCCTATATGATGCGAGAAATGTCATTAAAAATGGTGATTTTAATAATGGC2880 
TTATCCTGCTGGAACGTGAAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCG2940 
GTCCTTGTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGGT3000 
CGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAGGTTGCGTAACC3060 
ATTCATGAGATCGAGAACAATACAGACGAACTGAAGTTTAGCAACTGTGTAGAAGAGGAA3120 
GTATATCCAAACAACACGGTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAG3180 
GGTACGTACACTTCTCGTAATCGAGGATATGACGGAGCCTATGAAAGCAATTCTTCTGTA3240 
CCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACAGATGGACGAAGAGACAAT3300 
CCTTGTGAATCTAACAGAGGATATGGGGATTACACACCACTACCAGCTGGCTATGTGACA3360 
AAAGAATTAGAGTACTTCCCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAA3420 
GGAACATTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAA3468 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3468 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..3468 
(D) OTHER INFORMATION: /product="Full-length pure maize 
optimized synthetic Bt" 
/note= "Disclosed in Figure 3 as syn1T.mze" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTGAGCAACCCCGAG60 
GTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGCTACACCCCCATCGACATCAGCCTG120 
AGCCTGACCCAGTTCCTGCTGAGCGAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTG180 
GTGGACATCATCTGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCCATCAGCCGCCTG300 
GAGGGCCTGAGCAACCTGTACCAGATCTACGCCGAGAGCTTCCGCGAGTGGGAGGCCGAC360 
CCCACCAACCCCGCCCTGCGCGAGGAGATGCGCATCCAGTTCAACGACATGAACAGCGCC420 
CTGACCACCGCCATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTGAGCGTGTTCGGCCAG540 
CGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGCTACAACGACCTGACCCGCCTGATC600 
GGCAACTACACCGACCACGCCGTGCGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGC660 
CCCGACAGCCGCGACTGGATCCGCTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCCATCCGCACCGTG780 
AGCCAGCTGACCCGCGAGATCTACACCAACCCCGTGCTGGAGAACTTCGACGGCAGCTTC840 
CGCGGCAGCGCCCAGGGCATCGAGGGCAGCATCCGCAGCCCCCACCTGATGGACATCCTG900 
AACAGCATCACCATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
ATCATGGCCAGCCCCGTGGGCTTCAGCGGCCCCGAGTTCACCTTCCCCCTGTACGGCACC1020 
ATGGGCAACGCCGCCCCCCAGCAGCGCATCGTGGCCCAGCTGGGCCAGGGCGTGTACCGC1080 
ACCCTGAGCAGCACCCTGTACCGCCGCCCCTTCAACATCGGCATCAACAACCAGCAGCTG1140 
AGCGTGCTGGACGGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCCCAGAACAACAACGTG1260 
CCCCCCCGCCAGGGCTTCAGCCACCGCCTGAGCCACGTGAGCATGTTCCGCAGCGGCTTC1320 
AGCAACAGCAGCGTGAGCATCATCCGCGCCCCCATGTTCAGCTGGATCCACCGCAGCGCC1380 
GAGTTCAACAACATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGCGGCGACATCCTG1500 
CGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGCGTGAACATCACCGCCCCCCTGAGC1560 
CAGCGCTACCGCGTGCGCATCCGCTACGCCAGCACCACCAACCTGCAGTTCCACACCAGC1620 
ATCGACGGCCGCCCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAACTTCAGCAACGGC1740 
AGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAACAGCGGCAACGAGGTGTACATCGAC1800 
CGCATCGAGTTCGTGCCCGCCGAGGTGACCTTCGAGGCCGAGTACGACCTGGAGCGCGCC1860 
CAGAAGGCCGTGAACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
ACCGACTACCACATCGACCAGGTGAGCAACCTGGTGGAGTGCCTGAGCGACGAGTTCTGC1980 
CTGGACGAGAAGAAGGAGCTGAGCGAGAAGGTGAAGCACGCCAAGCGCCTGAGCGACGAG2040 
CGCAACCTGCTGCAGGACCCCAACTTCCGCGGCATCAACCGCCAGCTGGACCGCGGCTGG2100 
CGCGGCAGCACCGACATCACCATCCAGGGCGGCGACGACGTGTTCAAGGAGAACTACGTG2160 
ACCCTGCTGGGCACCTTCGACGAGTGCTACCCCACCTACCTGTACCAGAAGATCGACGAG2220 
AGCAAGCTGAAGGCCTACACCCGCTACCAGCTGCGCGGCTACATCGAGGACAGCCAGGAC2280 
CTGGAGATCTACCTGATCCGCTACAACGCCAAGCACGAGACCGTGAACGTGCCCGGCACC2340 
GGCAGCCTGTGGCCCCTGAGCGCCCCCAGCCCCATCGGCAAGTGCGCCCACCACAGCCAC2400 
CACTTCAGCCTGGACATCGACGTGGGCTGCACCGACCTGAACGAGGACCTGGGCGTGTGG2460 
GTGATCTTCAAGATCAAGACCCAGGACGGCCACGCCCGCCTGGGCAACCTGGAGTTCCTG2520 
GAGGAGAAGCCCCTGGTGGGCGAGGCCCTGGCCCGCGTGAAGCGCGCCGAGAAGAAGTGG2580 
CGCGACAAGCGCGAGAAGCTGGAGTGGGAGACCAACATCGTGTACAAGGAGGCCAAGGAG2640 
AGCGTGGACGCCCTGTTCGTGAACAGCCAGTACGACCGCCTGCAGGCCGACACCAACATC2700 
GCCATGATCCACGCCGCCGACAAGCGCGTGCACAGCATCCGCGAGGCCTACCTGCCCGAG2760 
CTGAGCGTGATCCCCGGCGTGAACGCCGCCATCTTCGAGGAGCTGGAGGGCCGCATCTTC2820 
ACCGCCTTCAGCCTGTACGACGCCCGCAACGTGATCAAGAACGGCGACTTCAACAACGGC2880 
CTGAGCTGCTGGAACGTGAAGGGCCACGTGGACGTGGAGGAGCAGAACAACCACCGCAGC2940 
GTGCTGGTGGTGCCCGAGTGGGAGGCCGAGGTGAGCCAGGAGGTGCGCGTGTGCCCCGGC3000 
CGCGGCTACATCCTGCGCGTGACCGCCTACAAGGAGGGCTACGGCGAGGGCTGCGTGACC3060 
ATCCACGAGATCGAGAACAACACCGACGAGCTGAAGTTCAGCAACTGCGTGGAGGAGGAG3120 
GTGTACCCCAACAACACCGTGACCTGCAACGACTACACCGCCACCCAGGAGGAGTACGAG3180 
GGCACCTACACCAGCCGCAACCGCGGCTACGACGGCGCCTACGAGAGCAACAGCAGCGTG3240 
CCCGCCGACTACGCCAGCGCCTACGAGGAGAAGGCCTACACCGACGGCCGCCGCGACAAC3300 
CCCTGCGAGAGCAACCGCGGCTACGGCGACTACACCCCCCTGCCCGCCGGCTACGTGACC3360 
AAGGAGCTGGAGTACTTCCCCGAGACCGACAAGGTGTGGATCGAGATCGGCGAGACCGAG3420 
GGCACCTTCATCGTGGACAGCGTGGAGCTGCTGCTGATGGAGGAGTAG3468 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1947 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..1947 
(D) OTHER INFORMATION: /product="Truncated synthetic 
maize optimized cryIA(b) gene" 
/note= "Disclosed in Figures 1, 2, 3, 4 and 5 as bssyn." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTGAGCAACCCCGAG60 
GTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGCTACACCCCCATCGACATCAGCCTG120 
AGCCTGACCCAGTTCCTGCTGAGCGAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTG180 
GTGGACATCATCTGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCCATCAGCCGCCTG300 
GAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAGAGCTTCCGCGAGTGGGAGGCCGAC360 
CCCACCAACCCCGCCCTGCGCGAGGAGATGCGCATCCAGTTCAACGACATGAACAGCGCC420 
CTGACCACCGCCATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGCGTGTTCGGCCAG540 
CGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGCTACAACGACCTGACCCGCCTGATC600 
GGCAACTACACCGACCACGCCGTGCGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGT660 
CCCGACAGCCGCGACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCCATCCGCACCGTG780 
AGCCAGCTGACCCGCGAGATTTACACCAACCCCGTGCTGGAGAACTTCGACGGCAGCTTC840 
CGCGGCAGCGCCCAGGGCATCGAGGGCAGCATCCGCAGCCCCCACCTGATGGACATCCTG900 
AACAGCATCACCATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCCCTGTACGGCACC1020 
ATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCACAGCTGGGCCAGGGAGTGTACCGC1080 
ACCCTGAGCAGCACCCTGTACCGTCGACCTTTCAACATCGGCATCAACAACCAGCAGCTG1140 
AGCGTGCTGGACGGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAGAACAACAACGTG1260 
CCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCACGTGAGCATGTTCCGCAGTGGCTTC1320 
AGCAACAGCAGCGTGAGCATCATCCGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCC1380 
GAGTTCAACAACATCATCCCCAGCAGCCAAATCACCCAGATCCCCCTGACCAAGAGCACC1440 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGCGGCGACATCCTG1500 
CGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGCGTGAACATCACCGCCCCCCTGAGC1560 
CAGCGCTACCGCGTCCGCATCCGCTACGCCAGCACCACCAACCTGCAGTTCCACACCAGC1620 
ATCGACGGCCGCCCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAACTTCAGCAACGGC1740 
AGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAACAGCGGCAACGAGGTGTACATCGAC1800 
CGCATCGAGTTCGTGCCCGCCGAGGTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCT1860 
CAGAAGGCCGTGAACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
ACCGACTACCACATCGATCAGGTGTAG1947 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3468 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..3468 
(D) OTHER INFORMATION: /product="Full length synthetic 
maize optimized" 
/note= "Disclosed in Figure 3 as synful.mod. This 
sequence is identical to flsynbt.fin as disclosed 
in Figure 1." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTGAGCAACCCCGAG60 
GTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGCTACACCCCCATCGACATCAGCCTG120 
AGCCTGACCCAGTTCCTGCTGAGCGAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTG180 
GTGGACATCATCTGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCCATCAGCCGCCTG300 
GAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAGAGCTTCCGCGAGTGGGAGGCCGAC360 
CCCACCAACCCCGCCCTGCGCGAGGAGATGCGCATCCAGTTCAACGACATGAACAGCGCC420 
CTGACCACCGCCATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGCGTGTTCGGCCAG540 
CGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGCTACAACGACCTGACCCGCCTGATC600 
GGCAACTACACCGACCACGCCGTGCGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGT660 
CCCGACAGCCGCGACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCCATCCGCACCGTG780 
AGCCAGCTGACCCGCGAGATTTACACCAACCCCGTGCTGGAGAACTTCGACGGCAGCTTC840 
CGCGGCAGCGCCCAGGGCATCGAGGGCAGCATCCGCAGCCCCCACCTGATGGACATCCTG900 
AACAGCATCACCATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCCCTGTACGGCACC1020 
ATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCACAGCTGGGCCAGGGAGTGTACCGC1080 
ACCCTGAGCAGCACCCTGTACCGTCGACCTTTCAACATCGGCATCAACAACCAGCAGCTG1140 
AGCGTGCTGGACGGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAGAACAACAACGTG1260 
CCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCACGTGAGCATGTTCCGCAGTGGCTTC1320 
AGCAACAGCAGCGTGAGCATCATCCGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCC1380 
GAGTTCAACAACATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGCGGCGACATCCTG1500 
CGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGCGTGAACATCACCGCCCCCCTGAGC1560 
CAGCGCTACCGCGTCCGCATCCGCTACGCCAGCACCACCAACCTGCAGTTCCACACCAGC1620 
ATCGACGGCCGCCCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAACTTCAGCAACGGC1740 
AGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAACAGCGGCAACGAGGTGTACATCGAC1800 
CGCATCGAGTTCGTGCCCGCCGAGGTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCT1860 
CAGAAGGCCGTGAACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
ACCGACTACCACATCGATCAGGTGAGCAACCTGGTGGAGTGCCTGAGCGACGAGTTCTGC1980 
CTGGACGAGAAGAAGGAGCTGAGCGAGAAGGTGAAGCACGCCAAGCGCCTGAGCGACGAG2040 
CGCAACCTGCTGCAGGACCCCAACTTCCGCGGCATCAACCGCCAGCTGGACCGCGGCTGG2100 
CGCGGCAGCACCGACATCACCATCCAGGGCGGCGACGACGTGTTCAAGGAGAACTACGTG2160 
ACCCTGCTGGGCACCTTCGACGAGTGCTACCCCACCTACCTGTACCAGAAGATCGACGAG2220 
AGCAAGCTGAAGGCCTACACCCGCTACCAGCTGCGCGGCTACATCGAGGACAGCCAGGAC2280 
CTGGAGATCTACCTGATCCGCTACAACGCCAAGCACGAGACCGTGAACGTGCCCGGCACC2340 
GGCAGCCTGTGGCCCCTGAGCGCCCCCAGCCCCATCGGCAAGTGCGCCCACCACAGCCAC2400 
CACTTCAGCCTGGACATCGACGTGGGCTGCACCGACCTGAACGAGGACCTGGGCGTGTGG2460 
GTGATCTTCAAGATCAAGACCCAGGACGGCCACGCCCGCCTGGGCAACCTGGAGTTCCTG2520 
GAGGAGAAGCCCCTGGTGGGCGAGGCCCTGGCCCGCGTGAAGCGCGCCGAGAAGAAGTGG2580 
CGCGACAAGCGCGAGAAGCTGGAGTGGGAGACCAACATCGTGTACAAGGAGGCCAAGGAG2640 
AGCGTGGACGCCCTGTTCGTGAACAGCCAGTACGACCGCCTGCAGGCCGACACCAACATC2700 
GCCATGATCCACGCCGCCGACAAGCGCGTGCACAGCATTCGCGAGGCCTACCTGCCCGAG2760 
CTGAGCGTGATCCCCGGCGTGAACGCCGCCATCTTCGAGGAGCTGGAGGGCCGCATCTTC2820 
ACCGCCTTCAGCCTGTACGACGCCCGCAACGTGATCAAGAACGGCGACTTCAACAACGGC2880 
CTGAGCTGCTGGAACGTGAAGGGCCACGTGGACGTGGAGGAGCAGAACAACCACCGCAGC2940 
GTGCTGGTGGTGCCCGAGTGGGAGGCCGAGGTGAGCCAGGAGGTGCGCGTGTGCCCCGGC3000 
CGCGGCTACATCCTGCGCGTGACCGCCTACAAGGAGGGCTACGGCGAGGGCTGCGTGACC3060 
ATCCACGAGATCGAGAACAACACCGACGAGCTCAAGTTCAGCAACTGCGTGGAGGAGGAG3120 
GTGTACCCCAACAACACCGTGACCTGCAACGACTACACCGCCACCCAGGAGGAGTACGAG3180 
GGCACCTACACCAGCCGCAACCGCGGCTACGACGGCGCCTACGAGAGCAACAGCAGCGTG3240 
CCCGCCGACTACGCCAGCGCCTACGAGGAGAAGGCCTACACCGACGGCCGCCGCGACAAC3300 
CCCTGCGAGAGCAACCGCGGCTACGGCGACTACACCCCCCTGCCCGCCGGCTACGTGACC3360 
AAGGAGCTGGAGTACTTCCCCGAGACCGACAAGGTGTGGATCGAGATCGGCGAGACCGAG3420 
GGCACCTTCATCGTGGACAGCGTGGAGCTGCTGCTGATGGAGGAGTAG3468 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1845 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..1845 
(D) OTHER INFORMATION: /note= "This is the synthetic Bt 
gene according to Perlak et al. as shown in Figures 4 
and 5 as PMONBT." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ATGGACAACAACCCAAACATCAACGAATGCATTCCATACAACTGCTTGAGTAACCCAGAA60 
GTTGAAGTACTTGGTGGAGAACGCATTGAAACCGGTTACACTCCCATCGACATCTCCTTG120 
TCCTTGACACAGTTTCTGCTCAGCGAGTTCGTGCCAGGTGCTGGGTTCGTTCTCGGACTA180 
GTTGACATCATCTGGGGTATCTTTGGTCCATCTCAATGGGATGCATTCCTGGTGCAAATT240 
GAGCAGTTGATCAACCAGAGGATCGAAGAGTTCGCCAGGAACCAGGCCATCTCTAGGTTG300 
GAAGGATTGAGCAATCTCTACCAAATCTATGCAGAGAGCTTCAGAGAGTGGGAAGCCGAT360 
CCTACTAACCCAGCTCTCCGCGAGGAAATGCGTATTCAATTCAACGACATGAACAGCGCC420 
TTGACCACAGCTATCCCATTGTTCGCAGTCCAGAACTACCAAGTTCCTCTCTTGTCCGTG480 
TACGTTCAAGCAGCTAATCTTCACCTCAGCGTGCTTCGAGACGTTAGCGTGTTTGGGCAA540 
AGGTGGGGATTCGATGCTGCAACCATCAATAGCCGTTACAACGACCTTACTAGGCTGATT600 
GGAAACTACACCGACCACGCTGTTCGTTGGTACAACACTGGCTTGGAGCGTGTCTGGGGT660 
CCTGATTCTAGAGATTGGATTAGATACAACCAGTTCAGGAGAGAATTGACCCTCACAGTT720 
TTGGACATTGTGTCTCTCTTCCCGAACTATGACTCCAGAACCTACCCTATCCGTACAGTG780 
TCCCAACTTACCAGAGAAATCTATACTAACCCAGTTCTTGAGAACTTCGACGGTAGCTTC840 
CGTGGTTCTGCCCAAGGTATCGAAGGCTCCATCAGGAGCCCACACTTGATGGACATCTTG900 
AACAGCATAACTATCTACAGCGATGCTCACAGAGGAGAGTATTACTGGTCTGGACACCAG960 
ATCATGGCCTCTCCAGTTGGATTCAGCGGGCCCGAGTTTACCTTTCCTCTCTATGGAACT1020 
ATGGGAAACGCCGCTCCACAACAACGTATCGTTGCTCAACTAGGTCAGGGTGTCTACAGA1080 
ACCTTGTCTTCCACCTTGTACAGAAGACCCTTCAATATCGGTATCAACAACCAGCAACTT1140 
TCCGTTCTTGACGGAACAGAGTTCGCCTATGGAACCTCTTCTAACTTGCCATCCGCTGTT1200 
TACAGAAAGAGCGGAACCGTTGATTCCTTGGACGAAATCCCACCACAGAACAACAATGTG1260 
CCACCCAGGCAAGGATTCTCCCACAGGTTGAGCCACGTGTCCATGTTCCGTTCCGGATTC1320 
AGCAACAGTTCCGTGAGCATCATCAGAGCTCCTATGTTCTCATGGATTCATCGTAGTGCT1380 
GAGTTCAACAATATCATTCCTTCCTCTCAAATCACCCAAATCCCATTGACCAAGTCTACT1440 
AACCTTGGATCTGGAACTTCTGTCGTGAAAGGACCAGGCTTCACAGGAGGTGATATTCTT1500 
AGAAGAACTTCTCCTGGCCAGATTAGCACCCTCAGAGTTAACATCACTGCACCACTTTCT1560 
CAAAGATATCGTGTCAGGATTCGTTACGCATCTACCACTAACTTGCAATTCCACACCTCC1620 
ATCGACGGAAGGCCTATCAATCAGGGTAACTTCTCCGCAACCATGTCAAGCGGCAGCAAC1680 
TTGCAATCCGGCAGCTTCAGAACCGTCGGTTTCACTACTCCTTTCAACTTCTCTAACGGA1740 
TCAAGCGTTTTCACCCTTAGCGCTCATGTGTTCAATTCTGGCAATGAAGTGTACATTGAC1800 
CGTATTGAGTTTGTGCCTGCCGAAGTTACCTTCGAGGCTGAGTAC1845 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3624 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3621 
(D) OTHER INFORMATION: /product="Full-length, maize 
optmized cryIB" 
/note= "Disclosed in Figure 6." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ATGGACCTGCTGCCCGACGCCCGCATCGAGGACAGCCTGTGCATCGCC48 
MetAspLeuLeuProAspAlaArgIleGluAspSerLeuCysIleAla 
151015 
GAGGGCAACAACATCGACCCCTTCGTGAGCGCCAGCACCGTGCAGACC96 
GluGlyAsnAsnIleAspProPheValSerAlaSerThrValGlnThr 
202530 
GGCATCAACATCGCCGGCCGCATCCTGGGCGTGCTGGGCGTGCCCTTC144 
GlyIleAsnIleAlaGlyArgIleLeuGlyValLeuGlyValProPhe 
354045 
GCCGGCCAGCTGGCCAGCTTCTACAGCTTCCTGGTGGGCGAGCTGTGG192 
AlaGlyGlnLeuAlaSerPheTyrSerPheLeuValGlyGluLeuTrp 
505560 
CCCCGCGGCCGCGACCAGTGGGAGATCTTCCTGGAGCACGTGGAGCAG240 
ProArgGlyArgAspGlnTrpGluIlePheLeuGluHisValGluGln 
65707580 
CTGATCAACCAGCAGATCACCGAGAACGCCCGCAACACCGCCCTGGCC288 
LeuIleAsnGlnGlnIleThrGluAsnAlaArgAsnThrAlaLeuAla 
859095 
CGCCTGCAGGGCCTGGGCGACAGCTTCCGCGCCTACCAGCAGAGCCTG336 
ArgLeuGlnGlyLeuGlyAspSerPheArgAlaTyrGlnGlnSerLeu 
100105110 
GAGGACTGGCTGGAGAACCGCGACGACGCCCGCACCCGCAGCGTGCTG384 
GluAspTrpLeuGluAsnArgAspAspAlaArgThrArgSerValLeu 
115120125 
TACACCCAGTACATCGCCCTGGAGCTGGACTTCCTGAACGCCATGCCC432 
TyrThrGlnTyrIleAlaLeuGluLeuAspPheLeuAsnAlaMetPro 
130135140 
CTGTTCGCCATCCGCAACCAGGAGGTGCCCCTGCTGATGGTGTACGCC480 
LeuPheAlaIleArgAsnGlnGluValProLeuLeuMetValTyrAla 
145150155160 
CAGGCCGCCAACCTGCACCTGCTGCTGCTGCGCGACGCCAGCCTGTTC528 
GlnAlaAlaAsnLeuHisLeuLeuLeuLeuArgAspAlaSerLeuPhe 
165170175 
GGCAGCGAGTTCGGCCTGACCAGCCAGGAGATCCAGCGCTACTACGAG576 
GlySerGluPheGlyLeuThrSerGlnGluIleGlnArgTyrTyrGlu 
180185190 
CGCCAGGTGGAGCGCACCCGCGACTACAGCGACTACTGCGTGGAGTGG624 
ArgGlnValGluArgThrArgAspTyrSerAspTyrCysValGluTrp 
195200205 
TACAACACCGGCCTGAACAGCCTGCGCGGCACCAACGCCGCCAGCTGG672 
TyrAsnThrGlyLeuAsnSerLeuArgGlyThrAsnAlaAlaSerTrp 
210215220 
GTGCGCTACAACCAGTTCCGCCGCGACCTGACCCTGGGCGTGCTGGAC720 
ValArgTyrAsnGlnPheArgArgAspLeuThrLeuGlyValLeuAsp 
225230235240 
CTGGTGGCCCTGTTCCCCAGCTACGACACCCGCACCTACCCCATCAAC768 
LeuValAlaLeuPheProSerTyrAspThrArgThrTyrProIleAsn 
245250255 
ACCAGCGCCCAGCTGACCCGCGAGGTGTACACCGACGCCATCGGCGCC816 
ThrSerAlaGlnLeuThrArgGluValTyrThrAspAlaIleGlyAla 
260265270 
ACCGGCGTGAACATGGCCAGCATGAACTGGTACAACAACAACGCCCCC864 
ThrGlyValAsnMetAlaSerMetAsnTrpTyrAsnAsnAsnAlaPro 
275280285 
AGCTTCAGCGCCATCGAGGCCGCCGCCATCCGCAGCCCCCACCTGCTG912 
SerPheSerAlaIleGluAlaAlaAlaIleArgSerProHisLeuLeu 
290295300 
GACTTCCTGGAGCAGCTGACCATCTTCAGCGCCAGCAGCCGCTGGAGC960 
AspPheLeuGluGlnLeuThrIlePheSerAlaSerSerArgTrpSer 
305310315320 
AACACCCGCCACATGACCTACTGGCGCGGCCACACCATCCAGAGCCGC1008 
AsnThrArgHisMetThrTyrTrpArgGlyHisThrIleGlnSerArg 
325330335 
CCCATCGGCGGCGGCCTGAACACCAGCACCCACGGCGCCACCAACACC1056 
ProIleGlyGlyGlyLeuAsnThrSerThrHisGlyAlaThrAsnThr 
340345350 
AGCATCAACCCCGTGACCCTGCGCTTCGCCAGCCGCGACGTGTACCGC1104 
SerIleAsnProValThrLeuArgPheAlaSerArgAspValTyrArg 
355360365 
ACCGAGAGCTACGCCGGCGTGCTGCTGTGGGGCATCTACCTGGAGCCC1152 
ThrGluSerTyrAlaGlyValLeuLeuTrpGlyIleTyrLeuGluPro 
370375380 
ATCCACGGCGTGCCCACCGTGCGCTTCAACTTCACCAACCCCCAGAAC1200 
IleHisGlyValProThrValArgPheAsnPheThrAsnProGlnAsn 
385390395400 
ATCAGCGACCGCGGCACCGCCAACTACAGCCAGCCCTACGAGAGCCCC1248 
IleSerAspArgGlyThrAlaAsnTyrSerGlnProTyrGluSerPro 
405410415 
GGCCTGCAGCTGAAGGACAGCGAGACCGAGCTGCCCCCCGAGACCACC1296 
GlyLeuGlnLeuLysAspSerGluThrGluLeuProProGluThrThr 
420425430 
GAGCGCCCCAACTACGAGAGCTACAGCCACCGCCTGAGCCACATCGGC1344 
GluArgProAsnTyrGluSerTyrSerHisArgLeuSerHisIleGly 
435440445 
ATCATCCTGCAGAGCCGCGTGAACGTGCCCGTGTACAGCTGGACCCAC1392 
IleIleLeuGlnSerArgValAsnValProValTyrSerTrpThrHis 
450455460 
CGCAGCGCCGACCGCACCAACACCATCGGCCCCAACCGCATCACCCAG1440 
ArgSerAlaAspArgThrAsnThrIleGlyProAsnArgIleThrGln 
465470475480 
ATCCCCATGGTGAAGGCCAGCGAGCTGCCCCAGGGCACCACCGTGGTG1488 
IleProMetValLysAlaSerGluLeuProGlnGlyThrThrValVal 
485490495 
CGCGGCCCCGGCTTCACCGGCGGCGACATCCTGCGCCGCACCAACACC1536 
ArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrAsnThr 
500505510 
GGCGGCTTCGGCCCCATCCGCGTGACCGTGAACGGCCCCCTGACCCAG1584 
GlyGlyPheGlyProIleArgValThrValAsnGlyProLeuThrGln 
515520525 
CGCTACCGCATCGGCTTCCGCTACGCCAGCACCGTGGACTTCGACTTC1632 
ArgTyrArgIleGlyPheArgTyrAlaSerThrValAspPheAspPhe 
530535540 
TTCGTGAGCCGCGGCGGCACCACCGTGAACAACTTCCGCTTCCTGCGC1680 
PheValSerArgGlyGlyThrThrValAsnAsnPheArgPheLeuArg 
545550555560 
ACCATGAACAGCGGCGACGAGCTGAAGTACGGCAACTTCGTGCGCCGC1728 
ThrMetAsnSerGlyAspGluLeuLysTyrGlyAsnPheValArgArg 
565570575 
GCCTTCACCACCCCCTTCACCTTCACCCAGATCCAGGACATCATCCGC1776 
AlaPheThrThrProPheThrPheThrGlnIleGlnAspIleIleArg 
580585590 
ACCAGCATCCAGGGCCTGAGCGGCAACGGCGAGGTGTACATCGACAAG1824 
ThrSerIleGlnGlyLeuSerGlyAsnGlyGluValTyrIleAspLys 
595600605 
ATCGAGATCATCCCCGTGACCGCCACCTTCGAGGCCGAGTACGACCTG1872 
IleGluIleIleProValThrAlaThrPheGluAlaGluTyrAspLeu 
610615620 
GAGCGCGCCCAGGAGGCCGTGAACGCCCTGTTCACCAACACCAACCCC1920 
GluArgAlaGlnGluAlaValAsnAlaLeuPheThrAsnThrAsnPro 
625630635640 
CGCCGCCTGAAGACCGACGTGACCGACTACCACATCGACCAGGTGAGC1968 
ArgArgLeuLysThrAspValThrAspTyrHisIleAspGlnValSer 
645650655 
AACCTGGTGGCCTGCCTGAGCGACGAGTTCTGCCTGGACGAGAAGCGC2016 
AsnLeuValAlaCysLeuSerAspGluPheCysLeuAspGluLysArg 
660665670 
GAGCTGCTGGAGAAGGTGAAGTACGCCAAGCGCCTGAGCGACGAGCGC2064 
GluLeuLeuGluLysValLysTyrAlaLysArgLeuSerAspGluArg 
675680685 
AACCTGCTGCAGGACCCCAACTTCACCAGCATCAACAAGCAGCCCGAC2112 
AsnLeuLeuGlnAspProAsnPheThrSerIleAsnLysGlnProAsp 
690695700 
TTCATCAGCACCAACGAGCAGAGCAACTTCACCAGCATCCACGAGCAG2160 
PheIleSerThrAsnGluGlnSerAsnPheThrSerIleHisGluGln 
705710715720 
AGCGAGCACGGCTGGTGGGGCAGCGAGAACATCACCATCCAGGAGGGC2208 
SerGluHisGlyTrpTrpGlySerGluAsnIleThrIleGlnGluGly 
725730735 
AACGACGTGTTCAAGGAGAACTACGTGACCCTGCCCGGCACCTTCAAC2256 
AsnAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAsn 
740745750 
GAGTGCTACCCCACCTACCTGTACCAGAAGATCGGCGAGAGCGAGCTG2304 
GluCysTyrProThrTyrLeuTyrGlnLysIleGlyGluSerGluLeu 
755760765 
AAGGCCTACACCCGCTACCAGCTGCGCGGCTACATCGAGGACAGCCAG2352 
LysAlaTyrThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGln 
770775780 
GACCTGGAGATCTACCTGATCCGCTACAACGCCAAGCACGAGACCCTG2400 
AspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrLeu 
785790795800 
GACGTGCCCGGCACCGAGAGCCTGTGGCCCCTGAGCGTGGAGAGCCCC2448 
AspValProGlyThrGluSerLeuTrpProLeuSerValGluSerPro 
805810815 
ATCGGCCGCTGCGGCGAGCCCAACCGCTGCGCCCCCCACTTCGAGTGG2496 
IleGlyArgCysGlyGluProAsnArgCysAlaProHisPheGluTrp 
820825830 
AACCCCGACCTGGACTGCAGCTGCCGCGACGGCGAGAAGTGCGCCCAC2544 
AsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHis 
835840845 
CACAGCCACCACTTCAGCCTGGACATCGACGTGGGCTGCACCGACCTG2592 
HisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeu 
850855860 
CACGAGAACCTGGGCGTGTGGGTGGTGTTCAAGATCAAGACCCAGGAG2640 
HisGluAsnLeuGlyValTrpValValPheLysIleLysThrGlnGlu 
865870875880 
GGCCACGCCCGCCTGGGCAACCTGGAGTTCATCGAGGAGAAGCCCCTG2688 
GlyHisAlaArgLeuGlyAsnLeuGluPheIleGluGluLysProLeu 
885890895 
CTGGGCGAGGCCCTGAGCCGCGTGAAGCGCGCCGAGAAGAAGTGGCGC2736 
LeuGlyGluAlaLeuSerArgValLysArgAlaGluLysLysTrpArg 
900905910 
GACAAGCGCGAGAAGCTGCAGCTGGAGACCAAGCGCGTGTACACCGAG2784 
AspLysArgGluLysLeuGlnLeuGluThrLysArgValTyrThrGlu 
915920925 
GCCAAGGAGGCCGTGGACGCCCTGTTCGTGGACAGCCAGTACGACCGC2832 
AlaLysGluAlaValAspAlaLeuPheValAspSerGlnTyrAspArg 
930935940 
CTGCAGGCCGACACCAACATCGGCATGATCCACGCCGCCGACAAGCTG2880 
LeuGlnAlaAspThrAsnIleGlyMetIleHisAlaAlaAspLysLeu 
945950955960 
GTGCACCGCATCCGCGAGGCCTACCTGAGCGAGCTGCCCGTGATCCCC2928 
ValHisArgIleArgGluAlaTyrLeuSerGluLeuProValIlePro 
965970975 
GGCGTGAACGCCGAGATCTTCGAGGAGCTGGAGGGCCACATCATCACC2976 
GlyValAsnAlaGluIlePheGluGluLeuGluGlyHisIleIleThr 
980985990 
GCCATCAGCCTGTACGACGCCCGCAACGTGGTGAAGAACGGCGACTTC3024 
AlaIleSerLeuTyrAspAlaArgAsnValValLysAsnGlyAspPhe 
99510001005 
AACAACGGCCTGACCTGCTGGAACGTGAAGGGCCACGTGGACGTGCAG3072 
AsnAsnGlyLeuThrCysTrpAsnValLysGlyHisValAspValGln 
101010151020 
CAGAGCCACCACCGCAGCGACCTGGTGATCCCCGAGTGGGAGGCCGAG3120 
GlnSerHisHisArgSerAspLeuValIleProGluTrpGluAlaGlu 
1025103010351040 
GTGAGCCAGGCCGTGCGCGTGTGCCCCGGCTGCGGCTACATCCTGCGC3168 
ValSerGlnAlaValArgValCysProGlyCysGlyTyrIleLeuArg 
104510501055 
GTGACCGCCTACAAGGAGGGCTACGGCGAGGGCTGCGTGACCATCCAC3216 
ValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHis 
106010651070 
GAGATCGAGAACAACACCGACGAGCTGAAGTTCAAGAACCGCGAGGAG3264 
GluIleGluAsnAsnThrAspGluLeuLysPheLysAsnArgGluGlu 
107510801085 
GAGGAGGTGTACCCCACCGACACCGGCACCTGCAACGACTACACCGCC3312 
GluGluValTyrProThrAspThrGlyThrCysAsnAspTyrThrAla 
109010951100 
CACCAGGGCACCGCCGGCTGCGCCGACGCCTGCAACAGCCGCAACGCC3360 
HisGlnGlyThrAlaGlyCysAlaAspAlaCysAsnSerArgAsnAla 
1105111011151120 
GGCTACGAGGACGCCTACGAGGTGGACACCACCGCCAGCGTGAACTAC3408 
GlyTyrGluAspAlaTyrGluValAspThrThrAlaSerValAsnTyr 
112511301135 
AAGCCCACCTACGAGGAGGAGACCTACACCGACGTGCGCCGCGACAAC3456 
LysProThrTyrGluGluGluThrTyrThrAspValArgArgAspAsn 
114011451150 
CACTGCGAGTACGACCGCGGCTACGTGAACTACCCCCCCGTGCCCGCC3504 
HisCysGluTyrAspArgGlyTyrValAsnTyrProProValProAla 
115511601165 
GGCTACGTGACCAAGGAGCTGGAGTACTTCCCCGAGACCGACACCGTG3552 
GlyTyrValThrLysGluLeuGluTyrPheProGluThrAspThrVal 
117011751180 
TGGATCGAGATCGGCGAGACCGAGGGCAAGTTCATCGTGGACAGCGTG3600 
TrpIleGluIleGlyGluThrGluGlyLysPheIleValAspSerVal 
1185119011951200 
GAGCTGCTGCTGATGGAGGAGTAG3624 
GluLeuLeuLeuMetGluGlu 
1205 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1207 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetAspLeuLeuProAspAlaArgIleGluAspSerLeuCysIleAla 
151015 
GluGlyAsnAsnIleAspProPheValSerAlaSerThrValGlnThr 
202530 
GlyIleAsnIleAlaGlyArgIleLeuGlyValLeuGlyValProPhe 
354045 
AlaGlyGlnLeuAlaSerPheTyrSerPheLeuValGlyGluLeuTrp 
505560 
ProArgGlyArgAspGlnTrpGluIlePheLeuGluHisValGluGln 
65707580 
LeuIleAsnGlnGlnIleThrGluAsnAlaArgAsnThrAlaLeuAla 
859095 
ArgLeuGlnGlyLeuGlyAspSerPheArgAlaTyrGlnGlnSerLeu 
100105110 
GluAspTrpLeuGluAsnArgAspAspAlaArgThrArgSerValLeu 
115120125 
TyrThrGlnTyrIleAlaLeuGluLeuAspPheLeuAsnAlaMetPro 
130135140 
LeuPheAlaIleArgAsnGlnGluValProLeuLeuMetValTyrAla 
145150155160 
GlnAlaAlaAsnLeuHisLeuLeuLeuLeuArgAspAlaSerLeuPhe 
165170175 
GlySerGluPheGlyLeuThrSerGlnGluIleGlnArgTyrTyrGlu 
180185190 
ArgGlnValGluArgThrArgAspTyrSerAspTyrCysValGluTrp 
195200205 
TyrAsnThrGlyLeuAsnSerLeuArgGlyThrAsnAlaAlaSerTrp 
210215220 
ValArgTyrAsnGlnPheArgArgAspLeuThrLeuGlyValLeuAsp 
225230235240 
LeuValAlaLeuPheProSerTyrAspThrArgThrTyrProIleAsn 
245250255 
ThrSerAlaGlnLeuThrArgGluValTyrThrAspAlaIleGlyAla 
260265270 
ThrGlyValAsnMetAlaSerMetAsnTrpTyrAsnAsnAsnAlaPro 
275280285 
SerPheSerAlaIleGluAlaAlaAlaIleArgSerProHisLeuLeu 
290295300 
AspPheLeuGluGlnLeuThrIlePheSerAlaSerSerArgTrpSer 
305310315320 
AsnThrArgHisMetThrTyrTrpArgGlyHisThrIleGlnSerArg 
325330335 
ProIleGlyGlyGlyLeuAsnThrSerThrHisGlyAlaThrAsnThr 
340345350 
SerIleAsnProValThrLeuArgPheAlaSerArgAspValTyrArg 
355360365 
ThrGluSerTyrAlaGlyValLeuLeuTrpGlyIleTyrLeuGluPro 
370375380 
IleHisGlyValProThrValArgPheAsnPheThrAsnProGlnAsn 
385390395400 
IleSerAspArgGlyThrAlaAsnTyrSerGlnProTyrGluSerPro 
405410415 
GlyLeuGlnLeuLysAspSerGluThrGluLeuProProGluThrThr 
420425430 
GluArgProAsnTyrGluSerTyrSerHisArgLeuSerHisIleGly 
435440445 
IleIleLeuGlnSerArgValAsnValProValTyrSerTrpThrHis 
450455460 
ArgSerAlaAspArgThrAsnThrIleGlyProAsnArgIleThrGln 
465470475480 
IleProMetValLysAlaSerGluLeuProGlnGlyThrThrValVal 
485490495 
ArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrAsnThr 
500505510 
GlyGlyPheGlyProIleArgValThrValAsnGlyProLeuThrGln 
515520525 
ArgTyrArgIleGlyPheArgTyrAlaSerThrValAspPheAspPhe 
530535540 
PheValSerArgGlyGlyThrThrValAsnAsnPheArgPheLeuArg 
545550555560 
ThrMetAsnSerGlyAspGluLeuLysTyrGlyAsnPheValArgArg 
565570575 
AlaPheThrThrProPheThrPheThrGlnIleGlnAspIleIleArg 
580585590 
ThrSerIleGlnGlyLeuSerGlyAsnGlyGluValTyrIleAspLys 
595600605 
IleGluIleIleProValThrAlaThrPheGluAlaGluTyrAspLeu 
610615620 
GluArgAlaGlnGluAlaValAsnAlaLeuPheThrAsnThrAsnPro 
625630635640 
ArgArgLeuLysThrAspValThrAspTyrHisIleAspGlnValSer 
645650655 
AsnLeuValAlaCysLeuSerAspGluPheCysLeuAspGluLysArg 
660665670 
GluLeuLeuGluLysValLysTyrAlaLysArgLeuSerAspGluArg 
675680685 
AsnLeuLeuGlnAspProAsnPheThrSerIleAsnLysGlnProAsp 
690695700 
PheIleSerThrAsnGluGlnSerAsnPheThrSerIleHisGluGln 
705710715720 
SerGluHisGlyTrpTrpGlySerGluAsnIleThrIleGlnGluGly 
725730735 
AsnAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAsn 
740745750 
GluCysTyrProThrTyrLeuTyrGlnLysIleGlyGluSerGluLeu 
755760765 
LysAlaTyrThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGln 
770775780 
AspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrLeu 
785790795800 
AspValProGlyThrGluSerLeuTrpProLeuSerValGluSerPro 
805810815 
IleGlyArgCysGlyGluProAsnArgCysAlaProHisPheGluTrp 
820825830 
AsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHis 
835840845 
HisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeu 
850855860 
HisGluAsnLeuGlyValTrpValValPheLysIleLysThrGlnGlu 
865870875880 
GlyHisAlaArgLeuGlyAsnLeuGluPheIleGluGluLysProLeu 
885890895 
LeuGlyGluAlaLeuSerArgValLysArgAlaGluLysLysTrpArg 
900905910 
AspLysArgGluLysLeuGlnLeuGluThrLysArgValTyrThrGlu 
915920925 
AlaLysGluAlaValAspAlaLeuPheValAspSerGlnTyrAspArg 
930935940 
LeuGlnAlaAspThrAsnIleGlyMetIleHisAlaAlaAspLysLeu 
945950955960 
ValHisArgIleArgGluAlaTyrLeuSerGluLeuProValIlePro 
965970975 
GlyValAsnAlaGluIlePheGluGluLeuGluGlyHisIleIleThr 
980985990 
AlaIleSerLeuTyrAspAlaArgAsnValValLysAsnGlyAspPhe 
99510001005 
AsnAsnGlyLeuThrCysTrpAsnValLysGlyHisValAspValGln 
101010151020 
GlnSerHisHisArgSerAspLeuValIleProGluTrpGluAlaGlu 
1025103010351040 
ValSerGlnAlaValArgValCysProGlyCysGlyTyrIleLeuArg 
104510501055 
ValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHis 
106010651070 
GluIleGluAsnAsnThrAspGluLeuLysPheLysAsnArgGluGlu 
107510801085 
GluGluValTyrProThrAspThrGlyThrCysAsnAspTyrThrAla 
109010951100 
HisGlnGlyThrAlaGlyCysAlaAspAlaCysAsnSerArgAsnAla 
1105111011151120 
GlyTyrGluAspAlaTyrGluValAspThrThrAlaSerValAsnTyr 
112511301135 
LysProThrTyrGluGluGluThrTyrThrAspValArgArgAspAsn 
114011451150 
HisCysGluTyrAspArgGlyTyrValAsnTyrProProValProAla 
115511601165 
GlyTyrValThrLysGluLeuGluTyrPheProGluThrAspThrVal 
117011751180 
TrpIleGluIleGlyGluThrGluGlyLysPheIleValAspSerVal 
1185119011951200 
GluLeuLeuLeuMetGluGlu 
1205 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3468 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3465 
(D) OTHER INFORMATION: /product="Full-length, hybrid, 
partially maize optimized cryIA(b)" 
/note= "Disclosed in Figure 7 as contained in pCIB4434." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTG48 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
121012151220 
AGCAACCCCGAGGTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGC96 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
122512301235 
TACACCCCCATCGACATCAGCCTGAGCCTGACCCAGTTCCTGCTGAGC144 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
1240124512501255 
GAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTGGTGGACATCATC192 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
126012651270 
TGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
127512801285 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCC288 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
129012951300 
ATCAGCCGCCTGGAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAG336 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
130513101315 
AGCTTCCGCGAGTGGGAGGCCGACCCCACCAACCCCGCCCTGCGCGAG384 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
1320132513301335 
GAGATGCGCATCCAGTTCAACGACATGAACAGCGCCCTGACCACCGCC432 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
134013451350 
ATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
135513601365 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGC528 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
137013751380 
GTGTTCGGCCAGCGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGC576 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
138513901395 
TACAACGACCTGACCCGCCTGATCGGCAACTACACCGACCACGCCGTG624 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
1400140514101415 
CGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGTCCCGACAGCCGC672 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
142014251430 
GACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
143514401445 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCC768 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
145014551460 
ATCCGCACCGTGAGCCAGCTGACCCGCGAGATTTACACCAACCCCGTG816 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
146514701475 
CTGGAGAACTTCGACGGCAGCTTCCGCGGCAGCGCCCAGGGCATCGAG864 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
1480148514901495 
GGCAGCATCCGCAGCCCCCACCTGATGGACATCCTGAACAGCATCACC912 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
150015051510 
ATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
151515201525 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCC1008 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
153015351540 
CTGTACGGCACCATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCA1056 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
154515501555 
CAGCTGGGCCAGGGAGTGTACCGCACCCTGAGCAGCACCCTGTACCGT1104 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
1560156515701575 
CGACCTTTCAACATCGGCATCAACAACCAGCAGCTGAGCGTGCTGGAC1152 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
158015851590 
GGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
159516001605 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAG1248 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
161016151620 
AACAACAACGTGCCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCAC1296 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
162516301635 
GTGAGCATGTTCCGCAGTGGCTTCAGCAACAGCAGCGTGAGCATCATC1344 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
1640164516501655 
CGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCCGAGTTCAACAAC1392 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
166016651670 
ATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
167516801685 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGC1488 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
169016951700 
GGCGACATCCTGCGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGC1536 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
170517101715 
GTGAACATCACCGCCCCCCTGAGCCAGCGCTACCGCGTCCGCATCCGC1584 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
1720172517301735 
TACGCCAGCACCACCAACCTGCAGTTCCACACCAGCATCGACGGCCGC1632 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
174017451750 
CCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
175517601765 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAAC1728 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
177017751780 
TTCAGCAACGGCAGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAAC1776 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
178517901795 
AGCGGCAACGAGGTGTACATCGACCGCATCGAGTTCGTGCCCGCCGAG1824 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
1800180518101815 
GTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCTCAGAAGGCCGTG1872 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
182018251830 
AACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
183518401845 
ACCGACTACCACATCGATCAAGTATCCAATTTAGTTGAGTGTTTATCT1968 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
185018551860 
GATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAA2016 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
186518701875 
CATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAAC2064 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
1880188518901895 
TTTAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACG2112 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
190019051910 
GATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTT2160 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
191519201925 
ACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAA2208 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
193019351940 
AAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTACCAATTAAGA2256 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
194519501955 
GGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTAC2304 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
1960196519701975 
AATGCCAAACACGAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGG2352 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
198019851990 
CCGCTTTCAGCCCCAAGTCCAATCGGAAAATGTGCCCATCATTCCCAT2400 
ProLeuSerAlaProSerProIleGlyLysCysAlaHisHisSerHis 
199520002005 
CATTTCTCCTTGGACATTGATGTTGGATGTACAGACTTAAATGAGGAC2448 
HisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAsp 
201020152020 
TTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGCCATGCA2496 
LeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAla 
202520302035 
AGACTAGGAAATCTAGAATTTCTCGAAGAGAAACCATTAGTAGGAGAA2544 
ArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGlu 
2040204520502055 
GCACTAGCTCGTGTGAAAAGAGCGGAGAAAAAATGGAGAGACAAACGT2592 
AlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArg 
206020652070 
GAAAAATTGGAATGGGAAACAAATATTGTTTATAAAGAGGCAAAAGAA2640 
GluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGlu 
207520802085 
TCTGTAGATGCTTTATTTGTAAACTCTCAATATGATAGATTACAAGCG2688 
SerValAspAlaLeuPheValAsnSerGlnTyrAspArgLeuGlnAla 
209020952100 
GATACCAACATCGCGATGATTCATGCGGCAGATAAACGCGTTCATAGC2736 
AspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSer 
210521102115 
ATTCGAGAAGCTTATCTGCCTGAGCTGTCTGTGATTCCGGGTGTCAAT2784 
IleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsn 
2120212521302135 
GCGGCTATTTTTGAAGAATTAGAAGGGCGTATTTTCACTGCATTCTCC2832 
AlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSer 
214021452150 
CTATATGATGCGAGAAATGTCATTAAAAATGGTGATTTTAATAATGGC2880 
LeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGly 
215521602165 
TTATCCTGCTGGAACGTGAAAGGGCATGTAGATGTAGAAGAACAAAAC2928 
LeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsn 
217021752180 
AACCACCGTTCGGTCCTTGTTGTTCCGGAATGGGAAGCAGAAGTGTCA2976 
AsnHisArgSerValLeuValValProGluTrpGluAlaGluValSer 
218521902195 
CAAGAAGTTCGTGTCTGTCCGGGTCGTGGCTATATCCTTCGTGTCACA3024 
GlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThr 
2200220522102215 
GCGTACAAGGAGGGATATGGAGAAGGTTGCGTAACCATTCATGAGATC3072 
AlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIle 
222022252230 
GAGAACAATACAGACGAACTGAAGTTTAGCAACTGTGTAGAAGAGGAA3120 
GluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGlu 
223522402245 
GTATATCCAAACAACACGGTAACGTGTAATGATTATACTGCGACTCAA3168 
ValTyrProAsnAsnThrValThrCysAsnAspTyrThrAlaThrGln 
225022552260 
GAAGAATATGAGGGTACGTACACTTCTCGTAATCGAGGATATGACGGA3216 
GluGluTyrGluGlyThrTyrThrSerArgAsnArgGlyTyrAspGly 
226522702275 
GCCTATGAAAGCAATTCTTCTGTACCAGCTGATTATGCATCAGCCTAT3264 
AlaTyrGluSerAsnSerSerValProAlaAspTyrAlaSerAlaTyr 
2280228522902295 
GAAGAAAAAGCATATACAGATGGACGAAGAGACAATCCTTGTGAATCT3312 
GluGluLysAlaTyrThrAspGlyArgArgAspAsnProCysGluSer 
230023052310 
AACAGAGGATATGGGGATTACACACCACTACCAGCTGGCTATGTGACA3360 
AsnArgGlyTyrGlyAspTyrThrProLeuProAlaGlyTyrValThr 
231523202325 
AAAGAATTAGAGTACTTCCCAGAAACCGATAAGGTATGGATTGAGATC3408 
LysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIle 
233023352340 
GGAGAAACGGAAGGAACATTCATCGTGGACAGCGTGGAATTACTTCTT3456 
GlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeu 
234523502355 
ATGGAGGAATAA3468 
MetGluGlu 
2360 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1155 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
151015 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
202530 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
354045 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
505560 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
65707580 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
859095 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
100105110 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
115120125 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
130135140 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
145150155160 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
165170175 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
180185190 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
195200205 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
210215220 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
225230235240 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
245250255 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
260265270 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
275280285 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
290295300 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
305310315320 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
325330335 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
340345350 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
355360365 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
370375380 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
385390395400 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
405410415 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
420425430 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
435440445 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
450455460 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
465470475480 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
485490495 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
500505510 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
515520525 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
530535540 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
545550555560 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
565570575 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
580585590 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
595600605 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
610615620 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
625630635640 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
645650655 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
660665670 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
675680685 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
690695700 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
705710715720 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
725730735 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
740745750 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
755760765 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
770775780 
ProLeuSerAlaProSerProIleGlyLysCysAlaHisHisSerHis 
785790795800 
HisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAsp 
805810815 
LeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAla 
820825830 
ArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGlu 
835840845 
AlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArg 
850855860 
GluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGlu 
865870875880 
SerValAspAlaLeuPheValAsnSerGlnTyrAspArgLeuGlnAla 
885890895 
AspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSer 
900905910 
IleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsn 
915920925 
AlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSer 
930935940 
LeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGly 
945950955960 
LeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsn 
965970975 
AsnHisArgSerValLeuValValProGluTrpGluAlaGluValSer 
980985990 
GlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThr 
99510001005 
AlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIle 
101010151020 
GluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGlu 
1025103010351040 
ValTyrProAsnAsnThrValThrCysAsnAspTyrThrAlaThrGln 
104510501055 
GluGluTyrGluGlyThrTyrThrSerArgAsnArgGlyTyrAspGly 
106010651070 
AlaTyrGluSerAsnSerSerValProAlaAspTyrAlaSerAlaTyr 
107510801085 
GluGluLysAlaTyrThrAspGlyArgArgAspAsnProCysGluSer 
109010951100 
AsnArgGlyTyrGlyAspTyrThrProLeuProAlaGlyTyrValThr 
1105111011151120 
LysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIle 
112511301135 
GlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeu 
114011451150 
MetGluGlu 
1155 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3546 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3543 
(D) OTHER INFORMATION: /product="Full-length, hybrid, 
maize optimized heat stable cryIA(b)" 
/note= "Disclosed in Figure 9 as contained in pCIB5511." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTG48 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
116011651170 
AGCAACCCCGAGGTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGC96 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
117511801185 
TACACCCCCATCGACATCAGCCTGAGCCTGACCCAGTTCCTGCTGAGC144 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
119011951200 
GAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTGGTGGACATCATC192 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
120512101215 
TGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
1220122512301235 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCC288 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
124012451250 
ATCAGCCGCCTGGAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAG336 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
125512601265 
AGCTTCCGCGAGTGGGAGGCCGACCCCACCAACCCCGCCCTGCGCGAG384 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
127012751280 
GAGATGCGCATCCAGTTCAACGACATGAACAGCGCCCTGACCACCGCC432 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
128512901295 
ATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
1300130513101315 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGC528 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
132013251330 
GTGTTCGGCCAGCGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGC576 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
133513401345 
TACAACGACCTGACCCGCCTGATCGGCAACTACACCGACCACGCCGTG624 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
135013551360 
CGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGTCCCGACAGCCGC672 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
136513701375 
GACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
1380138513901395 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCC768 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
140014051410 
ATCCGCACCGTGAGCCAGCTGACCCGCGAGATTTACACCAACCCCGTG816 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
141514201425 
CTGGAGAACTTCGACGGCAGCTTCCGCGGCAGCGCCCAGGGCATCGAG864 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
143014351440 
GGCAGCATCCGCAGCCCCCACCTGATGGACATCCTGAACAGCATCACC912 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
144514501455 
ATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
1460146514701475 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCC1008 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
148014851490 
CTGTACGGCACCATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCA1056 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
149515001505 
CAGCTGGGCCAGGGAGTGTACCGCACCCTGAGCAGCACCCTGTACCGT1104 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
151015151520 
CGACCTTTCAACATCGGCATCAACAACCAGCAGCTGAGCGTGCTGGAC1152 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
152515301535 
GGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
1540154515501555 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAG1248 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
156015651570 
AACAACAACGTGCCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCAC1296 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
157515801585 
GTGAGCATGTTCCGCAGTGGCTTCAGCAACAGCAGCGTGAGCATCATC1344 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
159015951600 
CGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCCGAGTTCAACAAC1392 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
160516101615 
ATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
1620162516301635 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGC1488 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
164016451650 
GGCGACATCCTGCGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGC1536 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
165516601665 
GTGAACATCACCGCCCCCCTGAGCCAGCGCTACCGCGTCCGCATCCGC1584 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
167016751680 
TACGCCAGCACCACCAACCTGCAGTTCCACACCAGCATCGACGGCCGC1632 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
168516901695 
CCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
1700170517101715 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAAC1728 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
172017251730 
TTCAGCAACGGCAGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAAC1776 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
173517401745 
AGCGGCAACGAGGTGTACATCGACCGCATCGAGTTCGTGCCCGCCGAG1824 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
175017551760 
GTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCTCAGAAGGCCGTG1872 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
176517701775 
AACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
1780178517901795 
ACCGACTACCACATCGATCAAGTATCCAATTTAGTTGAGTGTTTATCT1968 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
180018051810 
GATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAA2016 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
181518201825 
CATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAAC2064 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
183018351840 
TTTAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACG2112 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
184518501855 
GATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTT2160 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
1860186518701875 
ACGCTATTGGGTACCTTCGACGAGTGCTACCCCACCTACCTGTACCAG2208 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
188018851890 
AAGATCGACGAGAGCAAGCTGAAGGCCTACACCCGCTACCAGCTGCGC2256 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
189519001905 
GGCTACATCGAGGACAGCCAGGACCTGGAAATCTACCTGATCCGCTAC2304 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
191019151920 
AACGCCAAGCACGAGACCGTGAACGTGCCCGGCACCGGCAGCCTGTGG2352 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
192519301935 
CCCCTGAGCGCCCCCAGCCCCATCGGCAAGTGCGGGGAGCCGAATCGA2400 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
1940194519501955 
TGCGCTCCGCACCTGGAGTGGAACCCGGACCTAGACTGCAGCTGCAGG2448 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
196019651970 
GACGGGGAGAAGTGCGCCCACCACAGCCACCACTTCAGCCTGGACATC2496 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
197519801985 
GACGTGGGCTGCACCGACCTGAACGAGGACCTGGGCGTGTGGGTGATC2544 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
199019952000 
TTCAAGATCAAGACCCAGGACGGCCACGCCCGCCTGGGCAATCTAGAA2592 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
200520102015 
TTTCTCGAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAA2640 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
2020202520302035 
AGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAA2688 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
204020452050 
ACAAATATTGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTT2736 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
205520602065 
GTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATCGCGATG2784 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
207020752080 
ATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTG2832 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
208520902095 
CCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAA2880 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
2100210521102115 
TTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAAT2928 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
212021252130 
GTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTG2976 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
213521402145 
AAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCGGTCCTT3024 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
215021552160 
GTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGT3072 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
216521702175 
CCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATAT3120 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
2180218521902195 
GGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAA3168 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
220022052210 
CTGAAGTTTAGCAACTGTGTAGAAGAGGAAGTATATCCAAACAACACG3216 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
221522202225 
GTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAGGGTACG3264 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
223022352240 
TACACTTCTCGTAATCGAGGATATGACGGAGCCTATGAAAGCAATTCT3312 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
224522502255 
TCTGTACCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACA3360 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
2260226522702275 
GATGGACGAAGAGACAATCCTTGTGAATCTAACAGAGGATATGGGGAT3408 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
228022852290 
TACACACCACTACCAGCTGGCTATGTGACAAAAGAATTAGAGTACTTC3456 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
229523002305 
CCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAAGGAACA3504 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
231023152320 
TTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAA3546 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
232523302335 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
151015 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
202530 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
354045 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
505560 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
65707580 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
859095 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
100105110 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
115120125 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
130135140 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
145150155160 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
165170175 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
180185190 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
195200205 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
210215220 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
225230235240 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
245250255 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
260265270 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
275280285 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
290295300 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
305310315320 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
325330335 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
340345350 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
355360365 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
370375380 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
385390395400 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
405410415 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
420425430 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
435440445 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
450455460 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
465470475480 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
485490495 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
500505510 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
515520525 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
530535540 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
545550555560 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
565570575 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
580585590 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
595600605 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
610615620 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
625630635640 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
645650655 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
660665670 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
675680685 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
690695700 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
705710715720 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
725730735 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
740745750 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
755760765 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
770775780 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
785790795800 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
805810815 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
820825830 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
835840845 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
850855860 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
865870875880 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
885890895 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
900905910 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
915920925 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
930935940 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
945950955960 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
965970975 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
980985990 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
99510001005 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
101010151020 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
1025103010351040 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
104510501055 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
106010651070 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
107510801085 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
109010951100 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
1105111011151120 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
112511301135 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
114011451150 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
115511601165 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
117011751180 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3546 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3543 
(D) OTHER INFORMATION: /product="Full-length, hybrid, 
maize optimized heat stable cryIA(b)" 
/note= "Disclosed in Figure 11 as contained in pCIB5512" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTG48 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
118511901195 
AGCAACCCCGAGGTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGC96 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
120012051210 
TACACCCCCATCGACATCAGCCTGAGCCTGACCCAGTTCCTGCTGAGC144 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
121512201225 
GAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTGGTGGACATCATC192 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
1230123512401245 
TGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
125012551260 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCC288 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
126512701275 
ATCAGCCGCCTGGAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAG336 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
128012851290 
AGCTTCCGCGAGTGGGAGGCCGACCCCACCAACCCCGCCCTGCGCGAG384 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
129513001305 
GAGATGCGCATCCAGTTCAACGACATGAACAGCGCCCTGACCACCGCC432 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
1310131513201325 
ATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
133013351340 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGC528 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
134513501355 
GTGTTCGGCCAGCGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGC576 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
136013651370 
TACAACGACCTGACCCGCCTGATCGGCAACTACACCGACCACGCCGTG624 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
137513801385 
CGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGTCCCGACAGCCGC672 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
1390139514001405 
GACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
141014151420 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCC768 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
142514301435 
ATCCGCACCGTGAGCCAGCTGACCCGCGAGATTTACACCAACCCCGTG816 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
144014451450 
CTGGAGAACTTCGACGGCAGCTTCCGCGGCAGCGCCCAGGGCATCGAG864 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
145514601465 
GGCAGCATCCGCAGCCCCCACCTGATGGACATCCTGAACAGCATCACC912 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
1470147514801485 
ATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
149014951500 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCC1008 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
150515101515 
CTGTACGGCACCATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCA1056 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
152015251530 
CAGCTGGGCCAGGGAGTGTACCGCACCCTGAGCAGCACCCTGTACCGT1104 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
153515401545 
CGACCTTTCAACATCGGCATCAACAACCAGCAGCTGAGCGTGCTGGAC1152 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
1550155515601565 
GGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
157015751580 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAG1248 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
158515901595 
AACAACAACGTGCCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCAC1296 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
160016051610 
GTGAGCATGTTCCGCAGTGGCTTCAGCAACAGCAGCGTGAGCATCATC1344 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
161516201625 
CGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCCGAGTTCAACAAC1392 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
1630163516401645 
ATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
165016551660 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGC1488 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
166516701675 
GGCGACATCCTGCGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGC1536 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
168016851690 
GTGAACATCACCGCCCCCCTGAGCCAGCGCTACCGCGTCCGCATCCGC1584 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
169517001705 
TACGCCAGCACCACCAACCTGCAGTTCCACACCAGCATCGACGGCCGC1632 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
1710171517201725 
CCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
173017351740 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAAC1728 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
174517501755 
TTCAGCAACGGCAGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAAC1776 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
176017651770 
AGCGGCAACGAGGTGTACATCGACCGCATCGAGTTCGTGCCCGCCGAG1824 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
177517801785 
GTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCTCAGAAGGCCGTG1872 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
1790179518001805 
AACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
181018151820 
ACCGACTACCACATCGATCAGGTGAGCAACCTGGTGGAGTGCTTAAGC1968 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
182518301835 
GACGAGTTCTGCCTGGACGAGAAGAAGGAGCTGAGCGAGAAGGTGAAG2016 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
184018451850 
CACGCCAAGCGCCTGAGCGACGAGCGCAACCTGCTGCAGGACCCCAAC2064 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
185518601865 
TTCCGCGGCATCAACCGCCAGCTGGACCGCGGCTGGCGAGGCAGCACC2112 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
1870187518801885 
GATATCACCATCCAGGGCGGCGACGACGTGTTCAAGGAGAACTACGTG2160 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
189018951900 
ACCCTGCTGGGCACCTTCGACGAGTGCTACCCCACCTACCTGTACCAG2208 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
190519101915 
AAGATCGACGAGAGCAAGCTGAAGGCCTACACCCGCTACCAGCTGCGC2256 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
192019251930 
GGCTACATCGAGGACAGCCAGGACCTGGAAATCTACCTGATCCGCTAC2304 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
193519401945 
AACGCCAAGCACGAGACCGTGAACGTGCCCGGCACCGGCAGCCTGTGG2352 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
1950195519601965 
CCCCTGAGCGCCCCCAGCCCCATCGGCAAGTGCGGGGAGCCGAATCGA2400 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
197019751980 
TGCGCTCCGCACCTGGAGTGGAACCCGGACCTAGACTGCAGCTGCAGG2448 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
198519901995 
GACGGGGAGAAGTGCGCCCACCACAGCCACCACTTCAGCCTGGACATC2496 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
200020052010 
GACGTGGGCTGCACCGACCTGAACGAGGACCTGGGCGTGTGGGTGATC2544 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
201520202025 
TTCAAGATCAAGACCCAGGACGGCCACGCCCGCCTGGGCAATCTAGAA2592 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
2030203520402045 
TTTCTCGAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAA2640 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
205020552060 
AGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAA2688 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
206520702075 
ACAAATATTGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTT2736 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
208020852090 
GTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATCGCGATG2784 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
209521002105 
ATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTG2832 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
2110211521202125 
CCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAA2880 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
213021352140 
TTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAAT2928 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
214521502155 
GTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTG2976 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
216021652170 
AAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCGGTCCTT3024 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
217521802185 
GTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGT3072 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
2190219522002205 
CCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATAT3120 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
221022152220 
GGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAA3168 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
222522302235 
CTGAAGTTTAGCAACTGTGTAGAAGAGGAAGTATATCCAAACAACACG3216 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
224022452250 
GTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAGGGTACG3264 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
225522602265 
TACACTTCTCGTAATCGAGGATATGACGGAGCCTATGAAAGCAATTCT3312 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
2270227522802285 
TCTGTACCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACA3360 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
229022952300 
GATGGACGAAGAGACAATCCTTGTGAATCTAACAGAGGATATGGGGAT3408 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
230523102315 
TACACACCACTACCAGCTGGCTATGTGACAAAAGAATTAGAGTACTTC3456 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
232023252330 
CCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAAGGAACA3504 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
233523402345 
TTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAA3546 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
235023552360 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
151015 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
202530 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
354045 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
505560 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
65707580 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
859095 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
100105110 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
115120125 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
130135140 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
145150155160 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
165170175 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
180185190 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
195200205 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
210215220 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
225230235240 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
245250255 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
260265270 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
275280285 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
290295300 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
305310315320 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
325330335 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
340345350 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
355360365 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
370375380 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
385390395400 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
405410415 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
420425430 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
435440445 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
450455460 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
465470475480 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
485490495 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
500505510 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
515520525 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
530535540 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
545550555560 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
565570575 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
580585590 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
595600605 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
610615620 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
625630635640 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
645650655 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
660665670 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
675680685 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
690695700 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
705710715720 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
725730735 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
740745750 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
755760765 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
770775780 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
785790795800 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
805810815 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
820825830 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
835840845 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
850855860 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
865870875880 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
885890895 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
900905910 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
915920925 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
930935940 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
945950955960 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
965970975 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
980985990 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
99510001005 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
101010151020 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
1025103010351040 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
104510501055 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
106010651070 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
107510801085 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
109010951100 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
1105111011151120 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
112511301135 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
114011451150 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
115511601165 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
117011751180 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3546 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3543 
(D) OTHER INFORMATION: /product="Full-length, hybrid, 
maize optimized heat stable cryIA(b)" 
/note= "Disclosed in Figure 13 as contained in pCIB5513." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTG48 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
118511901195 
AGCAACCCCGAGGTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGC96 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
120012051210 
TACACCCCCATCGACATCAGCCTGAGCCTGACCCAGTTCCTGCTGAGC144 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
121512201225 
GAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTGGTGGACATCATC192 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
1230123512401245 
TGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
125012551260 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCC288 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
126512701275 
ATCAGCCGCCTGGAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAG336 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
128012851290 
AGCTTCCGCGAGTGGGAGGCCGACCCCACCAACCCCGCCCTGCGCGAG384 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
129513001305 
GAGATGCGCATCCAGTTCAACGACATGAACAGCGCCCTGACCACCGCC432 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
1310131513201325 
ATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
133013351340 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGC528 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
134513501355 
GTGTTCGGCCAGCGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGC576 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
136013651370 
TACAACGACCTGACCCGCCTGATCGGCAACTACACCGACCACGCCGTG624 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
137513801385 
CGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGTCCCGACAGCCGC672 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
1390139514001405 
GACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
141014151420 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCC768 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
142514301435 
ATCCGCACCGTGAGCCAGCTGACCCGCGAGATTTACACCAACCCCGTG816 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
144014451450 
CTGGAGAACTTCGACGGCAGCTTCCGCGGCAGCGCCCAGGGCATCGAG864 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
145514601465 
GGCAGCATCCGCAGCCCCCACCTGATGGACATCCTGAACAGCATCACC912 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
1470147514801485 
ATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
149014951500 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCC1008 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
150515101515 
CTGTACGGCACCATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCA1056 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
152015251530 
CAGCTGGGCCAGGGAGTGTACCGCACCCTGAGCAGCACCCTGTACCGT1104 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
153515401545 
CGACCTTTCAACATCGGCATCAACAACCAGCAGCTGAGCGTGCTGGAC1152 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
1550155515601565 
GGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
157015751580 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAG1248 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
158515901595 
AACAACAACGTGCCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCAC1296 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
160016051610 
GTGAGCATGTTCCGCAGTGGCTTCAGCAACAGCAGCGTGAGCATCATC1344 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
161516201625 
CGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCCGAGTTCAACAAC1392 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
1630163516401645 
ATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
165016551660 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGC1488 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
166516701675 
GGCGACATCCTGCGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGC1536 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
168016851690 
GTGAACATCACCGCCCCCCTGAGCCAGCGCTACCGCGTCCGCATCCGC1584 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
169517001705 
TACGCCAGCACCACCAACCTGCAGTTCCACACCAGCATCGACGGCCGC1632 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
1710171517201725 
CCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
173017351740 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAAC1728 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
174517501755 
TTCAGCAACGGCAGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAAC1776 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
176017651770 
AGCGGCAACGAGGTGTACATCGACCGCATCGAGTTCGTGCCCGCCGAG1824 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
177517801785 
GTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCTCAGAAGGCCGTG1872 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
1790179518001805 
AACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
181018151820 
ACCGACTACCACATCGACCAGGTGAGCAACCTGGTGGAGTGCTTAAGC1968 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
182518301835 
GACGAGTTCTGCCTGGACGAGAAGAAGGAGCTGAGCGAGAAGGTGAAG2016 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
184018451850 
CACGCCAAGCGCCTGAGCGACGAGCGCAACCTGCTGCAGGACCCCAAC2064 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
185518601865 
TTCCGCGGCATCAACCGCCAGCTGGACCGCGGCTGGCGAGGCAGCACC2112 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
1870187518801885 
GATATCACCATCCAGGGCGGCGACGACGTGTTCAAGGAGAACTACGTG2160 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
189018951900 
ACCCTGCAGGGCACCTTCGACGAGTGCTACCCCACCTACCTGTACCAG2208 
ThrLeuGlnGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
190519101915 
CCGATCGACGAGAGCAAGCTGAAGGCCTACACCCGCTACCAGCTGCGC2256 
ProIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
192019251930 
GGCTACATCGAGGACAGCCAGGACCTGGAAATCTACCTGATCCGCTAC2304 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
193519401945 
AACGCCAAGCACGAGACCGTGAACGTGCCCGGCACCGGCAGCCTGTGG2352 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
1950195519601965 
CCCCTGAGCGCCCCCAGCCCCATCGGCAAGTGCGGGGAGCCGAATCGA2400 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
197019751980 
TGCGCTCCGCACCTGGAGTGGAACCCGGACCTAGACTGCAGCTGCAGG2448 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
198519901995 
GACGGGGAGAAGTGCGCCCACCACAGCCACCACTTCAGCCTGGACATC2496 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
200020052010 
GACGTGGGCTGCACCGACCTGAACGAGGACCTGGGCGTGTGGGTGATC2544 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
201520202025 
TTCAAGATCAAGACCCAGGACGGCCACGCCCGCCTGGGCAATCTAGAG2592 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
2030203520402045 
TTCCTGGAGGAGAAGCCCCTGGTGGGCGAGGCCCTGGCCCGCGTGAAG2640 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
205020552060 
CGCGCCGAGAAGAAGTGGCGCGACAAGCGCGAGAAGCTGGAGTGGGAG2688 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
206520702075 
ACCAACATCGTGTACAAGGAGGCCAAGGAGAGCGTGGACGCCCTGTTC2736 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
208020852090 
GTGAACAGCCAGTACGACCGCCTGCAGGCCGACACCAACATCGCCATG2784 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
209521002105 
ATCCACGCCGCCGACAAGCGCGTGCACAGCATTCGCGAGGCCTACCTG2832 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
2110211521202125 
CCCGAGCTGAGCGTGATCCCCGGCGTGAACGCCGCCATCTTCGAGGAA2880 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
213021352140 
CTCGAGGGCCGCATCTTCACCGCCTTCAGCCTGTACGACGCCCGCAAC2928 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
214521502155 
GTGATCAAGAACGGCGACTTCAACAACGGCCTGAGCTGCTGGAACGTG2976 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
216021652170 
AAGGGCCACGTGGACGTGGAGGAGCAGAACAACCACCGCAGCGTGCTG3024 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
217521802185 
GTGGTGCCCGAGTGGGAGGCCGAGGTGAGCCAGGAGGTGCGCGTGTGC3072 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
2190219522002205 
CCCGGCCGCGGCTACATCCTGCGCGTGACCGCCTACAAGGAGGGCTAC3120 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
221022152220 
GGCGAGGGCTGCGTGACCATCCACGAGATCGAGAACAACACCGACGAG3168 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
222522302235 
CTCAAGTTCAGCAACTGCGTGGAGGAGGAGGTTTACCCCAACAACACC3216 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
224022452250 
GTGACCTGCAACGACTACACCGCGACCCAGGAGGAGTACGAAGGCACC3264 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
225522602265 
TACACCTCTCGCAACAGGGGTTACGACGGCGCCTACGAGTCCAACAGC3312 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
2270227522802285 
TCCGTGCCAGCCGACTACGCCAGCGCCTACGAGGAGAAAGCCTACACC3360 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
229022952300 
GACGGTAGACGCGACAACCCATGTGAGAGCAACAGAGGCTACGGCGAC3408 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
230523102315 
TACACCCCCCTGCCCGCTGGATACGTGACCAAGGAGCTGGAGTACTTC3456 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
232023252330 
CCCGAGACCGACAAGGTGTGGATCGAGATTGGCGAGACCGAGGGCACC3504 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
233523402345 
TTCATCGTGGACAGCGTGGAGCTGCTGCTGATGGAGGAGTAG3546 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
235023552360 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
151015 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
202530 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
354045 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
505560 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
65707580 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
859095 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
100105110 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
115120125 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
130135140 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
145150155160 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
165170175 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
180185190 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
195200205 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
210215220 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
225230235240 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
245250255 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
260265270 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
275280285 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
290295300 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
305310315320 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
325330335 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
340345350 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
355360365 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
370375380 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
385390395400 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
405410415 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
420425430 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
435440445 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
450455460 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
465470475480 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
485490495 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
500505510 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
515520525 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
530535540 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
545550555560 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
565570575 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
580585590 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
595600605 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
610615620 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
625630635640 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
645650655 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
660665670 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
675680685 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
690695700 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
705710715720 
ThrLeuGlnGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
725730735 
ProIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
740745750 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
755760765 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
770775780 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
785790795800 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
805810815 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
820825830 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
835840845 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
850855860 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
865870875880 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
885890895 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
900905910 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
915920925 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
930935940 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
945950955960 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
965970975 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
980985990 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
99510001005 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
101010151020 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
1025103010351040 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
104510501055 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
106010651070 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
107510801085 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
109010951100 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
1105111011151120 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
112511301135 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
114011451150 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
115511601165 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
117011751180 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3547 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3543 
(D) OTHER INFORMATION: /product="Full-length, hybrid, 
maize optimized heat stable cryIA(b)" 
/note= "Disclosed in Figure 15 as contained in pCIB5514." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTG48 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
118511901195 
AGCAACCCCGAGGTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGC96 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
120012051210 
TACACCCCCATCGACATCAGCCTGAGCCTGACCCAGTTCCTGCTGAGC144 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
121512201225 
GAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTGGTGGACATCATC192 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
1230123512401245 
TGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
125012551260 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCC288 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
126512701275 
ATCAGCCGCCTGGAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAG336 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
128012851290 
AGCTTCCGCGAGTGGGAGGCCGACCCCACCAACCCCGCCCTGCGCGAG384 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
129513001305 
GAGATGCGCATCCAGTTCAACGACATGAACAGCGCCCTGACCACCGCC432 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
1310131513201325 
ATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
133013351340 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGC528 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
134513501355 
GTGTTCGGCCAGCGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGC576 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
136013651370 
TACAACGACCTGACCCGCCTGATCGGCAACTACACCGACCACGCCGTG624 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
137513801385 
CGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGTCCCGACAGCCGC672 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
1390139514001405 
GACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
141014151420 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCC768 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
142514301435 
ATCCGCACCGTGAGCCAGCTGACCCGCGAGATTTACACCAACCCCGTG816 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
144014451450 
CTGGAGAACTTCGACGGCAGCTTCCGCGGCAGCGCCCAGGGCATCGAG864 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
145514601465 
GGCAGCATCCGCAGCCCCCACCTGATGGACATCCTGAACAGCATCACC912 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
1470147514801485 
ATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
149014951500 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCC1008 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
150515101515 
CTGTACGGCACCATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCA1056 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
152015251530 
CAGCTGGGCCAGGGAGTGTACCGCACCCTGAGCAGCACCCTGTACCGT1104 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
153515401545 
CGACCTTTCAACATCGGCATCAACAACCAGCAGCTGAGCGTGCTGGAC1152 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
1550155515601565 
GGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
157015751580 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAG1248 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
158515901595 
AACAACAACGTGCCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCAC1296 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
160016051610 
GTGAGCATGTTCCGCAGTGGCTTCAGCAACAGCAGCGTGAGCATCATC1344 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
161516201625 
CGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCCGAGTTCAACAAC1392 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
1630163516401645 
ATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
165016551660 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGC1488 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
166516701675 
GGCGACATCCTGCGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGC1536 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
168016851690 
GTGAACATCACCGCCCCCCTGAGCCAGCGCTACCGCGTCCGCATCCGC1584 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
169517001705 
TACGCCAGCACCACCAACCTGCAGTTCCACACCAGCATCGACGGCCGC1632 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
1710171517201725 
CCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
173017351740 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAAC1728 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
174517501755 
TTCAGCAACGGCAGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAAC1776 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
176017651770 
AGCGGCAACGAGGTGTACATCGACCGCATCGAGTTCGTGCCCGCCGAG1824 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
177517801785 
GTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCTCAGAAGGCCGTG1872 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
1790179518001805 
AACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
181018151820 
ACCGACTACCACATCGATCAAGTATCCAATTTAGTTGAGTGTTTATCT1968 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
182518301835 
GATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAA2016 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
184018451850 
CATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAAC2064 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
185518601865 
TTTAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACG2112 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
1870187518801885 
GATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTT2160 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
189018951900 
ACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAA2208 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
190519101915 
AAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTACCAATTAAGA2256 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
192019251930 
GGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTAC2304 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
193519401945 
AATGCCAAACACGAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGG2352 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
1950195519601965 
CCGCTTTCAGCCCCAAGTCCAATCGGCAAGTGCGGGGAGCCGAATCGA2400 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
197019751980 
TGCGCTCCGCACCTGGAGTGGAACCCGGACCTAGACTGCAGCTGCAGG2448 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
198519901995 
GACGGGGAGAAGTGCGCCCACCACAGCCACCACTTCAGCCTGGACATC2496 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
200020052010 
GACGTGGGCTGCACCGACCTGAACGAGGACCTGGGCGTGTGGGTGATC2544 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
201520202025 
TTCAAGATCAAGACCCAGGACGGCCACGCCCGCCTGGGCAATCTAGAA2592 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
2030203520402045 
TTTCTCGAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAA2640 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
205020552060 
AGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAA2688 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
206520702075 
ACAAATATTGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTT2736 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
208020852090 
GTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATCGCGATG2784 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
209521002105 
ATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTG2832 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
2110211521202125 
CCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAA2880 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
213021352140 
TTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAAT2928 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
214521502155 
GTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTG2976 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
216021652170 
AAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCGGTCCTT3024 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
217521802185 
GTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGT3072 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
2190219522002205 
CCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATAT3120 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
221022152220 
GGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAA3168 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
222522302235 
CTGAAGTTTAGCAACTGTGTAGAAGAGGAAGTATATCCAAACAACACG3216 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
224022452250 
GTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAGGGTACG3264 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
225522602265 
TACACTTCTCGTAATCGAGGATATGACGGAGCCTATGAAAGCAATTCT3312 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
2270227522802285 
TCTGTACCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACA3360 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
229022952300 
GATGGACGAAGAGACAATCCTTGTGAATCTAACAGAGGATATGGGGAT3408 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
230523102315 
TACACACCACTACCAGCTGGCTATGTGACAAAAGAATTAGAGTACTTC3456 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
232023252330 
CCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAAGGAACA3504 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
233523402345 
TTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAAG3547 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
235023552360 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
151015 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
202530 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
354045 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
505560 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
65707580 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
859095 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
100105110 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
115120125 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
130135140 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
145150155160 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
165170175 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
180185190 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
195200205 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
210215220 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
225230235240 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
245250255 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
260265270 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
275280285 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
290295300 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
305310315320 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
325330335 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
340345350 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
355360365 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
370375380 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
385390395400 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
405410415 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
420425430 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
435440445 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
450455460 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
465470475480 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
485490495 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
500505510 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
515520525 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
530535540 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
545550555560 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
565570575 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
580585590 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
595600605 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
610615620 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
625630635640 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
645650655 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
660665670 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
675680685 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
690695700 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
705710715720 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
725730735 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
740745750 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
755760765 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
770775780 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
785790795800 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
805810815 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
820825830 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
835840845 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
850855860 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
865870875880 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
885890895 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
900905910 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
915920925 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
930935940 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
945950955960 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
965970975 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
980985990 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
99510001005 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
101010151020 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
1025103010351040 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
104510501055 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
106010651070 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
107510801085 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
109010951100 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
1105111011151120 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
112511301135 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
114011451150 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
115511601165 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
117011751180 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4817 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(1839..2141, 2239..2547, 2641..2718, 2794 
..2871, 3001..3135, 3236..3370) 
(D) OTHER INFORMATION: /product="maize TrpA" 
/note= "Maize TrpA sequence as disclosed in Figure 24." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GAATTCGGATCCATTAAAGAAGTCTTTGAACAGATTCTAGAGATCTAGTTTAATGAGCTC60 
CCAAAAGTCTTGAAAAAATTCAGCGGGGAGGCCATTAGGGCAGGGGTACTGTTATGTTTT120 
AAAGAGAACACCACTTTCTTGATCTCTTCTAAAGAGAAATGTTTTGTAAGAAGGATCCTG180 
TCCTCCTCATCCAACCTTTTCATCGGCAAATTTTTCATAGAGATATTAGAGGCAAGAGAG240 
GGGCCAAAAAGATCCATGTAAATGGAAGTGGCCACCTGGTTGATACCTCCCTCATCTTCA300 
ACAGAAAATCCATTATGAAAAAGTGAATGGATTTTAAACTCTTCTTTTTCTTCCCTTTTG360 
CAATGAGCTGAAAATATCTGGTATTATTCTCATCACCCTCATTAATGAATCTGTCCCTAG420 
CAATTTGCTTTCTCTTGATCCCTTCTGCAGCCACCATGTTTCTTAAATTCCACTCCATAT480 
CAAGCTTTTCCAATCTATCAGAATCTGAGATGGCTGCAATCTCTCTCATTTTCTCAAGGA540 
TATCGATGTTATCCATAAGGTATTTCTTGAACTTCTTATATTTCCCTTCGACATTTATAT600 
TCCATCCTTTCAACATTTTTTTGTTCAATCTTTTTTGTTTTTTTCCTTTCCAAACATCGA660 
TACATTTCCTGCTCCTCACAGGTAAGGACGAGCTTTCAAAAAACCTTCTGCTTTAAAGTC720 
AGGTCTGAGCCTCCAGCAAAGCTCACATATCTAAAGTCCCTCTTCTTAGTTGGGACAGAG780 
TCAGTGCTAAGACACATGGGAACATGACCAGAAAAAAAAAATCATATTTAGCCCAGAGAC840 
AACAATATTCTTGTACTGCAAGTCTCGTTATGGGCTAGCAAAGGAATCTACCCAACTTCT900 
CAAATGTGTTGGGATGTCAAGTATATAGACTATTCATCAGTTCCAACTCTATCAAACTGT960 
GCAGCTCAATTATAGAGTTGAATAAAGTGCTCCATCTATTTGTTCTTATCCTCATATTTG1020 
GTTAAGATATTAAAATCACCTCCCACCAACATTTAAAGTGCACCATTTAAAGTGGCTCGC1080 
GAGCACCAAACCGCTGAAAACCGGAAATGTTTAGCACGTTGGCAGCGGGACCCTTTTCTA1140 
TCTCATCGTGTTCTTCGTTGTCCACCACGGCCCACGGGCCAACGCTCCTCCATCCTGTAG1200 
TGTAGAGTATATTCCATTTGCGACCGAGCCGAGCATCGATCCAGCCACACTGGCCACTGC1260 
CAGCCAGCCATGTGGCACTCCTACGTATACTACGTGAGGTGAGATTCACTCACATGGGAT1320 
GGGACCGAGATATTTTACTGCTGTGGTTGTGTGAGAGATAATAAAGCATTTATGACGATT1380 
GCTGAACAGCACACACCATGCGTCCAGATAGAGAAAGCTTTCTCTCTTTATTCGCATGCA1440 
TGTTTCATTATCTTTTATCATATATATATAACACATATTAAATGATTCTTCGTTCCAATT1500 
TATAATTCATTTGACTTTTTTATCCACCGATGCTCGTTTTATTAAAAAAAATATTATAAT1560 
TATTGTTACTTTTTGTTGTAATATTGTTTAGCATATAATAAACTTTGATACTAGTATGTT1620 
TCCGAGCAAAAAAAAATATTAATATTTAGATTACGAGCCCATTAATTAATTATATTCGAG1680 
ACAAGCGAAGCAAAGCAAAGCAAGCTAATGTTGCCCCTGCTGTGCATGCAGAGGCCCGCT1740 
CTTGCTATAAACGAGGCAGCTAGACGCGACTCGACTCATCAGCCTCATCAACCTCGACGA1800 
AGGAGGAACGAACGGACAGGTTGTTGCACAGAAGCGACATGGCTTTCGCGCCC1853 
MetAlaPheAlaPro 
15 
AAAACGTCCTCCTCCTCCTCGCTGTCCTCGGCGTTGCAGGCAGCTCAG1901 
LysThrSerSerSerSerSerLeuSerSerAlaLeuGlnAlaAlaGln 
101520 
TCGCCGCCGCTGCTCCTGAGGCGGATGTCGTCGACCGCAACACCGAGA1949 
SerProProLeuLeuLeuArgArgMetSerSerThrAlaThrProArg 
253035 
CGGAGGTACGACGCGGCCGTCGTCGTCACTACCACCACCACTGCTAGA1997 
ArgArgTyrAspAlaAlaValValValThrThrThrThrThrAlaArg 
404550 
GCTGCGGCGGCTGCTGTCACGGTTCCCGCCGCCCCGCCGCAGGCGGGC2045 
AlaAlaAlaAlaAlaValThrValProAlaAlaProProGlnAlaGly 
556065 
CGCCGCCGCCGGTGCCACCAAAGCAAGCGGCGGCACCCGCAGAGGAGG2093 
ArgArgArgArgCysHisGlnSerLysArgArgHisProGlnArgArg 
70758085 
AGCCGTCCGGTGTCGGACACCATGGCGGCGCTCATGGCCAAGGGCAAG2141 
SerArgProValSerAspThrMetAlaAlaLeuMetAlaLysGlyLys 
9095100 
GTTCGTATAGTACGCGCGCGTGTCGTCGTCGTTATTTTGCGCATAGGCGCGGACATACAC2201 
GTGCTTTAGCTAGCTAACAGCTAGATCATCGGTGCAGACGGCGTTCATCCCGTAC2256 
ThrAlaPheIleProTyr 
105 
ATCACCGCCGGCGACCCGGACCTAGCGACGACGGCCGAGGCGCTGCGT2304 
IleThrAlaGlyAspProAspLeuAlaThrThrAlaGluAlaLeuArg 
110115120 
CTGCTGGACGGCTGTGGCGCCGACGTCATCGAGCTGGGGGTACCCTGC2352 
LeuLeuAspGlyCysGlyAlaAspValIleGluLeuGlyValProCys 
125130135 
TCGGACCCCTACATCGACGGGCCCATCATCCAGGCGTCGGTGGCGCGG2400 
SerAspProTyrIleAspGlyProIleIleGlnAlaSerValAlaArg 
140145150155 
GCTCTGGCCAGCGGCACCACCATGGACGCCGTGCTGGAGATGCTGAGG2448 
AlaLeuAlaSerGlyThrThrMetAspAlaValLeuGluMetLeuArg 
160165170 
GAGGTGACGCCGGAGCTGTCGTGCCCCGTGGTGCTCCTCTCCTACTAC2496 
GluValThrProGluLeuSerCysProValValLeuLeuSerTyrTyr 
175180185 
AAGCCCATCATGTCTCGCAGCTTGGCCGAGATGAAAGAGGCGGGGGTC2544 
LysProIleMetSerArgSerLeuAlaGluMetLysGluAlaGlyVal 
190195200 
CACGGTAACTATAGCTAGCTCTTCCGATCCCCCTTCAATTAATTAATTTATAG2597 
His 
TAGTCCATTCATGTGATGATTTTTGTTTTTCTTTTTACTGACAGGTCTTATAGTG2652 
GlyLeuIleVal 
205 
CCTGATCTCCCGTACGTGGCCGCGCACTCGCTGTGGAGTGAAGCCAAG2700 
ProAspLeuProTyrValAlaAlaHisSerLeuTrpSerGluAlaLys 
210215220 
AACAACAACCTGGAGCTGGTAGGTTGAATTAAGTTGATGCATGTGATG2748 
AsnAsnAsnLeuGluLeu 
225230 
ATTTATGTAGCTAGATCGAGCTAGCTATAATTAGGAGCATATCAGGTGCTGCTG2802 
ValLeuLeu 
ACAACACCAGCCATACCAGAAGACAGGATGAAGGAGATCACCAAGGCT2850 
ThrThrProAlaIleProGluAspArgMetLysGluIleThrLysAla 
235240245 
TCAGAAGGCTTCGTCTACCTGGTAGTTATATGTATATATAGATGGACGACG2901 
SerGluGlyPheValTyrLeu 
250255 
TAACTCATTCCAGCCCCATGCATATATGGAGGCTTCAATTCTGCAGAGACGACGAAGACC2961 
ACGACGACGACTAACACTAGCTAGGGGCGTACGTTGCAGGTGAGCGTGAACGGA3015 
ValSerValAsnGly 
260 
GTGACAGGTCCTCGCGCAAACGTGAACCCACGAGTGGAGTCACTCATC3063 
ValThrGlyProArgAlaAsnValAsnProArgValGluSerLeuIle 
265270275 
CAGGAGGTTAAGAAGGTGACTAACAAGCCCGTTGCTGTTGGCTTCGGC3111 
GlnGluValLysLysValThrAsnLysProValAlaValGlyPheGly 
280285290 
ATATCCAAGCCCGAGCACGTGAAGCAGGTACGTACGTAGCTGACCAAAAAAAAC3165 
IleSerLysProGluHisValLys 
295300 
TGTTAACAAGTTTTGTTTGACAAGCCGGCTACTAGCTAGCTAACAGTGATCAGTGACACA3225 
CACACACACACAGATTGCGCAGTGGGGCGCTGACGGGGTGATCATCGGC3274 
GlnIleAlaGlnTrpGlyAlaAspGlyValIleIleGly 
305310 
AGCGCCATGGTGAGGCAGCTGGGCGAAGCGGCTTCTCCCAAGCAAGGC3322 
SerAlaMetValArgGlnLeuGlyGluAlaAlaSerProLysGlnGly 
315320325330 
CTGAGGAGGCTGGAGGAGTATGCCAGGGGCATGAAGAACGCGCTGCCA3370 
LeuArgArgLeuGluGluTyrAlaArgGlyMetLysAsnAlaLeuPro 
335340345 
TGAGTCCATGACAAAGTAAAACGTACAGAGACACTTGATAATATCTATCTATCATCTCGG3430 
AGAAGACGACCGACCAATAAAAATAAGCCAAGTGGAAGTGAAGCTTAGCTGTATATACAC3490 
CGTACGTCGTCGTCGTCGTTCCGGATCGATCTCGGCCGGCTAGCTAGCAGAACGTGTACG3550 
TAGTAGTATGTAATGCATGGAGTGTGGAGCTACTAGCTAGCTGGCCGTTCATTCGATTAT3610 
AATTCTTCGCTCTGCTGTGGTAGCAGATGTACCTAGTCGATCTTGTACGACGAAGAAGCT3670 
GGCTAGCTAGCCGTCTCGATCGTATATGTACTGATTAATCTGCAGATTGAATAAAAACTA3730 
CAGTACGCATATGATGCGTACGTACGTGTGTATAGTTTGTGCTCATATATGCTCCTCATC3790 
ACCTGCCTGATCTGCCCATCGATCTCTCTCGTACTCCTTCCTGTTAAATGCCTTCTTTGA3850 
CAGACACACCACCACCAGCAGCAGTGACGCTCTGCACGCCGCCGCTTTAAGACATGTAAG3910 
ATATTTTAAGAGGTATAAGATACCAAGGAGCACAAATCTGGAGCACTGGGATATTGCAAA3970 
GACAAAAAAAAAACAAAATTAAAGTCCCACCAAAGTAGAGATAGTAAAGAGGTGGATGGA4030 
TTAAAATTATCTCATGATTTTTGGATCTGCTCAAATAGATCGATATGGTATTCAGATCTA4090 
TGTTGTATAGCCTTTTCATTAGCTTTCTGAAAAAAAAATGGTATGATGAGTGCGGAGTAG4150 
CTAGGGCTGTGAAGGAGTCGGATGGGCTTCCACGTACTTGTTTGTGGCCCTAGTCCGGTT4210 
CTATTTAGGTCCGATCCGAGTCCGGCATGGTCCGGTTCCATACGGGCTAGGACCAAGCTC4270 
GGCACGTGAGTTTTAGGCCCGTCGGCTAGCCCGAGCACGACCCGTTTTTAAACTGGCTAG4330 
GACTCGCCCATTTAATAAGACAAACATTGCAAAAAATAGCTCTATTTTTTATTTAAAATA4390 
TATTGTTTATTTGTGAAATGTGTATTATTTGTAATATATATTATTGTATATAGTTATATC4450 
TTCAATTATGATTTATAAATATGTTTTTTATTATGAACTCAATTTTAAGTTTGATTTATG4510 
CGTTGGCGGGCTCGAGGAGGCACGGTGAACATTTTTGGGTCGGGCTTAACGGGTCGGCCC4570 
GGCCCGGTTCGGCCCATCCACGGCCCATCCCGTGTCGGCCTCGTTCGGTGAGTTCAGCCC4630 
GTCGGACAACCCGTCCCCGGCCCGGATAATTAATCGGGCCTAACCGTGGCGTGCTTAAAC4690 
GGTCCGTGCCTCAACGGACCGGGCCGCGGGCGGCCCGTTTGACATCTCTAGTGGTGTGAT4750 
TAGAGATGGCGATGGGAACCGATCACTGATTCCGTGTGGAGAATTCGATATCAAGCTTAT4810 
CGATACC4817 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 346 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
MetAlaPheAlaProLysThrSerSerSerSerSerLeuSerSerAla 
151015 
LeuGlnAlaAlaGlnSerProProLeuLeuLeuArgArgMetSerSer 
202530 
ThrAlaThrProArgArgArgTyrAspAlaAlaValValValThrThr 
354045 
ThrThrThrAlaArgAlaAlaAlaAlaAlaValThrValProAlaAla 
505560 
ProProGlnAlaGlyArgArgArgArgCysHisGlnSerLysArgArg 
65707580 
HisProGlnArgArgSerArgProValSerAspThrMetAlaAlaLeu 
859095 
MetAlaLysGlyLysThrAlaPheIleProTyrIleThrAlaGlyAsp 
100105110 
ProAspLeuAlaThrThrAlaGluAlaLeuArgLeuLeuAspGlyCys 
115120125 
GlyAlaAspValIleGluLeuGlyValProCysSerAspProTyrIle 
130135140 
AspGlyProIleIleGlnAlaSerValAlaArgAlaLeuAlaSerGly 
145150155160 
ThrThrMetAspAlaValLeuGluMetLeuArgGluValThrProGlu 
165170175 
LeuSerCysProValValLeuLeuSerTyrTyrLysProIleMetSer 
180185190 
ArgSerLeuAlaGluMetLysGluAlaGlyValHisGlyLeuIleVal 
195200205 
ProAspLeuProTyrValAlaAlaHisSerLeuTrpSerGluAlaLys 
210215220 
AsnAsnAsnLeuGluLeuValLeuLeuThrThrProAlaIleProGlu 
225230235240 
AspArgMetLysGluIleThrLysAlaSerGluGlyPheValTyrLeu 
245250255 
ValSerValAsnGlyValThrGlyProArgAlaAsnValAsnProArg 
260265270 
ValGluSerLeuIleGlnGluValLysLysValThrAsnLysProVal 
275280285 
AlaValGlyPheGlyIleSerLysProGluHisValLysGlnIleAla 
290295300 
GlnTrpGlyAlaAspGlyValIleIleGlySerAlaMetValArgGln 
305310315320 
LeuGlyGluAlaAlaSerProLysGlnGlyLeuArgArgLeuGluGlu 
325330335 
TyrAlaArgGlyMetLysAsnAlaLeuPro 
340345 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1349 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 3..1226 
(D) OTHER INFORMATION: /note= "cDNA sequence for maize 
pollen- specific calcium dependent protein kinase gene as 
disclosed in Figure 30." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
TGCAGATCATGCACCACCTCTCCGGCCAGCCCAACGTGGTGGGCCTC47 
GlnIleMetHisHisLeuSerGlyGlnProAsnValValGlyLeu 
350355360 
CGCGGCGCGTACGAGGACAAGCAGAGCGTGCACCTCGTCATGGAGCTG95 
ArgGlyAlaTyrGluAspLysGlnSerValHisLeuValMetGluLeu 
365370375 
TGCGCGGGCGGGGAGCTCTTCGACCGCATCATCGCCCGGGGCCAGTAC143 
CysAlaGlyGlyGluLeuPheAspArgIleIleAlaArgGlyGlnTyr 
380385390 
ACGGAGCGCGGCGCCGCGGAGCTGCTGCGCGCCATCGTGCAGATCGTG191 
ThrGluArgGlyAlaAlaGluLeuLeuArgAlaIleValGlnIleVal 
395400405 
CACACCTGCCACTCCATGGGGGTGATGCACCGGGACATCAAGCCCGAG239 
HisThrCysHisSerMetGlyValMetHisArgAspIleLysProGlu 
410415420425 
AACTTCCTGCTGCTCAGCAAGGACGAGGACGCGCCGCTCAAGGCCACC287 
AsnPheLeuLeuLeuSerLysAspGluAspAlaProLeuLysAlaThr 
430435440 
GACTTCGGCCTCTCCGTCTTCTTCAAGGAGGGCGAGCTGCTCAGGGAC335 
AspPheGlyLeuSerValPhePheLysGluGlyGluLeuLeuArgAsp 
445450455 
ATCGTCGGCAGCGCCTACTACATCGCGCCCGAGGTGCTCAAGAGGAAG383 
IleValGlySerAlaTyrTyrIleAlaProGluValLeuLysArgLys 
460465470 
TACGGCCCGGAGGCCGACATCTGGAGCGTCGGCGTCATGCTCTACATC431 
TyrGlyProGluAlaAspIleTrpSerValGlyValMetLeuTyrIle 
475480485 
TTCCTCGCCGGCGTGCCTCCCTTCTGGGCAGAGAACGAGAACGGCATC479 
PheLeuAlaGlyValProProPheTrpAlaGluAsnGluAsnGlyIle 
490495500505 
TTCACCGCCATCCTGCGAGGGCAGCTTGACCTCTCCAGCGAGCCATGG527 
PheThrAlaIleLeuArgGlyGlnLeuAspLeuSerSerGluProTrp 
510515520 
CCACACATCTCGCCGGGAGCCAAGGATCTCGTCAAGAAGATGCTCAAC575 
ProHisIleSerProGlyAlaLysAspLeuValLysLysMetLeuAsn 
525530535 
ATCAACCCCAAGGAGCGGCTCACGGCGTTCCAGGTCCTCAATCACCCA623 
IleAsnProLysGluArgLeuThrAlaPheGlnValLeuAsnHisPro 
540545550 
TGGATCAAAGAAGACGGAGACGCGCCTGACACGCCGCTTGACAACGTT671 
TrpIleLysGluAspGlyAspAlaProAspThrProLeuAspAsnVal 
555560565 
GTTCTCGACAGGCTCAAGCAGTTCAGGGCCATGAACCAGTTCAAGAAA719 
ValLeuAspArgLeuLysGlnPheArgAlaMetAsnGlnPheLysLys 
570575580585 
GCAGCATTGAGGATCATAGCTGGGTGCCTATCCGAAGAGGAGATCACA767 
AlaAlaLeuArgIleIleAlaGlyCysLeuSerGluGluGluIleThr 
590595600 
GGGCTGAAGGAGATGTTCAAGAACATTGACAAGGATAACAGCGGGACC815 
GlyLeuLysGluMetPheLysAsnIleAspLysAspAsnSerGlyThr 
605610615 
ATTACCCTCGACGAGCTCAAACACGGGTTGGCAAAGCACGGGCCCAAG863 
IleThrLeuAspGluLeuLysHisGlyLeuAlaLysHisGlyProLys 
620625630 
CTGTCAGACAGCGAAATGGAGAAACTAATGGAAGCAGCTGACGCTGAC911 
LeuSerAspSerGluMetGluLysLeuMetGluAlaAlaAspAlaAsp 
635640645 
GGCAACGGGTTAATTGACTACGACGAATTCGTCACCGCAACAGTGCAT959 
GlyAsnGlyLeuIleAspTyrAspGluPheValThrAlaThrValHis 
650655660665 
ATGAACAAACTGGATAGAGAAGAGCACCTTTACACAGCATTCCAGTAT1007 
MetAsnLysLeuAspArgGluGluHisLeuTyrThrAlaPheGlnTyr 
670675680 
TTCGACAAGGACAACAGCGGGTACATTACTAAAGAAGAGCTTGAGCAC1055 
PheAspLysAspAsnSerGlyTyrIleThrLysGluGluLeuGluHis 
685690695 
GCCTTGAAGGAGCAAGGGTTGTATGACGCCGATAAAATCAAAGACATC1103 
AlaLeuLysGluGlnGlyLeuTyrAspAlaAspLysIleLysAspIle 
700705710 
ATCTCCGATGCCGACTCTGACAATGATGGAAGGATAGATTATTCAGAG1151 
IleSerAspAlaAspSerAspAsnAspGlyArgIleAspTyrSerGlu 
715720725 
TTTGTGGCGATGATGAGGAAAGGGACGGCTGGTGCCGAGCCAATGAAC1199 
PheValAlaMetMetArgLysGlyThrAlaGlyAlaGluProMetAsn 
730735740745 
ATCAAGAAGAGGCGAGACATAGTCCTATAGTGAAGTGAAGCAGCAAG1246 
IleLysLysArgArgAspIleValLeu 
750 
TGTGTAATGTAATGTGTATAGCAGCTCAAACAAGCAAATTTGTACATCTGTACACAAATG1306 
CAATGGGGTTACTTTTGCAAAAAAAAAAAAAAAAAAAAAAAAA1349 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 408 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GlnIleMetHisHisLeuSerGlyGlnProAsnValValGlyLeuArg 
151015 
GlyAlaTyrGluAspLysGlnSerValHisLeuValMetGluLeuCys 
202530 
AlaGlyGlyGluLeuPheAspArgIleIleAlaArgGlyGlnTyrThr 
354045 
GluArgGlyAlaAlaGluLeuLeuArgAlaIleValGlnIleValHis 
505560 
ThrCysHisSerMetGlyValMetHisArgAspIleLysProGluAsn 
65707580 
PheLeuLeuLeuSerLysAspGluAspAlaProLeuLysAlaThrAsp 
859095 
PheGlyLeuSerValPhePheLysGluGlyGluLeuLeuArgAspIle 
100105110 
ValGlySerAlaTyrTyrIleAlaProGluValLeuLysArgLysTyr 
115120125 
GlyProGluAlaAspIleTrpSerValGlyValMetLeuTyrIlePhe 
130135140 
LeuAlaGlyValProProPheTrpAlaGluAsnGluAsnGlyIlePhe 
145150155160 
ThrAlaIleLeuArgGlyGlnLeuAspLeuSerSerGluProTrpPro 
165170175 
HisIleSerProGlyAlaLysAspLeuValLysLysMetLeuAsnIle 
180185190 
AsnProLysGluArgLeuThrAlaPheGlnValLeuAsnHisProTrp 
195200205 
IleLysGluAspGlyAspAlaProAspThrProLeuAspAsnValVal 
210215220 
LeuAspArgLeuLysGlnPheArgAlaMetAsnGlnPheLysLysAla 
225230235240 
AlaLeuArgIleIleAlaGlyCysLeuSerGluGluGluIleThrGly 
245250255 
LeuLysGluMetPheLysAsnIleAspLysAspAsnSerGlyThrIle 
260265270 
ThrLeuAspGluLeuLysHisGlyLeuAlaLysHisGlyProLysLeu 
275280285 
SerAspSerGluMetGluLysLeuMetGluAlaAlaAspAlaAspGly 
290295300 
AsnGlyLeuIleAspTyrAspGluPheValThrAlaThrValHisMet 
305310315320 
AsnLysLeuAspArgGluGluHisLeuTyrThrAlaPheGlnTyrPhe 
325330335 
AspLysAspAsnSerGlyTyrIleThrLysGluGluLeuGluHisAla 
340345350 
LeuLysGluGlnGlyLeuTyrAspAlaAspLysIleLysAspIleIle 
355360365 
SerAspAlaAspSerAspAsnAspGlyArgIleAspTyrSerGluPhe 
370375380 
ValAlaMetMetArgLysGlyThrAlaGlyAlaGluProMetAsnIle 
385390395400 
LysLysArgArgAspIleValLeu 
405 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 464 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..464 
(D) OTHER INFORMATION: /note= "derived protein sequence of 
pollen specific CDPK as disclosed in Figure 34." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
ValLeuGlyArgProMetGluAspValArgAlaThrTyrSerMetGly 
151015 
LysGluLeuGlyArgGlyGlnPheGlyValThrHisLeuCysThrHis 
202530 
ArgThrSerGlyGluLysLeuAlaCysLysThrIleAlaLysArgLys 
354045 
LeuAlaAlaArgGluAspValAspAspValArgArgGluValGlnIle 
505560 
MetHisHisLeuSerGlyGlnProAsnValValGlyLeuArgGlyAla 
65707580 
TyrGluAspLysGlnSerValHisLeuValMetGluLeuCysAlaGly 
859095 
GlyGluLeuPheAspArgIleIleAlaArgGlyGlnTyrThrGluArg 
100105110 
GlyAlaAlaGluLeuLeuArgAlaIleValGlnIleValHisThrCys 
115120125 
HisSerMetGlyValMetHisArgAspIleLysProGluAsnPheLeu 
130135140 
LeuLeuSerLysAspGluAspAlaProLeuLysAlaThrAspPheGly 
145150155160 
LeuSerValPhePheLysGluGlyGluLeuLeuArgAspIleValGly 
165170175 
SerAlaTyrTyrIleAlaProGluValLeuLysArgLysTyrGlyPro 
180185190 
GluAlaAspIleTrpSerValGlyValMetLeuTyrIlePheLeuAla 
195200205 
GlyValProProPheTrpAlaGluAsnGluAsnGlyIlePheThrAla 
210215220 
IleLeuArgGlyGlnLeuAspLeuSerSerGluProTrpProHisIle 
225230235240 
SerProGlyAlaLysAspLeuValLysLysMetLeuAsnIleAsnPro 
245250255 
LysGluArgLeuThrAlaPheGlnValLeuAsnHisProTrpIleLys 
260265270 
GluAspGlyAspAlaProAspThrProLeuAspAsnValValLeuAsp 
275280285 
ArgLeuLysGlnPheArgAlaMetAsnGlnPheLysLysAlaAlaLeu 
290295300 
ArgIleIleAlaGlyCysLeuSerGluGluGluIleThrGlyLeuLys 
305310315320 
GluMetPheLysAsnIleAspLysAspAsnSerGlyThrIleThrLeu 
325330335 
AspGluLeuLysHisGlyLeuAlaLysHisGlyProLysLeuSerAsp 
340345350 
SerGluMetGluLysLeuMetGluAlaAlaAspAlaAspGlyAsnGly 
355360365 
LeuIleAspTyrAspGluPheValThrAlaThrValHisMetAsnLys 
370375380 
LeuAspArgGluGluHisLeuTyrThrAlaPheGlnTyrPheAspLys 
385390395400 
AspAsnSerGlyTyrIleThrLysGluGluLeuGluHisAlaLeuLys 
405410415 
GluGlnGlyLeuTyrAspAlaAspLysIleLysAspIleIleSerAsp 
420425430 
AlaAspSerAspAsnAspGlyArgIleAspTyrSerGluPheValAla 
435440445 
MetMetArgLysGlyThrAlaGlyAlaGluProMetAsnIleLysLys 
450455460 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 295 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..295 
(D) OTHER INFORMATION: /note= "rat protein kinase II 
protein sequence as shown in Figure 32." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
TyrGlnLeuPheGluGluLeuGlyLysGlyAlaPheSerValValArg 
151015 
ArgCysValLysLysThrSerThrGlnGluTyrAlaAlaLysIleIle 
202530 
AsnThrLysLysLeuSerAlaArgAspHisGlnLysLeuGluArgGlu 
354045 
AlaArgIleCysArgLeuLeuLysHisProAsnIleValArgLeuHis 
505560 
AspSerIleSerGluGluGlyPheHisTyrLeuValPheAspLeuVal 
65707580 
ThrGlyGlyGluLeuPheGluAspIleValAlaArgGluTyrTyrSer 
859095 
GluAlaAspAlaSerHisCysIleHisGlnIleLeuGluSerValAsn 
100105110 
HisIleHisGlnHisAspIleValHisArgAspLeuLysProGluAsn 
115120125 
LeuLeuLeuAlaSerLysCysLysGlyAlaAlaValLysLeuAlaAsp 
130135140 
PheGlyLeuAlaIleGluValGlnGlyGluGlnGlnAlaTrpPheGly 
145150155160 
PheAlaGlyThrProGlyTyrLeuSerProGluValLeuArgLysAsp 
165170175 
ProTyrGlyLysProValAspIleTrpAlaCysGlyValIleLeuTyr 
180185190 
IleLeuLeuValGlyTyrProProPheTrpAspGluAspGlnHisLys 
195200205 
LeuTyrGlnGlnIleLysAlaGlyAlaTyrAspPheProSerProGlu 
210215220 
TrpAspThrValThrProGluAlaLysAsnLeuIleAsnGlnMetLeu 
225230235240 
ThrIleAsnProAlaLysArgIleThrAlaAspGlnAlaLeuLysHis 
245250255 
ProTrpValCysGlnArgSerThrValAlaSerMetMetHisArgGln 
260265270 
GluThrValGluCysLeuArgLysPheAsnAlaArgArgLysLeuLys 
275280285 
GlyAlaIleLeuThrThrMet 
290295 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 142 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..142 
(D) OTHER INFORMATION: /note= "human calmodulin protein 
sequence as shown in Figure 33." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
LeuThrGluGluGlnIleAlaGluPheLysGluAlaPheSerLeuPhe 
151015 
AspLysAspGlyAspGlyThrIleThrThrLysGluLeuGlyThrVal 
202530 
MetArgSerLeuGlyGlnAsnProThrGluAlaGluLeuGlnAspMet 
354045 
IleAsnGluValAspAlaAspGlyAsnGlyThrIleAspPheProGlu 
505560 
PheLeuThrMetMetAlaArgLysMetLysAspThrAspSerGluGlu 
65707580 
GluIleArgGluAlaPheArgValLysAspLysAspGlyAsnGlyTyr 
859095 
IleSerAlaAlaGluLeuArgHisValMetThrAsnLeuGlyGluLys 
100105110 
LeuThrAspGluGluValAspGluMetIleArgGluAlaAspIleAsp 
115120125 
GlyAspGlyGlnValAsnTyrGluGluPheValGlnMetMet 
130135140 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 463 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..463 
(D) OTHER INFORMATION: /note= "protein sequence for 
soybean CDPK as shown in Figure 34." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
ValLeuProGlnArgThrGlnAsnIleArgGluValTyrGluValGly 
151015 
ArgLysLeuGlyGlnGlyGlnPheGlyThrThrPheGluCysThrArg 
202530 
ArgAlaSerGlyGlyLysPheAlaCysLysSerIleProLysArgLys 
354045 
LeuLeuCysLysGluAspTyrGluAspValTrpArgGluIleGlnIle 
505560 
MetHisHisLeuSerGluHisAlaAsnValValArgIleGluGlyThr 
65707580 
TyrGluAspSerThrAlaValHisLeuValMetGluLeuCysGluGly 
859095 
GlyGluLeuPheAspArgIleValGlnLysGlyHisTyrSerGluArg 
100105110 
GlnAlaAlaArgLeuIleLysThrIleValGluValValGluAlaCys 
115120125 
HisSerLeuGlyValMetHisArgAspLeuLysProGluAsnPheLeu 
130135140 
PheAspThrIleAspGluAspAlaLysLeuLysAlaThrAspPheGly 
145150155160 
LeuSerValPheTyrLysProGlyGluSerPheCysAspValValGly 
165170175 
SerProTyrTyrValAlaProGluValLeuArgLysLeuTyrGlyPro 
180185190 
GluSerAspValTrpSerAlaGlyValIleLeuTyrIleLeuLeuSer 
195200205 
GlyValProProPheTrpAlaGluSerGluProGlyIlePheArgGln 
210215220 
IleLeuLeuGlyLysLeuAspPheHisSerGluProTrpProSerIle 
225230235240 
SerAspSerAlaLysAspLeuIleArgLysMetLeuAspGlnAsnPro 
245250255 
LysThrArgLeuThrAlaHisGluValLeuArgHisProTrpIleVal 
260265270 
AspAspAsnIleAlaProAspLysProLeuAspSerAlaValLeuSer 
275280285 
ArgLeuLysGlnPheSerAlaMetAsnLysLeuLysLysMetAlaLeu 
290295300 
ArgValIleAlaGluArgLeuSerGluGluGluIleGlyGlyLeuLys 
305310315320 
GluLeuPheLysMetIleAspThrAspAsnSerGlyThrIleThrPhe 
325330335 
AspGluLeuLysAspGlyLeuLysArgValGlySerGluLeuMetGlu 
340345350 
SerGluIleLysAspLeuMetAspAlaAlaAspIleAspLysSerGly 
355360365 
ThrIleAspTyrGlyGluPheIleAlaAlaThrValHisLeuAsnLys 
370375380 
LeuGluArgGluGluAsnLeuValSerAlaPheSerTyrPheAspLys 
385390395400 
AspGlySerGlyTyrIleThrLeuAspGluIleGlnGlnAlaCysLys 
405410415 
AspPheGlyLeuAspAspIleHisIleAspAspMetIleLysGluIle 
420425430 
AspGlnAspAsnAspGlyGlnIleAspTyrGlyGluPheAlaAlaMet 
435440445 
MetArgLysGlyAsnGlyGlyIleGlyArgArgThrMetArgLys 
450455460 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4165 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1416..1425 
(D) OTHER INFORMATION: /note= "start of mRNA" 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 1481..2366 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 2367..2449 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 2450..2602 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 2603..2688 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 2689..2804 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 2805..2906 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 2907..3074 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 3075..3177 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 3178..3305 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 3306..3397 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 3398..3497 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 3498..3712 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 3713..3811 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
TTAGTAACACCTCTCCAATCGCTTGGGTTGGCACATTCTTAGCTTTTATCACATTTTAAG60 
AAATAGAGTTCACCACCTTCAAAATATGCCTATACAATGAATGATGCTTGGATGCAATAT120 
AGCTAGATTCAACTAGCTATATATGGTCAATAGAACCCTGTGAGCACCTCACAAACACGA180 
CTTCAATTTTGAGACCCTAAGCGAGTAAATGGTTAAAGTCCTCTTATTATTAGTCTTAGG240 
ACTTCTCCTTGCTAAATGCTTGTCAGCGATCTATATATCTTCCCCACTGCGGGAGATACT300 
ATATATAGGGCCTTGGACCTCTAGGGTATCTCAAAGGCCTAGTCACAACAATTCTCAACA360 
GTATTTAATTTTATACATGTATGAACAGTGTAGGAATTTGAGTGCCCAACCCAAGAGTGG420 
GAGGTGTAAATTGGGTAGCTAAACTTAAATAGGGCTCTTCTTATTTAGGTTTATCTAGTC480 
TCTACTTAGACTAATTCAGAAAGAATTTTACAACCTATGGTTAATCATATCTCTAGTCTA540 
AGCAAATTTAGGAAAGTTAAAAGCACACAATTAGGCACATGTGAAAGATGTGTATGGTAA600 
GTAAAAGACTTATAAGGAAAAAGTGGGTGAATCCTCAAGATGTGGTGGTATATCCCAATG660 
ATATTAGATGCCAGAATATAGGGGGGAAATCGATGTATACCATCTCTACCAGGATACCTG720 
TGCGGACTGTGCAACTGACACATGGACCATGGTGTCTTCTTAGATTTGGTTATTAGCTAA780 
TTGCGCTACAACTTGTTCAAGGCTAGACCAAATTAAAAAACTAATATTAAACATAAAAAG840 
TTAGGCAAACTATAGTAAATTATGCAGCGATCCAACAACAAGCCATGTCTCGTGGGTCAT900 
GAGCCACGCGTCGGCCATACACCCACATGATGTTTCCATACGGATGGTCCTTATGCAATT960 
TTGTCTGCAAAACACAAGCCTTAATACAGCCACGCGACAATCATGGAAGTGGTCGTTTTA1020 
GGTCCTCATCATGAAGTTCAGGGAAAACGCATCAAATGTAATGCAGAGAAATGGTATTTC1080 
TTCTCTTGTAAATCAGGGAGAGGAGTACCATCAGTACAGATTCAGAATCAGAATTCAGTC1140 
TTCCAACGACAATAATCGCAGCATCTTGTAAAAATTTGCAGAAACTTCTGTTTGACTTGT1200 
AGCCCTGACCTTTGCAAATATTTGAAGTTGTGCCTGCTGACACAACTTCAATCTGGAAGT1260 
GCTGTTGATCAGTTTTGCCAGAAACAGCAAGCAGCCTATATATATCTGTCACGAGACACC1320 
CTGCCGCCCTCTTCTTTCCCGCCATTCCCTCCCTACCCTTCAAAATCTAGAAACCTTTTT1380 
TTTTCCTCCCGATACGCCCCTCCATCTCTCGCCGTTCATGTCCGTGGCTGGCTGCCCTCC1440 
GTGGGAGCAGGCGGCCGCACTCGTTCCCCGCCGCAGCCATGGGCCAGTGCTGCTCCAAGG1500 
GCGCCGGAGAGGCCCCGCCACCGAGGCGCCAAACGGCAGGCGCCAAGCCGCGGGCGTCCG1560 
CGAACAACGCCGACGGACAACGGGCGTCGTCCTCGTCCGCGGTGGCTGCTGCCGCTGCTG1620 
CTGCCGGTGGTGGTGGCGGCGGCACGACGAAGCCGGCCTCACCCACCGGCGGCGCCAGGG1680 
CCAGCTCCGGCAGCAAACCGGCGGCGGCCGTGGGCACGGTGCTGGGCCGGCCCATGGAGG1740 
ACGTGCGCGCGACCTACTCGATGGGCAAGGAGCTCGGGCGCGGGCAGTTCGGCGTGACGC1800 
ACCTGTGCACGCACCGGACGAGCGGCGAGAAGCTGGCGTGCAAGACGATCGCGAAGCGGA1860 
AGCTGGCGGCCAGGGAGGACGTGGACGACGTGCGGCGGGAGGTGCAGATCATGCACCACC1920 
TCTCCGGCCAGCCCAACGTGGTGGGCCTCCGCGGCGCGTACGAGGACAAGCAGAGCGTGC1980 
ACCTCGTCATGGAGCTGTGCGCGGGCGGGGAGCTCTTCGACCGCATCATCGCCCGGGGCC2040 
AGTACACGGAGCGCGGCGCCGCGGAGCTGCTGCGCGCCATCGTGCAGATCGTGCACACCT2100 
GCCACTCCATGGGGGTGATGCACCGGGACATCAAGCCCGAGAACTTCCTGCTGCTCAGCA2160 
AGGACGAGGACGCGCCGCTCAAGGCCACCGACTTCGGCCTCTCCGTCTTCTTCAAGGAGG2220 
GCGAGCTGCTCAGGGACATCGTCGGCAGCGCCTACTACATCGCGCCCGAGGTGCTCAAGA2280 
GGAAGTACGGCCCGGAGGCCGACATCTGGAGCGTCGGCGTCATGCTCTACATCTTCCTCG2340 
CCGGCGTGCCTCCCTTCTGGGCAGGTCGGATCCGTCCGTGTTCGTCCTAGACGATATACA2400 
GAACCCGACGATGGATTTGCTTCTCAGCCCTGTTCTTGCATCACCAGAGAACGAGAACGG2460 
CATCTTCACCGCCATCCTGCGAGGGCAGCTTGACCTCTCCAGCGAGCCATGGCCACACAT2520 
CTCGCCGGGAGCCAAGGATCTCGTCAAGAAGATGCTCAACATCAACCCCAAGGAGCGGCT2580 
CACGGCGTTCCAGGTCCTCAGTAAGTACCCAGATCGTTGCTGTCATACACTCATATGAAT2640 
TGTATCGTTCATGAGCAACGATCGAGCGGATTTGGTGAACTTGTAGATCACCCATGGATC2700 
AAAGAAGACGGAGACGCGCCTGACACGCCGCTTGACAACGTTGTTCTCGACAGGCTCAAG2760 
CAGTTCAGGGCCATGAACCAGTTCAAGAAAGCAGCATTGAGGGTACATTATCTGATAAAA2820 
GCTCCACAAATACAACTTCTGAAGAACAGCAATGCTTACACGGCAGAATTTTCATTATAA2880 
ATGCTCTTGATGACATAATGTTAGATCATAGCTGGGTGCCTATCCGAAGAGGAGATCACA2940 
GGGCTGAAGGAGATGTTCAAGAACATTGACAAGGATAACAGCGGGACCATTACCCTCGAC3000 
GAGCTCAAACACGGGTTGGCAAAGCACGGGCCCAAGCTGTCAGACAGCGAAATGGAGAAA3060 
CTAATGGAAGCAGTGAGTTTTCAGAGTACAATCTTAAAAAAAGGAATTGTGATTCTTTTC3120 
AAAATGAAGAAGTAATCTGAAAACATCCCTGCTGAAATGCTTTATACATTTCCAGGCTGA3180 
CGCTGACGGCAACGGGTTAATTGACTACGACGAATTCGTCACCGCAACAGTGCATATGAA3240 
CAAACTGGATAGAGAAGAGCACCTTTACACAGCATTCCAGTATTTCGACAAGGACAACAG3300 
CGGGTAAGTTGAACGTTAAAATGATACAGCTGGTACCTGAATTCTGGACAACACATATCA3360 
TAACAGGACACATATATAATTCGTTTATCTCACAGGTACATTACTAAAGAAGAGCTTGAG3420 
CACGCCTTGAAGGAGCAAGGGTTGTATGACGCCGATAAAATCAAAGACATCATCTCCGAT3480 
GCCGACTCTGACAATGTAAGGAACAAACATTATTTAAATTTCAGCCGACAAACTAAACTA3540 
TAGAAACCACATCATGATATCAAATTTTGAGGTGGCGGTGCTACAGAAATAGAACCCAGT3600 
ACACCAAAATGACTAACTTGTCATGATTAGTTGTTCCTCGTAACTGAACATTTGTGTTCT3660 
TAGTTTCTTATTGTTAAACCAAAGACTTAAATTCACTTTTGCACATGCAGGATGGAAGGA3720 
TAGATTATTCAGAGTTTGTGGCGATGATGAGGAAAGGGACGGCTGGTGCCGAGCCAATGA3780 
ACATCAAGAAGAGGCGAGACATAGTCCTATAGTGAAGTGAAGCAGWAAGTGTGTAATGTA3840 
ATGTGTATAGCAGCTCAAACAAGCAAATTTGTACATCTGTACACAAATGCAATGGGGTTA3900 
CTTTTGCAACTTAGTTCATGGATGGTTGTGTACGTTGTGCTATTGATTGCAAGTGATTTG3960 
AAAGACATGCATACTTAGGAACTGAGAAAGATAGATCTACTACTGCTAGAGACAGAACAA4020 
TAGGATKKYAATTCAGYAAGTGYGTATTTCAGAAGACTACAGCTGGCATCTATTATTCTC4080 
ATTGTCCTCGCAAAAATACTGATGATGCATTTGAGAGAACAATATGCAACAAGATCGAGC4140 
TCCCTATAGTGAGTCGTATTAGGCC4165 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3546 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Synthetic DNA" 
(iii) HYPOTHETICAL: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3543 
(D) OTHER INFORMATION: /product="Full-length, hybrid 
maize optimized heat stable cryIA(b)" 
/note= "DNA sequence as disclosed in Figure 37 as 
contained in pCIB5515." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
ATGGACAACAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTG48 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
410415420 
AGCAACCCCGAGGTGGAGGTGCTGGGCGGCGAGCGCATCGAGACCGGC96 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
425430435440 
TACACCCCCATCGACATCAGCCTGAGCCTGACCCAGTTCCTGCTGAGC144 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
445450455 
GAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTGGTGGACATCATC192 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
460465470 
TGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAGATC240 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
475480485 
GAGCAGCTGATCAACCAGCGCATCGAGGAGTTCGCCCGCAACCAGGCC288 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
490495500 
ATCAGCCGCCTGGAGGGCCTGAGCAACCTGTACCAAATCTACGCCGAG336 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
505510515520 
AGCTTCCGCGAGTGGGAGGCCGACCCCACCAACCCCGCCCTGCGCGAG384 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
525530535 
GAGATGCGCATCCAGTTCAACGACATGAACAGCGCCCTGACCACCGCC432 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
540545550 
ATCCCCCTGTTCGCCGTGCAGAACTACCAGGTGCCCCTGCTGAGCGTG480 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
555560565 
TACGTGCAGGCCGCCAACCTGCACCTGAGCGTGCTGCGCGACGTCAGC528 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
570575580 
GTGTTCGGCCAGCGCTGGGGCTTCGACGCCGCCACCATCAACAGCCGC576 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
585590595600 
TACAACGACCTGACCCGCCTGATCGGCAACTACACCGACCACGCCGTG624 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
605610615 
CGCTGGTACAACACCGGCCTGGAGCGCGTGTGGGGTCCCGACAGCCGC672 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
620625630 
GACTGGATCAGGTACAACCAGTTCCGCCGCGAGCTGACCCTGACCGTG720 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
635640645 
CTGGACATCGTGAGCCTGTTCCCCAACTACGACAGCCGCACCTACCCC768 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
650655660 
ATCCGCACCGTGAGCCAGCTGACCCGCGAGATTTACACCAACCCCGTG816 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
665670675680 
CTGGAGAACTTCGACGGCAGCTTCCGCGGCAGCGCCCAGGGCATCGAG864 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
685690695 
GGCAGCATCCGCAGCCCCCACCTGATGGACATCCTGAACAGCATCACC912 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
700705710 
ATCTACACCGACGCCCACCGCGGCGAGTACTACTGGAGCGGCCACCAG960 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
715720725 
ATCATGGCCAGCCCCGTCGGCTTCAGCGGCCCCGAGTTCACCTTCCCC1008 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
730735740 
CTGTACGGCACCATGGGCAACGCTGCACCTCAGCAGCGCATCGTGGCA1056 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
745750755760 
CAGCTGGGCCAGGGAGTGTACCGCACCCTGAGCAGCACCCTGTACCGT1104 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
765770775 
CGACCTTTCAACATCGGCATCAACAACCAGCAGCTGAGCGTGCTGGAC1152 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
780785790 
GGCACCGAGTTCGCCTACGGCACCAGCAGCAACCTGCCCAGCGCCGTG1200 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
795800805 
TACCGCAAGAGCGGCACCGTGGACAGCCTGGACGAGATCCCCCCTCAG1248 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
810815820 
AACAACAACGTGCCACCTCGACAGGGCTTCAGCCACCGTCTGAGCCAC1296 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
825830835840 
GTGAGCATGTTCCGCAGTGGCTTCAGCAACAGCAGCGTGAGCATCATC1344 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
845850855 
CGTGCACCTATGTTCAGCTGGATTCACCGCAGTGCCGAGTTCAACAAC1392 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
860865870 
ATCATCCCCAGCAGCCAGATCACCCAGATCCCCCTGACCAAGAGCACC1440 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
875880885 
AACCTGGGCAGCGGCACCAGCGTGGTGAAGGGCCCCGGCTTCACCGGC1488 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
890895900 
GGCGACATCCTGCGCCGCACCAGCCCCGGCCAGATCAGCACCCTGCGC1536 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
905910915920 
GTGAACATCACCGCCCCCCTGAGCCAGCGCTACCGCGTCCGCATCCGC1584 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
925930935 
TACGCCAGCACCACCAACCTGCAGTTCCACACCAGCATCGACGGCCGC1632 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
940945950 
CCCATCAACCAGGGCAACTTCAGCGCCACCATGAGCAGCGGCAGCAAC1680 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
955960965 
CTGCAGAGCGGCAGCTTCCGCACCGTGGGCTTCACCACCCCCTTCAAC1728 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
970975980 
TTCAGCAACGGCAGCAGCGTGTTCACCCTGAGCGCCCACGTGTTCAAC1776 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
9859909951000 
AGCGGCAACGAGGTGTACATCGACCGCATCGAGTTCGTGCCCGCCGAG1824 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
100510101015 
GTGACCTTCGAGGCCGAGTACGACCTGGAGAGGGCTCAGAAGGCCGTG1872 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
102010251030 
AACGAGCTGTTCACCAGCAGCAACCAGATCGGCCTGAAGACCGACGTG1920 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
103510401045 
ACCGACTACCACATCGATCAAGTATCCAATTTAGTTGAGTGTTTATCT1968 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
105010551060 
GATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAA2016 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
1065107010751080 
CATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAAC2064 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
108510901095 
TTTAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACG2112 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
110011051110 
GATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTT2160 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
111511201125 
ACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAA2208 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
113011351140 
AAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTACCAATTAAGA2256 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
1145115011551160 
GGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTAC2304 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
116511701175 
AATGCCAAACACGAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGG2352 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
118011851190 
CCGCTTTCAGCCCCAAGTCCAATCGGAAAATGTGGGGAGCCGAATCGA2400 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
119512001205 
TGCGCTCCGCACCTGGAGTGGAACCCGGACCTAGACTGCAGCTGCAGG2448 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
121012151220 
GACGGGGAGAAGTGCGCCCATCATTCCCATCATTTCTCCTTGGACATT2496 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
1225123012351240 
GATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATA2544 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
124512501255 
TTCAAGATTAAGACGCAAGATGGCCATGCAAGACTAGGAAATCTAGAA2592 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
126012651270 
TTTCTCGAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAA2640 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
127512801285 
AGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAA2688 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
129012951300 
ACAAATATTGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTT2736 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
1305131013151320 
GTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATCGCGATG2784 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
132513301335 
ATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTG2832 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
134013451350 
CCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAA2880 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
135513601365 
TTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAAT2928 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
137013751380 
GTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTG2976 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
1385139013951400 
AAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCGGTCCTT3024 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
140514101415 
GTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGT3072 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
142014251430 
CCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATAT3120 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
143514401445 
GGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAA3168 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
145014551460 
CTGAAGTTTAGCAACTGTGTAGAAGAGGAAGTATATCCAAACAACACG3216 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
1465147014751480 
GTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAGGGTACG3264 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
148514901495 
TACACTTCTCGTAATCGAGGATATGACGGAGCCTATGAAAGCAATTCT3312 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
150015051510 
TCTGTACCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACA3360 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
151515201525 
GATGGACGAAGAGACAATCCTTGTGAATCTAACAGAGGATATGGGGAT3408 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
153015351540 
TACACACCACTACCAGCTGGCTATGTGACAAAAGAATTAGAGTACTTC3456 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
1545155015551560 
CCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAAGGAACA3504 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
156515701575 
TTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAA3546 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
15801585 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu 
151015 
SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly 
202530 
TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer 
354045 
GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle 
505560 
TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle 
65707580 
GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 
859095 
IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu 
100105110 
SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu 
115120125 
GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla 
130135140 
IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal 
145150155160 
TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer 
165170175 
ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg 
180185190 
TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaVal 
195200205 
ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg 
210215220 
AspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal 
225230235240 
LeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrPro 
245250255 
IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal 
260265270 
LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu 
275280285 
GlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr 
290295300 
IleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGln 
305310315320 
IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 
325330335 
LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla 
340345350 
GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg 
355360365 
ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp 
370375380 
GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal 
385390395400 
TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln 
405410415 
AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis 
420425430 
ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle 
435440445 
ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn 
450455460 
IleIleProSerSerGlnIleThrGlnIleProLeuThrLysSerThr 
465470475480 
AsnLeuGlySerGlyThrSerValValLysGlyProGlyPheThrGly 
485490495 
GlyAspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArg 
500505510 
ValAsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArg 
515520525 
TyrAlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArg 
530535540 
ProIleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsn 
545550555560 
LeuGlnSerGlySerPheArgThrValGlyPheThrThrProPheAsn 
565570575 
PheSerAsnGlySerSerValPheThrLeuSerAlaHisValPheAsn 
580585590 
SerGlyAsnGluValTyrIleAspArgIleGluPheValProAlaGlu 
595600605 
ValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal 
610615620 
AsnGluLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspVal 
625630635640 
ThrAspTyrHisIleAspGlnValSerAsnLeuValGluCysLeuSer 
645650655 
AspGluPheCysLeuAspGluLysLysGluLeuSerGluLysValLys 
660665670 
HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsn 
675680685 
PheArgGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThr 
690695700 
AspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal 
705710715720 
ThrLeuLeuGlyThrPheAspGluCysTyrProThrTyrLeuTyrGln 
725730735 
LysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGlnLeuArg 
740745750 
GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr 
755760765 
AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrp 
770775780 
ProLeuSerAlaProSerProIleGlyLysCysGlyGluProAsnArg 
785790795800 
CysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg 
805810815 
AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle 
820825830 
AspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIle 
835840845 
PheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu 
850855860 
PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLys 
865870875880 
ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGlu 
885890895 
ThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe 
900905910 
ValAsnSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleAlaMet 
915920925 
IleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeu 
930935940 
ProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu 
945950955960 
LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsn 
965970975 
ValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnVal 
980985990 
LysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeu 
99510001005 
ValValProGluTrpGluAlaGluValSerGlnGluValArgValCys 
101010151020 
ProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyr 
1025103010351040 
GlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu 
104510501055 
LeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThr 
106010651070 
ValThrCysAsnAspTyrThrAlaThrGlnGluGluTyrGluGlyThr 
107510801085 
TyrThrSerArgAsnArgGlyTyrAspGlyAlaTyrGluSerAsnSer 
109010951100 
SerValProAlaAspTyrAlaSerAlaTyrGluGluLysAlaTyrThr 
1105111011151120 
AspGlyArgArgAspAsnProCysGluSerAsnArgGlyTyrGlyAsp 
112511301135 
TyrThrProLeuProAlaGlyTyrValThrLysGluLeuGluTyrPhe 
114011451150 
ProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThr 
115511601165 
PheIleValAspSerValGluLeuLeuLeuMetGluGlu 
117011751180 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 88 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE74A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GCAGATCTGGATCCATGCACGCCGTGAAGGGCCCTTCTAGAAGGCCTATCGATAAAGAGC60 
TCCCCGGGGATGGATTGCACGCAGGTTC88 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE72A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
GCGTTAACATGTCGACTCAGAAGAACTCGTCAAGAAGGCG40 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P1(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
GTCGACAAGGATCCAACAATGG22 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P1(b)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
AATTGTCGACAAGGATCCAACAATGG26 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P2(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ACACGCTGACGTCGCGCAGCACG23 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P2(b)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
AGCTACACGCTGACGTCGCGCAG23 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer A1" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
AATTGTCGAC10 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer A2" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
GCGTGTAGCT10 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P3(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
GCTGCGCGACGTCAGCGTGTTCGG24 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P3(b)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
AATTGCTGCGCGACGTCAGCGTG23 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P4(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
GGCGTTGCCCATGGTGCCGTACAGG25 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P4(b)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
AGCTGGCGTTGCCCATGGTGCCG23 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer B1" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
AATTGCTGCG10 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer B2" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
AACGCCAGCT10 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P5(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
TTCCCCCTGTACGGCACCATGGGCAACGCCGC32 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P5(b)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
AATTGTACGGCACCATGGGCAAC23 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P6(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
GAAGCCGGGGCCCTTCACCACGCTGG26 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P6(b)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
AGCTGAAGCCGGGGCCCTTCACC23 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer C1" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
AATTGTACGG10 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer C2 - first half" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
TTCCCCTGTACGG13 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer C1 - second half" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
GGCTTCAGCT10 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer PEPCivs#9 - forward" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
GTACAAAAACCAGCAACTC19 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer PEPCivs#9 reverse" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
CTGCACAAAGTGGAGTAGT19 
(2) INFORMATION FOR SEQ ID NO:52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P7(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
TGGTGAAGGGCCCCGGCTTCACCGG25 
(2) INFORMATION FOR SEQ ID NO:53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer P8(a)" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
ATCATCGATGAGCTCCTACACCTGATCGATGTGGTA36 
(2) INFORMATION FOR SEQ ID NO:54: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer for fourth quarter - 
second half" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
ATCAGGAGCTCATCGATGAT20 
(2) INFORMATION FOR SEQ ID NO:55: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer for third quarter - 
first half" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
TTCCCCCTGTA11 
(2) INFORMATION FOR SEQ ID NO:56: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer MK23A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
GGGGCTGCGGATGCTGCCCT20 
(2) INFORMATION FOR SEQ ID NO:57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer MK25A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
GAGCTGACCCTGACCGTGCT20 
(2) INFORMATION FOR SEQ ID NO:58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer MK26A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
CACCTGATGGACATCCTGAA20 
(2) INFORMATION FOR SEQ ID NO:59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "sequence in pCIB3073 prior 
to deletion" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
TATAAGGATCCCGGGGGCAAGATCTGAGATATG33 
(2) INFORMATION FOR SEQ ID NO:60: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE134A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
CGTGACCGACTACCACATCGATCAAGTATCCAATTTAGTTGAGT44 
(2) INFORMATION FOR SEQ ID NO:61: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE135A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
ACTCAACTAAATTGGATACTTGATCGATGTGGTAGTCGGTCACG44 
(2) INFORMATION FOR SEQ ID NO:62: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE136A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
GCAGATCTGAGCTCTTAGGTACCCAATAGCGTAACGT37 
(2) INFORMATION FOR SEQ ID NO:63: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE137A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
GCTGATTATGCATCAGCCTAT21 
(2) INFORMATION FOR SEQ ID NO:64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE138A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
GCAGATCTGAGCTCTTATTCCTCCATAAGAAGTAATTC38 
(2) INFORMATION FOR SEQ ID NO:65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer MK05A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
CAAAGGTACCCAATAGCGTAACG23 
(2) INFORMATION FOR SEQ ID NO:66: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer MK35A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
AACGAGGTGTACATCGACCG20 
(2) INFORMATION FOR SEQ ID NO:67: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "forward primer for 
pCIB4434" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
GCACCGATATCACCATCCAAGGAGGCGATGACGTATTCAAAG42 
(2) INFORMATION FOR SEQ ID NO:68: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "reverse primer for 
pCIB4434" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
AGCGCATCGATTCGGCTCCCCGCACTTGCCGATTGGACTTGGGGCTGAAAG51 
(2) INFORMATION FOR SEQ ID NO:69: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #1" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
ATTACGTTACGCTATTGGGTACCTTTGATG30 
(2) INFORMATION FOR SEQ ID NO:70: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #2" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
TCCCCGTCCCTGCAGCTGCAGTCTAGGTCCGGGTTCCACTCCAGGTGCGGAGCGCATCGA60 
TTCGGCTCCCCGCACTTGCCGATTGGACTTGGGGCTGA98 
(2) INFORMATION FOR SEQ ID NO:71: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #3" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
CAAGTGCGGGGAGCCGAATCGATGCGCTCCGCACCTGGAGTGGAACCCGGACCTAGACTG60 
CAGCTGCAGGGACGGGGAAAAATGTGCCCATCATTCCC98 
(2) INFORMATION FOR SEQ ID NO:72: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #4" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
TGGTTTCTCTTCGAGAAATTCTAGATTTCC30 
(2) INFORMATION FOR SEQ ID NO:73: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer used to map 
transcriptional start site for TrpA gene" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
CCGTTCGTTCCTCCTTCGTCGAGG24 
(2) INFORMATION FOR SEQ ID NO:74: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: N-terminal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..26 
(D) OTHER INFORMATION: /note= "N-terminal peptide from 
pollen specific protein" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
ThrThrProLeuThrPheGlnValGlyLysGlySerLysProGlyHis 
151015 
LeuIleLeuThrProAsnValAlaThrIle 
2025 
(2) INFORMATION FOR SEQ ID NO:75: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..20 
(D) OTHER INFORMATION: /note= "internal peptide of pollen 
specific protein" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
LysProGlyHisLeuIleLeuThrProAsnValAlaThrIleSerAsp 
151015 
ValValIleLys 
20 
(2) INFORMATION FOR SEQ ID NO:76: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..16 
(D) OTHER INFORMATION: /note= "internal peptide from 
pollen specific protein" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
SerGlyGlyThrArgIleAlaAspAspValIleProAlaAspPheLys 
151015 
(2) INFORMATION FOR SEQ ID NO:77: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "internal peptide from 
pollen specific protein" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
GluHisGlyGlyAspAspPheSerPheThrLeuLys 
1510 
(2) INFORMATION FOR SEQ ID NO:78: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "internal peptide from 
pollen specific protein" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
GluGlyProThrGlyThrTrpThrLeuAspThrLys 
1510 
(2) INFORMATION FOR SEQ ID NO:79: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "oligonucleotide #51" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
AARTCRTCABCACCRTGYTC20 
(2) INFORMATION FOR SEQ ID NO:80: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "oligonucleotide #58" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
CCYTTNCCCACYTGRAA17 
(2) INFORMATION FOR SEQ ID NO:81: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "oligonucleotide PE51" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
TGGCCCATGGCTGCGGCGGGGAACGAGTGCGGC33 
(2) INFORMATION FOR SEQ ID NO:82: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #42" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
AGCGGTCGACCTGCAGGCATGCGATCTGCACCTCCCGCCG40 
(2) INFORMATION FOR SEQ ID NO:83: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #43" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
ATGGGCAAGGAGCTCGGG18 
(2) INFORMATION FOR SEQ ID NO:84: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #SK50" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
CCCTTCAAAATCTAGAAACCT21 
(2) INFORMATION FOR SEQ ID NO:85: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer #SK49" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
TAATGTCGACGAACGGCGAGAGATGGA27 
(2) INFORMATION FOR SEQ ID NO:86: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE99A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 
TGCGGTTACCGCCGATCACATG22 
(2) INFORMATION FOR SEQ ID NO:87: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE97A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 
GCGGTACCGCGTCGACGCGGATCCCGCGGCGGGAAGCTAAG41 
(2) INFORMATION FOR SEQ ID NO:88: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE100A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 
GTCGTCGACCGCAACA16 
(2) INFORMATION FOR SEQ ID NO:89: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE98A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: 
GCGGTACCGCGTTAACGCGGATCCTGTCCGACACCGGAC39 
(2) INFORMATION FOR SEQ ID NO:90: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE104A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: 
GATGTCGTCGACCGCAACAC20 
(2) INFORMATION FOR SEQ ID NO:91: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE103A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: 
GCGGTACCGCGGATCCTGTCCGACACCGGACGGCT35 
(2) INFORMATION FOR SEQ ID NO:92: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE127" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: 
GCGGATCCGGCTGCGGCGGGGAACGA26 
(2) INFORMATION FOR SEQ ID NO:93: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE150A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: 
ATTCGCATGCATGTTTCATTATC23 
(2) INFORMATION FOR SEQ ID NO:94: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer KE151A28" 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 
GCTGGTACCACGGATCCGTCGCTTCTGTGCAACAACC37 
__________________________________________________________________________