Barley .beta. amylase structural gene

The present invention provides a barley .beta.-amylase structural gene consisting of the nucleotide sequence shown in SEQ. ID. No. 1 in attached sequence listing, a barley .beta.-amylase structural gene consisting of the nucleotide sequence shown in SEQ. ID. No. 2 in attached sequence listing, and a plasmid containing the nucleotide sequence shown in SEQ. ID. NO. 1 or 2 in attached sequence listing. The present invention can be utilized in breeding of barley and other plants and industrial production of .beta.-amylase and can contribute in the technical fields of plant breeding, production of brewed products such as beer, production of distilled liquors, food production and enzyme industry.

FIELD OF TECHNOLOGY 
The present invention relates to barley .beta.-amylase structural genes, to 
.beta.-amylase genes subjected to nucleotide substitution in order to 
allow amino acid replacements effective for enhancement of heat stability 
of .beta.-amylase, and to plasmids containing such genes. 
BACKGROUND TECHNOLOGY 
.beta.-Amylase (.alpha.-1,4-glucan maltohydrolase; EC 3.2.1.2) catalyzes 
the liberation of .beta.-maltose from the nonreducing ends of starch and 
related 1,4-.alpha.-glucans. The enzyme has been purified from various 
higher plants, e.g. barley and soybean, and microorganisms, and it is a 
major protein in the starchy endsperm of ungerminated barley seeds. 
Barley .beta.-amylase and soybean .beta.-amylase are known as an enzyme 
useful in industrial production of maltose for infusion and food. 
Barley malt is also known to be used as one of materials for beer or 
distilled liquors, and .beta.-amylase in the malts is known as one of the 
most important enzymes for saccharification of starch at the mashing 
stage. 
As a structural gene of barley .beta.-amylase, a full length cDNA 
consisting of 1754 bases of cultivar Hiproly was reported, and it was 
coded for a polypeptide of 535 amini acids (Eur. J. Biochem., 169, 517 
(1987)). A full length cDNA consisting of 1775 nucleotide cultivar of 
Haruna NIJO (two-rows) was also reported, and it was coded for a 
polypeptide of 535 amini acids (J. Biochem., 115, 47 (1994), Japanese 
Patent Kokai Hei 6-303983). 
In the study of .beta.-amylase of cultivar Haruna NIJO, an expression 
vector (pBETA92) was constructed by deleting 5'-terminal 55 base pairs of 
the full length cDNA and ligating a SmaI linker to form a DNA which was 
then inserted into the SmaI site of a plasmid pKK 223-3 (produced by 
Pharmacia Biotech). 
Also by transforming this expression vector into Escherichia coli JM 109 
(produced by Toyoho), recombinant .beta.-amylase was produced. Recombinant 
.beta.-amylase consisted of 531 amino acids, and had almost the same 
properties as .beta.-amylase from barley (Biosci. Biotech. Biochem., 58, 
1080 (1994), Japanese Patent Kokai Hei 6-303983). 
However, it is not satisfactory that recombinant .beta.-amylase had almost 
same properties as .beta.-amylase from barley, since soybean 
.beta.-amylase is employed more frequently in practice than barley 
.beta.-amylase because of somewhat higher heat stability of the former. 
Accordingly, in order to enhance the utility of barley-derived recombinant 
.beta.-amylase, its thermostability at least equivalent to that of soybean 
.beta.-amylase should be needed. 
As an example of .beta.-amylases whose heat stability was enhanced by 
protein engineering, sevenfold-mutant .beta.-amylase in which methionine 
at 181 position of the original recombinant .beta.-amylase is replaced 
with leucine, serine at 291 position with alanine, isoleucine at 293 
position with valine, serine at 346 position with proline, serine at 347 
position with proline, glutamine at 348 position with aspartic acid and 
alanine at 372 position with serine was proven to have the thermostability 
higher than that of the original recombinant .beta.-amylase (Japanese 
Patent Kokai Hei 6-233086). The original recombinant .beta.-amylase lacked 
four amino acids at positions 2-5 in comparison with the presumed amino 
acid sequence of barley .beta.-amylase. Therefore, amino acid positions 
described above correspond to amino acid positions higher by four in 
barley .beta.-amylase(Biosci. Biotech. Biochem., 58, 1080 (1994), Japanese 
Patent Kokai Hei 6-303983). 
Barley .beta.-amylase genes are only known to be present not only on the 
short arm of chromosome 2 but also on the long arm of chromosome 4 (Genet. 
Res. Camb., 51, 13 (1988)). But no studies of the isolation and sequence 
of a nuclear gene encoding .beta.-amylase have been reported to data. 
Furthermore, new technique of genetic manipulation will be applied in 
addition to conventional mating methods in the field of breeding barley 
cultivars, and .beta.-amylase gene is considered to be a candidate of the 
genes to be introduced to barley cells (Brewers' Guardian, 123, 31 
(1994)). 
When considering mass production of .beta.-amylase gene in barley or 
expression in other plants, not only the .beta.-amylase cDNA which has 
already been isolated but also a nuclear gene containing introns must be a 
leading candidate of the gene to be introduced because of the following 
reason. In chromosomal DNA of an eucaryote there are intervening sequences 
called as introns which are spliced when converted into a mature mRNA and 
thus are not present in a mature mRNA, and there is a possibility that 
these introns play an important role in the expression of a large amount 
of .beta.-amylase in barley or the expression on other plants. It was 
reported that, especially in the plants such as rice, by using a gene 
containing an intron, the amount of the expression in a plant cell was 
increased by 10 times or more ("Creation of dream plants", TOKYO KAGAKU 
DOJIN, page 57 (1994)). 
The present invention was established in view of the subject mentioned 
above, and the object of the invention is to isolate the barley 
.beta.-amylase structural gene containing introns and to provide a plasmid 
comprising such gene. It is another object of the invention to construct a 
gene in which nucleotide substitutions capable of causing amino acid 
replacements serving to enhance heat stability of .beta.-amylase is 
introduced by site-directed mutagenesis, whereby providing a plasmid 
containing such gene. 
The present inventors made much effort to isolate barley .beta.-amylase 
structural gene containing introns to which the method of enhancing heat 
stability disclosed in the former patent application (Japanese Patent 
Kokai Hei 6-233086) (i.e. introduction of nucleotide substitutions by 
site-directed mutagenesis causing amino acid replacements serving to 
enhance heat stability of .beta.-amylase) is applied, and as a result the 
present invention is established. 
Thus, according to the present invention, barley .beta.-amylase structural 
gene containing introns can be sequenced, and can be expressed in a large 
amount in barley or can be expressed also in plants other than barley 
while ligating appropriate promoter or transcription terminator to this 
gene, whereby being utilized to breed barley or other plants. 
In addition, the present invention enables expression of .beta.-amylase 
having enhanced heat stability in barley or other plants by ligating an 
appropriate promoter or transcription terminator to Barley .beta.-amylase 
gene in which amino acid replacements serving to enhance heat stability 
has occurred, whereby being utilized to breed barley or other plants. 
DISCLOSURE OF THE INVENTION 
The first aspect of the present invention is a barley .beta.-amylase 
structural gene consisting of the nucleotide sequence shown in SEQ. ID. 
No. 1 in attached sequence listing. 
The second aspect of the present invention is a barley .beta.-amylase 
structural gene consisting of the nucleotide sequence shown in SEQ. ID. 
No. 2 in attached sequence listing. 
The third aspect of the present invention is a plasmid containing the 
nucleotide sequence shown in SEQ. ID. NO. 1 or 2 in attached sequence 
listing.

BEST MODE TO PRACTICE THE INVENTION 
Practical procedures to construct barley .beta.-amylase structural genes, 
genes coding barley .beta.-amylase having enhanced heat stability and 
plasmids containing such genes are described below. 
(1) Preparation of barley chromosomal DNA 
Barley seeds are germinated in darkness at 20.degree. C. for 7 days in 
vermiculite. From young leaves chromosomal DNA can be isolated in a known 
manner, for example, by the method described in "Cloning and 
sequence--Plant biotechnology experiment manual" (NOSON BUNKASHA, page 252 
(1989)). 
(2) Cloning of barley .beta.-amylase structural gene 
The barley .beta.-amylase structural gene can be cloned using Ex Taq kit 
(produced by Takara Shuzo). DNA obtained is ligated with various linkers 
or adapters using DNA ligases or, alternatively, a restriction enzyme site 
has previously been introduced in primers and amplified DNA is digested 
with the restriction enzyme, and then inserted into an appropriate plasmid 
to construct a recombinant DNA. 
As a host microorganism, any of those in which the recombinant DNA can be 
kept stably and which can proliferates spontaneously may be employed. For 
example, Escherichia coli may be employed. 
The recombinant DNA may be transfered into the host microorganism by a 
known method, for example, employing competent cell method (J. Mol. Biol., 
53, 159 (1970)) when the host microorganism is Escherichia coli. 
(3) Nucleotide sequencing 
DNA sequencing can be done using the chemical modification method by 
MAXAM-GILBERT (Methods in Enzymology, 65, 499 (1980)) or the dideoxy 
method (Gene, 19, 269 (1982)). 
The barley .beta.-amylase amino acid sequence can be deduced from the 
nucleotide sequence determined as described below. 
(4) Nucleotide substitution in .beta.-amylase structural gene 
The nucleotide substitutions can be done by site-directed mutagenesis 
(Anal. Biochem., 200, 81 (1992)). 
In order to utilize the barley .beta.-amylase structural gene in breeding, 
a plasmid containing said gene is constructed and introduced into barley 
or other plants according to a standard manner. To introduce any gene into 
plant cell directly, any of known methods such as electroporation, 
polyethylene glycol method, particle gun method, laser puncture method and 
the like may be employed. As described above, the present invention 
relates to the barley .beta.-amylase structural gene which can be utilized 
in plant breeding and industrial manufacturing of .beta.-amylase, and thus 
is very useful in the technical fields of plant breeding, production of 
brewed products such as beer, production of distilled liquors, food 
production and enzyme industry. 
EXAMPLE 
The present invention is further described in the following examples, by 
which the present invention is not restricted in any way. 
Example 1 
Preparation of Barley Chromosomal DNA 
About 1000 barley seeds (Haruna NIJO) were germinated in darkness at 
20.degree. C. for 7 days in vermiculite. Young leaves obtained (about 65 
g) were cut into pieces of about 1 cm, from which then chromosomal DNA was 
prepared. As a result, about 1 mg of DNA was isolated from 10 g of the 
ypung leaves. 
Example 2 
Cloning of Barley .beta.-amylase Structural Gene 
Polymerase chain reaction (PCR) amplification was used. The reaction 
mixture contained 10 .mu.g of DNA, 20 pmol of each primer, 2.5 mM of dNTP, 
and 2.5 units of Ex Taq DNA polymerase. The 5'-end primer sequence was 
shown in SEQ. ID. No. 3 in the sequence listing and the 3'-end primer 
sequence was shown in SEQ. ID No. 4 in the sequence listing. 
The restriction map of the DNA obtained by PCR amplification is shown in 
FIGURE 1. Open boxes and the boxes with oblique lines indicate exon and 
intron, respectively. 
Example 3 
.beta.-amylase Structural Gene Nucleotide Sequencing 
DNA obtained was digested with various restriction enzymes and were 
subcloned into pUC 118 and pUC 119 for DNA sequencing. DNA sequencing was 
done using the dideoxy method (See SEQ. ID. No. 1 in the sequence 
listing). 
The structural gene was 3825 base pairs in length. In the sequence of the 
structural gene, a sequence identical to that of the cDNA (J. Biochem., 
115, 47 (1994), Japanese Patent Kokai Hei 6-303983) was found to contain 
seven exons and six introns. 
Nucleotide sequence of the structural gene and deduced amino acids are 
shown below (SEQ ID No. 1 and 10). Small letters indicate the sequences of 
introns, and the intron sequences are boxed. Asterisks mark a stop codon. 
##STR1## 
Example 4 
Site-Directed Mutagenesis 
Site-directed mutagenesis was done with transfer site directed mutagenesis 
kit (produced by CLONETECH LABORATORIES). 
Mutagenesis primers employed were: the nucleotide sequence shown in SEQ. 
ID. No. 5 in the sequence listing for substitution of Met at 185 position 
with Leu, the nucleotide sequence shown in SEQ. ID. No. 6 for substitution 
of Ser at 295 position with Ala and Ile at 297 position with Val, the 
nucleotide sequence shown in SEQ. ID. No. 7 for substitution of Ser at 350 
position with Pro, Ser at 351 position with Pro and Gln at 352 position 
with Asp, and the nucleotide sequence shown in SEQ. ID. No. 8 for 
substitution of Ala at 376 position with Ser. 
As a selection primer, the nucleotide sequence shown in SEQ. ID. No. 9 in 
the sequence listing was used. 
With the primers described above the site-directed mutagenesis was done to 
substitute the nucleotides which allows amino acid replacements serving to 
enhance heat stability of .beta.-amylase to occur, whereby obtaining the 
gene coding .beta.-amylase consisting of the nucleotide sequence shown in 
SEQ. ID. No. 2 in the sequence listing. 
Example 5 
Confirmation of Nucleotide Sequence 
When examining the nucleotide sequence of the gene obtained in Example 4, 
as indicated in SEQ. ID. No.2 in the sequence listing, substitution of A 
at 1195 position with T, T at 2699 position with G, A at 2705 position 
with G, AG at 2864 to 2865 positions with CC, T at 2867 position with C, C 
at 2870 position with G, G at 2872 position with C and G at 3118 position 
with T were confirmed. Accordingly, the gene which had been subjected to 
the nucleotide substitution which causes amino acid replacements serving 
to enhance heat stability of .beta.-amylase consisted of the nucleotide 
sequence shown in SEQ. ID. No. 2. 
Corresponding to the nucleotide sequence described above, amino acid 
replacements serving to enhance heat stability of .beta.-amylase was done 
as follows: Met at 185 position with Leu, Set at 295 position with Ala, 
Ile at 297 position with Val, Set at 350 position with Pro, Set at 351 
position with Pro, Gin at 352 position with Asp and Ala at 376 position 
with Set. 
Possibility of Industrial Utilization 
According to the present invention, the nucleotides of the barley 
.beta.-amylase structural genes containing introns were sequenced, and the 
.beta.-amylase genes, in which amino acid replacements serving to enhance 
heat stability of .beta.-amylase has occurred, can be constructed. By 
ligating an appropriate promoter or transcription terminator to such genes 
to construct recombinant plasmids and introducing the plasmids into barley 
or other plants, expression of .beta.-amylase in a large amount in barley 
or expression thereof in other plants and expression of .beta.-amylase 
having enhanced heat stability in barley or other plants are possible. 
Accordingly, the present invention can be utilized in breeding of plants 
and industrial production of .beta.-amylase and can contribute in the 
technical fields of plant breeding, production of brewed products such as 
beer, production of distilled liquors, food production and enzyme 
industry. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 10 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3825 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GATATCCAACAAACCATTTGAAGTTGTAGAGCATCATCCATAGCCAGCATCCACAATGGA60 
GGTGAACGTGAAAGGCAACTATGTCCAAGTCTACGTCATGCTCCCTGTAAGCTCCATCCA120 
TTCAGACCAATCGCTGAGAACCACACACTAAAACTATTTCAAGGATCTAGTGCACACATA180 
TACATTATTGTTGTACATATAACATTGATACTTCTTGTAAAACTCTAATTCAAAGGGTGA240 
AGAACAAGATCTGAGGCCTCAAATGAGTATTTTATTTGTACTAACCTTGACTACACTTCC300 
ATTGTTGAAATAAATAAATAGCTGGACGCCGTGAGCGTGAACAACAGGTTCGAGAAGGGC360 
GACGAGCTGAGGGCGCAATTGAGGAAGCTGGTAGAGGCCGGTGTGGATGGTGTCATGGTA420 
GACGTCTGGTGGGGCTTGGTGGAGGGCAAGGGCCCCAAGGCGTATGACTGGTCCGCCTAC480 
AAGCAGTTGTTTGAGCTGGTGCAGAAGGCTGGGCTGAAGCTACAGGCCATCATGTCGTTC540 
CACCAGTGTGGTGGCAACGTCGGCGACGCCGTCAACATCCCAATCCCACAGTGGGTGCGG600 
GACGTCGGCACGCGTGATCCCGACATTTTCTACACCGACGGTCACGGGACTAGGAACATT660 
GAGTACCTCACTCTTGGAGTTGATAACCAGCCTCTCTTCCATGGAAGATCTGCCGTCCAG720 
GTTACTTTAAACCACCACTCTAGTTCTCTGATGCATATTTATATAGAAGTTCAAGATGAC780 
ACCAAATACAAGCAAAAGGTTAAAGGTGCCAAAAACAGATAAGCAAAGAAACAAAACCTA840 
GCTAATGAAACAGTCTAGAGCCTATCAAAAAAAAAAAAAAAACATCGAGAAGGTGCCTAG900 
AGCGGATGGGTTTCGACAACCCTTTAGCTTTCATGCATCTTTTTGGGAAAGGGTGAAAAA960 
CACCGTCCTTTAAGTCGATTGATGCAGGCAGCCTTCTATTGTTTGTAAGCTATCAGGAAA1020 
TACAAAATTAATAGCTAGTTGTCATTTTAATAGTTGTAGCAAGCTTTGATTCTTCTTTTG1080 
TGGCTGTGACAGATGTATGCCGATTACATGACAAGCTTCAGGGAGAACATGAAAGACTTC1140 
TTGGATGCTGGTGTTATCGTCGACATTGAAGTGGGACTTGGCCCAGCTGGAGAGATGAGG1200 
TACCCATCATATCCTCAGAGCCACGGATGGTCGTTCCCAGGCATCGGAGAATTCATCGTG1260 
AGTGTTTGTTTCCAAACTAATAATCTTTCCTCTTCTGTTCCGATCAAATATAATTTTAGA1320 
TGTAACTCAACATGTGAATATGTGATGGCCAAGTCACGATCTACATTTAGAAAAGTTTTT1380 
TCAGGAAACAAACACCCAAATGAAAAATGATTCTTAAAGGAAAAAAGTGCATGGATAAAA1440 
TGGCAGTTTCAGATTAGGACAAGGCGTGGTAAACCTGACTTGATCATTTCTGTTACCCAA1500 
TATACCCCCGCCCAATTTTTGTTTTTTCTTATTCCTCCCAAATAAGACATCATATAAACT1560 
TGACACATTCGTATTACAATATGTGAAATATATAGGATTTATCTTTGCAACTTAAATACT1620 
TAAATTGACCTTTTTTATTGGAAAAGACTAATTTTATATATTTATGGTACACCAAAAATC1680 
CAAAATGTTTTCGGCACATTGTAGTCTCTATGATTCATTGACCCCACACGTGCGGTTCCC1740 
TCAGGCCTAAACGTGGATTGAAAGTAGGCTGCAATTTTAAAATTTTAAATTTAGTGTGTC1800 
AGGTCTTGAATTTAAGATCTTTTGACTCGGATACCATGTAAAACTGCACCAGACAGTTCA1860 
CCCATAAGCTCTTGCTTATGGGGAAAGGTGGGCTATGCATTTATACTTCAACAATAAAAA1920 
TAGTGATTTAGCACAAACCTTGATGCAAGGCTAGATGACACAGATGTGTGTGTGTGTGTG1980 
TGTGGGGGGGGGGGGGGGGGTGATGCACCTGAATCCGAGCTCAGGAAGTTTGGCACTACT2040 
TTTCCCTTCCGGGAGACCATATATGTTATTGCTTTGAGCAAAGTATCATAGCAAATACAA2100 
GACCTTCTTAAAATACATGACATGAAATAGTTAAAACAAATGCACGATAATATATACCAT2160 
TGCCATTACAGAAAAATGGCTCTACTTAACTGTTTGAACTAATAGTACAAATAAAAATAA2220 
AATTGCAGTGCTATGATAAATACCTACAAGCAGACTTCAAAGCAGCAGCAGCGGCGGTCG2280 
GCCATCCTGAGTGGGAATTTCCTAACGATGCCGGACAGTACAATGACACTCCCGAGAGAA2340 
CTCAATTCTTCAGGGACAACGGGACATACCTAAGTGAGAAGGGGAGGTTTTTCCTTGCAT2400 
GGTACTCCAACAATCTGATCAAGCACGGTGACAGGATCTTGGATGAAGCAAACAAGGTCT2460 
TCTTGGGATACAAGGTGCAATTGGCAATCAAGGTATAAGCACTTTCATGCCTCCTAAAGA2520 
TCTCGGTTTATTACTACAGTAGTAGATAGGATTTGAGAAACCATGATTCAGTTGAGAAGT2580 
TGTGTATGATAAACAACAAAAAAAATACACAAACTATCCAGGCTAAGGGAACTCGCATTG2640 
CTTAATAGCTAGAATGTAAATGAGACATGGCCGGCCAAATAATGTTTGGTTGCAGATCTC2700 
TGGCATTCACTGGTGGTACAAGGTTCCAAGCCATGCAGCCGAGCTCACAGCTGGGTACTA2760 
TAACTTACATGATAGAGACGGCTACAGAACCATAGCACGCATGCTCAAAAGGCACCGTGC2820 
TAGCATTAACTTCACTTGCGCGGAGATGAGGGATTCGGAGCAAAGCTCGCAGGCGATGAG2880 
CGCACCAGAAGAACTAGTCCAACAGGTAGGTAATAACTTATGCGTTCAGATATATTACGC2940 
TTATATATCTACGTATATACTATGATGGAAACACCTTTTTCTTTAGAAAAGGAGGCTTAG3000 
CCCCGGCCTCTGCATCGAAAGATGCATACGGCCATGATGATGGAAACACCTAAATCACTT3060 
GTCGTCAAAATAATTTCTCAGGTGTTGAGTGCTGGATGGAGAGAGGGCCTAAATGTGGCA3120 
TGCGAAAACGCGCTTCCACGATATGATCCAACTGCTTACAACACCATACTCAGGAATGCG3180 
AGGCCTCATGGAATCAACCAGAGCGGCCCTCCTGAGCACAAGCTGTTTGGATTCACCTAC3240 
CTTCGGCTGTCGAATCAGCTGGTGGAGGGACAAAACTATGTCAACTTCAAGACCTTTGTC3300 
GACAGAATGCATGCCAACCTGGTTAGTGCCACAACCACTTACTAACGCATGTCAAAAATT3360 
AAACATATACAAGAACCATTTGTTGATTTGCAGGTGCCTATTATATACTAATAATTTAAT3420 
TTTATTGTTTTCAGCCTCGTGACCCATATGTTGATCCAATGGCGCCTTTGCCAAGATCAG3480 
GGCCAGAAATATCGATTGAGATGATCCTACAAGCAGCACAGCCAAAACTGCAGCCATTCC3540 
CCTTCCAGGAGCACACCGACCTGCCAGTAGGCCCTACTGGTGGCATGGGTGGGCAGGCTG3600 
AAGGCCCCACCTGTGGCATGGGTGGGCAAGTTAAAGGCCCTACTGGTGGCATGGGTGGGC3660 
AGGCTGAAGACCCTACTAGTGGCATGGGTGGGGAGCTCCCTGCCACCATGTAATGGAACC3720 
TTTATGATTTACTACCCTTTATGTTGTGTGTGAGTGTGACAGAGAAACCTTTCTCTGCCT3780 
TATTAATAATAAATAAAGCACATCACTTGTGTGTGTTCTGAAAAG3825 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3825 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GATATCCAACAAACCATTTGAAGTTGTAGAGCATCATCCATAGCCAGCATCCACAATGGA60 
GGTGAACGTGAAAGGCAACTATGTCCAAGTCTACGTCATGCTCCCTGTAAGCTCCATCCA120 
TTCAGACCAATCGCTGAGAACCACACACTAAAACTATTTCAAGGATCTAGTGCACACATA180 
TACATTATTGTTGTACATATAACATTGATACTTCTTGTAAAACTCTAATTCAAAGGGTGA240 
AGAACAAGATCTGAGGCCTCAAATGAGTATTTTATTTGTACTAACCTTGACTACACTTCC300 
ATTGTTGAAATAAATAAATAGCTGGACGCCGTGAGCGTGAACAACAGGTTCGAGAAGGGC360 
GACGAGCTGAGGGCGCAATTGAGGAAGCTGGTAGAGGCCGGTGTGGATGGTGTCATGGTA420 
GACGTCTGGTGGGGCTTGGTGGAGGGCAAGGGCCCCAAGGCGTATGACTGGTCCGCCTAC480 
AAGCAGTTGTTTGAGCTGGTGCAGAAGGCTGGGCTGAAGCTACAGGCCATCATGTCGTTC540 
CACCAGTGTGGTGGCAACGTCGGCGACGCCGTCAACATCCCAATCCCACAGTGGGTGCGG600 
GACGTCGGCACGCGTGATCCCGACATTTTCTACACCGACGGTCACGGGACTAGGAACATT660 
GAGTACCTCACTCTTGGAGTTGATAACCAGCCTCTCTTCCATGGAAGATCTGCCGTCCAG720 
GTTACTTTAAACCACCACTCTAGTTCTCTGATGCATATTTATATAGAAGTTCAAGATGAC780 
ACCAAATACAAGCAAAAGGTTAAAGGTGCCAAAAACAGATAAGCAAAGAAACAAAACCTA840 
GCTAATGAAACAGTCTAGAGCCTATCAAAAAAAAAAAAAAAACATCGAGAAGGTGCCTAG900 
AGCGGATGGGTTTCGACAACCCTTTAGCTTTCATGCATCTTTTTGGGAAAGGGTGAAAAA960 
CACCGTCCTTTAAGTCGATTGATGCAGGCAGCCTTCTATTGTTTGTAAGCTATCAGGAAA1020 
TACAAAATTAATAGCTAGTTGTCATTTTAATAGTTGTAGCAAGCTTTGATTCTTCTTTTG1080 
TGGCTGTGACAGATGTATGCCGATTACATGACAAGCTTCAGGGAGAACATGAAAGACTTC1140 
TTGGATGCTGGTGTTATCGTCGACATTGAAGTGGGACTTGGCCCAGCTGGAGAGATGAGG1200 
TACCCATCATATCCTCAGAGCCACGGATGGTCGTTCCCAGGCATCGGAGAATTCATCGTG1260 
AGTGTTTGTTTCCAAACTAATAATCTTTCCTCTTCTGTTCCGATCAAATATAATTTTAGA1320 
TGTAACTCAACATGTGAATATGTGATGGCCAAGTCACGATCTACATTTAGAAAAGTTTTT1380 
TCAGGAAACAAACACCCAAATGAAAAATGATTCTTAAAGGAAAAAAGTGCATGGATAAAA1440 
TGGCAGTTTCAGATTAGGACAAGGCGTGGTAAACCTGACTTGATCATTTCTGTTACCCAA1500 
TATACCCCCGCCCAATTTTTGTTTTTTCTTATTCCTCCCAAATAAGACATCATATAAACT1560 
TGACACATTCGTATTACAATATGTGAAATATATAGGATTTATCTTTGCAACTTAAATACT1620 
TAAATTGACCTTTTTTATTGGAAAAGACTAATTTTATATATTTATGGTACACCAAAAATC1680 
CAAAATGTTTTCGGCACATTGTAGTCTCTATGATTCATTGACCCCACACGTGCGGTTCCC1740 
TCAGGCCTAAACGTGGATTGAAAGTAGGCTGCAATTTTAAAATTTTAAATTTAGTGTGTC1800 
AGGTCTTGAATTTAAGATCTTTTGACTCGGATACCATGTAAAACTGCACCAGACAGTTCA1860 
CCCATAAGCTCTTGCTTATGGGGAAAGGTGGGCTATGCATTTATACTTCAACAATAAAAA1920 
TAGTGATTTAGCACAAACCTTGATGCAAGGCTAGATGACACAGATGTGTGTGTGTGTGTG1980 
TGTGGGGGGGGGGGGGGGGGTGATGCACCTGAATCCGAGCTCAGGAAGTTTGGCACTACT2040 
TTTCCCTTCCGGGAGACCATATATGTTATTGCTTTGAGCAAAGTATCATAGCAAATACAA2100 
GACCTTCTTAAAATACATGACATGAAATAGTTAAAACAAATGCACGATAATATATACCAT2160 
TGCCATTACAGAAAAATGGCTCTACTTAACTGTTTGAACTAATAGTACAAATAAAAATAA2220 
AATTGCAGTGCTATGATAAATACCTACAAGCAGACTTCAAAGGAGCAGCAGCGGCGGTCG2280 
GCCATCCTGAGTGGGAATTTCCTAACGATGCCGGACAGTACAATGACACTCCCGAGAGAA2340 
CTCAATTCTTCAGGGACAACGGGACATACCTAAGTGAGAAGGGGAGGTTTTTCCTTGCAT2400 
GGTACTCCAACAATCTGATCAAGCACGGTGACAGGATCTTGGATGAAGCAAACAAGGTCT2460 
TCTTGGGATACAAGGTGCAATTGGCAATCAAGGTATAAGCACTTTCATGCCTCCTAAAGA2520 
TCTCGGTTTATTACTACAGTAGTAGATAGGATTTGAGAAACCATGATTCAGTTGAGAAGT2580 
TGTGTATGATAAACAACAAAAAAAATACACAAACTATCCAGGCTAAGGGAACTCGCATTG2640 
CTTAATAGCTAGAATGTAAATGAGACATGGCCGGCCAAATAATGTTTGGTTGCAGATCTC2700 
TGGCATTCACTGGTGGTACAAGGTTCCAAGCCATGCAGCCGAGCTCACAGCTGGGTACTA2760 
TAACTTACATGATAGAGACGGCTACAGAACCATAGCACGCATGCTCAAAAGGCACCGTGC2820 
TAGCATTAACTTCACTTGCGCGGAGATGAGGGATTCGGAGCAAAGCTCGCAGGCGATGAG2880 
CGCACCAGAAGAACTAGTCCAACAGGTAGGTAATAACTTATGCGTTCAGATATATTACGC2940 
TTATATATCTACGTATATACTATGATGGAAACACCTTTTTCTTTAGAAAAGGAGGCTTAG3000 
CCCCGGCCTCTGCATCGAAAGATGCATACGGCCATGATGATGGAAACACCTAAATCACTT3060 
GTCGTCAAAATAATTTCTCAGGTGTTGAGTGCTGGATGGAGAGAGGGCCTAAATGTGGCA3120 
TGCGAAAACGCGCTTCCACGATATGATCCAACTGCTTACAACACCATACTCAGGAATGCG3180 
AGGCCTCATGGAATCAACCAGAGCGGCCCTCCTGAGCACAAGCTGTTTGGATTCACCTAC3240 
CTTCGGCTGTCGAATCAGCTGGTGGAGGGACAAAACTATGTCAACTTCAAGACCTTTGTC3300 
GACAGAATGCATGCCAACCTGGTTAGTGCCACAACCACTTACTAACGCATGTCAAAAATT3360 
AAACATATACAAGAACCATTTGTTGATTTGCAGGTGCCTATTATATACTAATAATTTAAT3420 
TTTATTGTTTTCAGCCTCGTGACCCATATGTTGATCCAATGGCGCCTTTGCCAAGATCAG3480 
GGCCAGAAATATCGATTGAGATGATCCTACAAGCAGCACAGCCAAAACTGCAGCCATTCC3540 
CCTTCCAGGAGCACACCGACCTGCCAGTAGGCCCTACTGGTGGCATGGGTGGGCAGGCTG3600 
AAGGCCCCACCTGTGGCATGGGTGGGCAAGTTAAAGGCCCTACTGGTGGCATGGGTGGGC3660 
AGGCTGAAGACCCTACTAGTGGCATGGGTGGGGAGCTCCCTGCCACCATGTAATGGAACC3720 
TTTATGATTTACTACCCTTTATGTTGTGTGTGAGTGTGACAGAGAAACCTTTCTCTGCCT3780 
TATTAATAATAAATAAAGCACATCACTTGTGTGTGTTCTGAAAAG3825 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AAGGATCCGATATCCAACAAACCATTTG28 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AAGGATCCCTTTTCAGAACACACACAAG28 
(2) INFORMATION FOR SEQ ID NO:5: 
(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 = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AGCTGGAGAGTTGAGGTACCC21 
(2) INFORMATION FOR SEQ ID NO:6: 
(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 = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GTTGCAGATCGCTGGCGTTCACTGGTG27 
(2) INFORMATION FOR SEQ ID NO:7: 
(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 = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AGGGATTCGGAGCAACCCCCGGACGCGATGAGCGCACCA39 
(2) INFORMATION FOR SEQ ID NO:8: 
(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 = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CCTAAATGTGTCATGCGAAAA21 
(2) INFORMATION FOR SEQ ID NO:9: 
(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 = "SYNTHETIC DNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTGCATGCCTCCAGGTCGACT21 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 535 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
MetGluValAsnValLysGlyAsnTyrValGlnValTyrValMetLeu 
151015 
ProLeuAspAlaValSerValAsnAsnArgPheGluLysGlyAspGlu 
202530 
LeuArgAlaGlnLeuArgLysLeuValGluAlaGlyValAspGlyVal 
354045 
MetValAspValTrpTrpGlyLeuValGluGlyLysGlyProLysAla 
505560 
TyrAspTrpSerAlaTyrLysGlnLeuPheGluLeuValGlnLysAla 
65707580 
GlyLeuLysLeuGlnAlaIleMetSerPheHisGlnCysGlyGlyAsn 
859095 
ValGlyAspAlaValAsnIleProIleProGlnTrpValArgAspVal 
100105110 
GlyThrArgAspProAspIlePheTyrThrAspGlyHisGlyThrArg 
115120125 
AsnIleGluTyrLeuThrLeuGlyValAspAsnGlnProLeuPheHis 
130135140 
GlyArgSerAlaValGlnMetTyrAlaAspTyrMetThrSerPheArg 
145150155160 
GluAsnMetLysAspPheLeuAspAlaGlyValIleValAspIleGlu 
165170175 
ValGlyLeuGlyProAlaGlyGluMetArgTyrProSerTyrProGln 
180185190 
SerHisGlyTrpSerPheProGlyIleGlyGluPheIleCysTyrAsp 
195200205 
LysTyrLeuGlnAlaAspPheLysAlaAlaAlaAlaAlaValGlyHis 
210215220 
ProGluTrpGluPheProAsnAspAlaGlyGlnTyrAsnAspThrPro 
225230235240 
GluArgThrGlnPhePheArgAspAsnGlyThrTyrLeuSerGluLys 
245250255 
GlyArgPhePheLeuAlaTrpTyrSerAsnAsnLeuIleLysHisGly 
260265270 
AspArgIleLeuAspGluAlaAsnLysValPheLeuGlyTyrLysVal 
275280285 
GlnLeuAlaIleLysIleSerGlyIleHisTrpTrpTyrLysValPro 
290295300 
SerHisAlaAlaGluLeuThrAlaGlyTyrTyrAsnLeuHisAspArg 
305310315320 
AspGlyTyrArgThrIleAlaArgMetLeuLysArgHisArgAlaSer 
325330335 
IleAsnPheThrCysAlaGluMetArgAspSerGluGlnSerSerGln 
340345350 
AlaMetSerAlaProGluGluLeuValGlnGlnValLeuSerAlaGly 
355360365 
TrpArgGluGlyLeuAsnValAlaCysGluAsnAlaLeuProArgTyr 
370375380 
AspProThrAlaTyrAsnThrIleLeuArgAsnAlaArgProHisGly 
385390395400 
IleAsnGlnSerGlyProProGluHisLysLeuPheGlyPheThrTyr 
405410415 
LeuArgLeuSerAsnGlnLeuValGluGlyGlnAsnTyrValAsnPhe 
420425430 
LysThrPheValAspArgMetHisAlaAsnLeuProArgAspProTyr 
435440445 
ValAspProMetAlaProLeuProArgSerGlyProGluIleSerIle 
450455460 
GluMetIleLeuGlnAlaAlaGlnProLysLeuGlnProPheProPhe 
465470475480 
GlnGluHisThrAspLeuProValGlyProThrGlyGlyMetGlyGly 
485490495 
GlnAlaGluGlyProThrCysGlyMetGlyGlyGlnValLysGlyPro 
500505510 
ThrGlyGlyMetGlyGlyGlnAlaGluAspProThrSerGlyMetGly 
515520525 
GlyGluLeuProAlaThrMet 
530535 
__________________________________________________________________________