Crucifer AFT proteins and uses thereof

Purified DNA encoding crucifer AFT proteins and chimeric transcriptional activator proteins from such DNA are disclosed. Such proteins are also involved in plant defense mechanisms by interacting with proteins involved in protecting plants from pathogens. The recombinant polypeptides and fragments are useful in methods of modulating plant gene expression.

BACKGROUND OF THE INVENTION 
This invention relates to recombinant plant nucleic acids and polypeptides. 
Improved means to manipulate plant gene expression is desired for a variety 
of industrial, agricultural, and commercial food uses. To produce new 
plant varieties, it is necessary to change the genetic makeup of the crop 
or plant in question. Desirable genes have to be incorporated into the 
crop or plant, and undesirable genes have to be eliminated or replaced. In 
other words, one needs to genetically engineer the plant to meet the 
demands of agriculture. Accordingly, genetic engineering of crop plants 
necessitates methods of identifying potentially valuable genes and 
transferring these to the crop that one desires to improve. 
SUMMARY OF THE INVENTION 
We have identified and describe herein a novel plant transcriptional 
activator from the crucifer, Arabidopsis thaliana. In addition to its role 
as a transcriptional activator, we have also determined that this protein 
plays a role in plant defense mechanisms by interacting with proteins, 
e.g., 3-O-methyltransferase and ascorbate peroxidase, involved in 
protecting plants from pathogens. We named this protein AFT1 (Arabidopsis 
Fourteen-Three-three 1) because it shows sequence homology to the 
widespread 14-3-3 protein family. 
The AFT1 protein provides a means to enhance, control, modify or otherwise 
alter plant gene expression, e.g, as a transcription activator or as a 
chimeric transcriptional activator, or even to modulate events during 
plant cell-signalling processes, e.g., signal transduction events involved 
in plant defense responses to pathogens such as fungi, nematodes, insects, 
bacteria, and viruses. Of special interest are the nucleic acid sequences 
corresponding to not only other AFT1 proteins found in the plant kingdom, 
but also sequences corresponding to proteins which interact with AFT1 
during plant signal transduction events, e.g., those pathways which 
operate during a plant's response to a pathogen, for applications in 
genetic engineering, especially as related to agricultural biotechnology. 
Accordingly, in general, the invention features recombinant AFT1 
polypeptides, preferably, including an amino acid sequence substantially 
identical to the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). The 
invention also features a recombinant polypeptide which is a fragment or 
analog of an AFT1 polypeptide that includes a domain capable of activating 
transcription, e.g., AFT1 (34-248)(SEQ ID NO: 27) or AFT1 (122-248)(SEQ ID 
NO: 28). Transcription activation may be assayed, for example, according 
to the methods described herein. 
In various preferred embodiments, the polypeptide is derived from a plant 
(e.g., a monocot or dicot), and preferably from a crucifer such as 
Arabidopsis. 
In a second aspect, the invention features a chimeric AFT1 transcriptional 
activation protein including an AFT1 polypeptide fused to a DNA-binding 
polypeptide. In preferred embodiments, the DNA-binding polypeptide 
includes, without limitation, Gal4 or LexA. 
In a third aspect, the invention features a transgenic plant containing a 
transgene comprising an AFT1 protein operably linked to a constitutive 
(e.g., the 35S CaMV promoter) or regulated or inducible promoter (e.g., 
rbcS promoter). In other related aspects, the invention also features a 
transgenic plant containing a transgene containing a chimeric AFT1 
transcriptional activator protein. In related aspects, the invention 
features a seed and a cell from a transgenic plant containing the AFT1 
protein, fragment or analog, or a chimeric AFT1 transcriptional activator 
protein. 
In a fourth aspect, the invention features a transgenic plant expressing a 
polypeptide of interest which involves: (a) a nucleic acid sequence 
encoding a chimeric AFT1 transcriptional activator protein; and (b) a 
nucleic acid sequence encoding a polypeptide of interest in an expressible 
genetic construction, wherein the binding of the chimeric protein 
regulates the expression of the polypeptide of interest. In preferred 
embodiments the polypeptide of interest is, without limitation, a storage 
protein, e.g., napin, legumin, or phaseolin, or any other protein of 
agricultural significance. 
In a fifth aspect, the invention features substantially pure DNA (for 
example, genomic DNA, cDNA, or synthetic DNA) encoding an AFT1 protein. 
Accordingly, the invention features a nucleotide sequence substantially 
identical to the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1). In 
related aspects, the invention also features substantially pure DNA 
encoding a recombinant polypeptide including an amino acid sequence 
substantially identical to the amino acid sequence of AFT1 polypeptide 
shown in FIG. 1 (SEQ ID NO: 2). Such DNA may, if desired, be operably 
linked to a constitutive or regulated or inducible promoter as described 
herein. In preferred embodiments, the DNA sequence is from a crucifer 
(e.g., Arabidopsis). In related aspects, the invention also features a 
vector, a cell (e.g., a plant cell), and a transgenic plant or seed 
thereof which includes such substantially pure AFT1 DNA. In various 
preferred embodiments, the cell is a prokaryotic cell, for example, E. 
coli or Agrobacterium, or more preferably, a eukaryotic cell, for example, 
a transformed plant cell derived from a cell of a transgenic plant. 
In a sixth aspect, the invention features a recombinant polypeptide which 
is a fragment or analog of an AFT1 polypeptide (SEQ ID NO: 2) including a 
domain capable of interacting with a plant defense related protein. 
Preferably, the polypeptide is AFT1(33-194)(SEQ ID NO: 29). In related 
aspects, the invention also features substantially pure DNA encoding an 
AFT1 polypeptide fragment or analog, preferably the DNA is substantially 
identical to the DNA sequence shown in FIG. 1 (SEQ ID NO: 1). In other 
aspects, the DNA is operably linked to a constitutive or regulated or 
inducible promoter. 
By "crucifer" is meant any plant that is classified within the Cruciferae 
family as commonly described in, e.g., Gray's Manual of Botany American 
Book Company, N.Y., 1950; Hortus Third: A Concise Dictionary of Plants 
Cultivated in the U.S. and Canada, Macmillan, 1976; or Simmons, N.W., 
Evolution of Crop Plants, 1986. The Cruciferae include many agricultural 
crops, including, broccoli, cabbage, brussel sprouts, rapeseed, kale, 
Chinese kale, cauliflower, horseradish, and Arabidopsis. 
By "AFT1" is meant a crucifer polypeptide capable of effecting 
transcriptional activation or interacting with a polypeptide involved with 
a plant defense polypeptide. Such an AFT1 polypeptide has the sequence 
shown in FIG. 1 (SEQ ID NO.: 1). 
By "protein" and "polypeptide" is meant any chain of amino acids, 
regardless of length or post-translational modification (e.g., 
glycosylation or phosphorylation). 
By "substantially identical" is meant a polypeptide or nucleic acid 
exhibiting at least 90%, preferably 93%, more preferably 95%, and most 
preferably 97% homology to a reference amino acid or nucleic acid 
sequence. For polypeptides, the length of comparison sequences will 
generally be at least 16 amino acids, preferably at least 20 amino acids, 
more preferably at least 25 amino acids, and most preferably 35 amino 
acids. For nucleic acids, the length of comparison sequences will 
generally be at least 50 nucleotides, preferably at least 60 nucleotides, 
more preferably at least 75 nucleotides, and most preferably 110 
nucleotides. 
Homology is typically measured using sequence analysis software (e.g., 
Sequence Analysis Software Package of the Genetics Computer Group, 
University of Wisconsin Biotechnology Center, 1710 University Avenue, 
Madison, Wis. 53705). Such software matches similar sequences by assigning 
degrees of homology to various substitutions, deletions, substitutions, 
and other modifications. Conservative substitutions typically include 
substitutions within the following groups: glycine, alanine; valine, 
isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; 
serine, threonine; lysine, arginine; and phenylalanine, tyrosine. 
By a "substantially pure polypeptide" is meant an AFT1 protein which has 
been separated from components which naturally accompany it. Typically, 
the polypeptide is substantially pure when it is at least 60%, by weight, 
free from the proteins and naturally-occurring organic molecules with 
which it is naturally associated. Preferably, the preparation is at least 
75%, more preferably at least 90%, and most preferably at least 99%, by 
weight, AFT1 polypeptide. A substantially pure AFT1 polypeptide may be 
obtained, for example, by extraction from a natural source (e.g., a plant 
cell); by expression of a recombinant nucleic acid encoding an AFT1 
polypeptide; or by chemically synthesizing the protein. Purity can be 
measured by any appropriate method, e.g., those described in column 
chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. 
A protein is substantially free of naturally associated components when it 
is separated from those contaminants which accompany it in its natural 
state. Thus, a protein which is chemically synthesized or produced in a 
cellular system different from the cell from which it naturally originates 
will be substantially free from its naturally associated components. 
Accordingly, substantially pure polypeptides include those derived from 
eukaryotic organisms but synthesized in E. coli or other prokaryotes. 
By "substantially pure DNA" is meant DNA that is free of the genes which, 
in the naturally-occurring genome of the organism from which the DNA of 
the invention is derived, flank the gene. The term therefore includes, for 
example, a recombinant DNA which is incorporated into a vector; into an 
autonomously replicating plasmid or virus; or into the genomic DNA of a 
prokaryote or eukaryote; or which exists as a separate molecule (e.g., a 
cDNA or a genomic or cDNA fragment produced by PCR or restriction 
endonuclease digestion) independent of other sequences. It also includes a 
recombinant DNA which is part of a hybrid gene encoding additional 
polypeptide sequence. 
By "transformed cell" is meant a cell into which (or into an ancestor of 
which) has been introduced, by means of recombinant DNA techniques, a DNA 
molecule encoding (as used herein) an AFT1 protein or an AFT1 chimeric 
transcriptional activator. 
By "promoter" is meant a DNA sequence sufficient to direct transcription; 
such elements may be located in the 5' or 3' regions of the gene. By 
"constitutive" promoter is meant a promoter capable of mediating gene 
expression without regulation, i.e., the promoter is always 
transcriptionally active. By "regulated or inducible" promoter is meant a 
promoter capable of mediating gene expression in response to a variety of 
developmental (e.g., cell-specific, tissue-specific, and organ-specific 
promoters), environmental, and hormonal cues including, but not limited 
to, promoters such as the rbcS, wunI, chlorophyll a/b, or E.sub.2 
promoters described herein. 
By "operably linked" is meant that a gene and a regulatory sequence(s) 
(e.g., a promoter) are connected in such a way as to permit gene 
expression when the appropriate molecules (e.g., transcriptional activator 
proteins) are bound to the regulatory sequence(s). 
By "plant cell" is meant any self-propagating cell bounded by a 
semi-permeable membrane and containing a plastid. Such a cell also 
requires a cell wall if further propagation is desired. Plant cell, as 
used herein includes, without limitation, algae, cyanobacteria, seeds 
suspension cultures, embryos, meristematic regions, callus tissue, leaves, 
roots, shoots, gametophytes, sporophytes, pollen, and microspores. 
By "transgene" is meant any piece of DNA which is inserted by artifice into 
a cell, and becomes part of the genome of the organism which develops from 
that cell. Such a transgene may include a gene which is partly or entirely 
heterologous (i.e., foreign) to the transgenic organism, or may represent 
a gene homologous to an endogenous gene of the organism. 
By "transgenic" is meant any cell which includes a DNA sequence which is 
inserted by artifice into a cell and becomes part of the genome of the 
organism which develops from that cell. As used herein, the transgenic 
organisms are generally transgenic plants and the DNA (transgene) is 
inserted by artifice into either the nuclear or plastidic genome. 
By "plant defense related protein" is meant any protein which is involved 
in the protection or resistance to plant pests (e.g., bacteria, insects, 
nematodes, fungi, and viruses). Such proteins include, without limitation, 
3-O-methyltransferases, ascorbate peroxidases, chalcone synthases, 
hydroxyproline rich glycoproteins, glucanases, chitanases, and proteinase 
inhibitors. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof, and from the 
claims.

POLYPETIDES ACCORDING TO THE INVENTION 
Polypeptides according to the invention include the entire Arabidopsis AFT1 
protein (as described in FIG. 1; SEQ ID No: 2). These polypeptides are 
used, e.g., to manipulate plant gene expression at the transcriptional 
level (as discussed infra) or to manipulate the plant signal transduction 
pathway by providing plants with the potential of resisting pathogens such 
as fungi, insects, nematodes, bacteria, and viruses. Polypeptides of the 
invention also include any analog or fragment of the Arabidopsis AFT1 
protein capable of activating transcription in a host plant. The efficacy 
of an AFT1 analog or fragment to activate transcription is dependent upon 
its ability to interact with the transcription complex; such an 
interaction may be readily assayed using any number of standard in vivo 
methods, e.g., the interaction trap mechanism described infra. Similarly, 
the polypeptides of the invention include chimeric AFT1 transcriptional 
activator proteins capable of selectively activating transcription of a 
specified gene. 
Specific AFT1 analogs of interest include full-length or partial (described 
infra) AFT1 proteins, including amino acid sequences which differ only by 
conservative amino acid substitutions, for example, substitutions of one 
amino acid for another of the same class (e.g., valine for glycine, 
arginine for lysine, etc.) or by one or more non-conservative amino acid 
substitutions, deletions, or insertions at positions of the amino acid 
sequence which will not destroy AFT1's ability to activate transcription 
(e.g., as assayed infra). 
Specific AFT1 fragments of interest include any portions of the AFT1 
protein which are capable of interaction with an AFT1 ligand, e.g., a 
member of the transcriptional complex or a protein involved in plant 
defense mechanisms, such as 3-O-methyltransferase, and ascorbate 
peroxidase. Identification of such ligands may be readily assayed using 
any number of standard in vivo methods, e.g., the interaction trap 
mechanism described infra. 
There now follows a description of the cloning and characterization of an 
Arabidopsis AFT-encoding cDNA useful in the instant invention, and a 
characterization of its ability to activate transcription, and its protein 
interacting properties. This example is provided for the purpose of 
illustrating the invention and should not be construed as limiting. 
Isolation of an Arabidopsis Gene Encodidng an AFT Protein 
The Arabidopsis AFT1 gene was isolated as follows. A yeast interaction trap 
system (Zervos et al., Cell 72: 223-232, 1993; Gyuris et al., Cell 75: 
791-803, 1993) was modified for the isolation of an Arabidopsis AFT 
protein. The yeast strain EGY48 (MATa trp1 ura3 his3 LEU2::plexAop6-LEU2) 
containing a plasmid pJK103 (Zervos et al., supra) that directs expression 
of a Gall-lacZ gene from two high affinity ColE1 LexA operators, was used 
in the interaction trap experiment. A "bait" (LexA/AKR1-261, residues 
1-261 of AKRP (Arabidopsis anKyrin repeat protein) fused to DNA binding 
protein LexA) was introduced into the strain and then an Arabidopsis cDNA 
expression library was introduced (see, e.g., Zhang et al., Plant Cell 4: 
1575-1588, 1992). Selection was first carried out on leucine minus plates, 
and Leu.sup.+ colonies were analyzed on X-gal plates. The clones which 
activated transcription of reporter genes in the presence of, but not in 
the absence of, the LexA protein or its fusion derivatives were isolated. 
The oligo(dT)-primed activation-tagged cDNA expression library in vector 
pJG4-5 (Gyuris et al., supra) was made from mRNA of four week-old 
Arabidopsis leaves. The yeast strain EGY48, the vector plasmids pJG4-5 and 
pEG202, and the plasmids pHM1--1, pHM7-3, pHM12, pHM.o slashed., and 
pSH18-34 were provided by Dr. Roger Brent. The LexA/AKR fusion proteins 
were constructed as follows. The oligonucleotides used to amplify desired 
AKR fragments which were later subcloned into pEG202 are shown below. 
__________________________________________________________________________ 
OAB-9: 
GCGGAATTCATGAGGCCCATTAAAATT 
(SEQ ID NO: 3) 
OAB-10: 
GTAGGATCCGGTCGGATTTCTTGTCGC 
(SEQ ID NO: 4) 
OAB-11: 
CGCGAATTCAATAGCGACAAGTACGAT 
(SEQ ID NO: 5) 
OAB-12: 
GTAGGATCCGTCTCTCTTCCAAGGTAGA 
(SEQ ID NO: 6) 
OAB-20: 
GATCCTAGAATTCAAGAAGAATCGGCGTGGC 
(SEQ ID NO: 7) 
__________________________________________________________________________ 
The combination of oligonucleotides used for fusion proteins are: OAB-9 and 
OAB-10 (LexA/AKR1-261); OAB-11 and OAB-12 (LexA/AKR249-434); OAB-20 and 
OAB-12 (LexA/AKR114-434). Normally, with this technique, a library that 
expresses cDNA-encoded proteins fused to a transcription activator domain 
(B42) is introduced into a special yeast strain. This strain also contains 
a plasmid which directs constitutive production of a transcriptionally 
inert LexA fusion protein which is called the "bait" (LexA fused to the 
protein of interest) and two reporter genes. The transcription of these 
two reporter genes can be stimulated if the cDNA-encoded protein complexes 
with the bait. One reporter gene LEU2 allows growth in the absence of 
leucine and the other reporter gene LacZ codes for .beta.-galactosidase. 
We found that many proteins encoded by Arabidopsis cDNAs activated 
transcription with LexA protein alone, or with many different baits, 
although all of these proteins required a LexA binding domain. This 
results in the isolation of cDNA clones which are not true interaction 
partners of the "bait" and requires further analysis to separate these 
"false positive" clones from the desired partner clones. Examples of 
activation by AFT1 which is dependent upon the presence of LexA are shown 
in FIG. 2. To further understand such activation, we characterized 81 cDNA 
clones which encoded proteins capable of activating the expression of the 
reporter genes. Among the cDNAs sequenced, 36 clones were derived from the 
same gene which encodes a 14-3-3-like protein. This gene was named AFT1 
(Arabidopsis Fourteen-Three-three 1), and the protein AFT1 encodes is 
designated as AFT1. AFT1 contains 248 amino acids with a molecular weight 
of about 28 kD. 
Transcription Activation by AFT1 
A series of experiments were performed to determine which AFT1 sequences 
were required for transcriptional activation in the yeast interaction trap 
system. Accordingly, a series of deletion constructs were made and 
analyzed according to methods known in the art as follows. To test 
activation by B42/AFT1 fusion proteins, a series of AFT1 derivatives fused 
to B42 in the plasmid pJG4-5 were constructed. These plasmids were 
introduced into the strain EGY48 containing the plasmid pEG202 which 
directs the constitutive production of LexA protein and the plasmid 
pSH18-34 which contains the LexAop-LacZ reporter gene. To test activation 
by LexA/AFT1 fusion proteins, a series of AFT1 derivatives were fused to 
LexA in the plasmid pEG202 were constructed and were introduced into the 
strain EGY48 containing the plasmid pSH18-34. Transcription activation by 
AFT1 and its derivatives was measured by the growth of yeast on leucine 
minus plates and the activity of .beta.-galactosidase. The assay for 
.beta.-galactosidase was conducted as described by Zervos et al., supra. 
The oligonucleotides used to amplify desired AFT1 fragments which were 
later subcloned into pJG4-5 and pEG202 are shown below. 
__________________________________________________________________________ 
JW-5: 
CTGACTGAATTCATGGCGGCGACATTAGG 
(SEQ ID NO: 8 
JW-6: 
GACTGAGTCGACCCTTCATCTAGATCCTC 
(SEQ ID NO: 9) 
JW-7: 
GACTGACTCGAGCCTTCATCTAGATCCTCA 
(SEQ ID NO: 10) 
JW-8: 
CTGACTGAATTCGAGTCTAAGGTCTTTAC 
(SEQ ID NO: 11) 
JW-9: 
GACTGACTCGAGACTCGCTCCAGCAGATGG 
(SEQ ID NO: 12) 
JW-10: 
GACTGACTCGAGTGAAGAATTGAGAATCTC 
(SEQ ID NO: 13) 
JW-11: 
GACTGAGTCGACACTCGCTCCAGCAGATGG 
(SEQ ID NO: 14) 
JW-12: 
GACTGAGTCGACTGAAGAATTGAGAATCTC 
(SEQ ID NO: 15) 
JW-13: 
CTGACTGAATTCGTTACAGGCGCTACTCCAG 
(SEQ ID NO: 16) 
__________________________________________________________________________ 
The combinations of oligonucleotides used for fusion proteins were: JW-5 
and JW-6 (LexA/1-248); JW-5 and JW-12 (LexA/1-194); JW-5 and JW-11 
(LexA/1-121); JW-13 and JW-6 (LexA/34-248); JW-8 and JW-6 (LexA/122-248); 
JW-5 and JW-7 (B42/1-248); JW-5 and JW-9 (B42/1-121); JW-13 and JW-7 
(B42/34-248); JW-8 and JW-7 (B42/122-248); JW-13 and JW-10 (B42/34-194). 
Results from such experiments revealed that deletion of the C-terminal half 
of AFT1 (B42/1-121) completely abolished AFT1's ability to activate, 
whereas deletion of either 33 or 121 residues from the N-terminus 
(B42/34-248 and B42/122-248) increased activation (FIG. 3A). The reason 
for the increased activation is not known, but might be due to the 
tertiary structures of these two fusion proteins (B42/34-248 and 
B42/122-248) which could result in stronger interactions with the 
transcriptional machinery. Nevertheless, it is the C-terminal half that is 
responsible for the observed activation when AFT1 is fused to B42, e.g., 
AFT1 residues 34-248 (SEQ ID NO: 27) and 122-248 (SEQ ID NO: 28). However, 
since B42 is an activator domain, the observed transcription activation 
may be due to the direct interaction of AFT1 with LexA, thereby bringing 
B42 into the proximity of the reporter gene promoter. An alternate 
possibility is suggested by the acidic nature of AFT1 (pI=4.6), namely, 
AFT1 itself might be a transcription activator, since it shares this 
acidic feature with many transcription activators. 
AFT1 was also fused directly to LexA to test if AFT1 can activate 
transcription. The results shown in FIG. 3B demonstrate that AFT1 does 
activate transcription. To determine which portion of AFT1 was important 
for activation, 54 amino acids were deleted from the AFT C-terminus 
(LexA/1-194). This deletion caused AFT1 to lose its ability to activate 
completely; whereas deletion of 33 amino acids from the N-terminus, 
(LexA/34-248) decreased activation by about 75%. As shown in Panel B of 
FIG. 3, when the N-terminal half of AFT1 (LexA/122-248) was deleted, 
activation dropped to basal levels. Thus, even though the C-terminal half 
is critical for activation and is more acidic than the N-terminal half, 
the N-terminal half also plays a role in activation. 
AFT1 Copy Number 
The copy number of the AFT1 gene was determined by genomic DNA (Southern) 
blot analysis. Genomic DNA was prepared according to the method of 
Dellaporta et al. (Plant Mol. Biol. Rep. 4: 19-21, 1983), digested with 
restriction enzymes, electrophoresed (5 .mu.g per lane), blotted to a 
Biotrans.TM. Nylon membrane, and hybridized with labeled ATF1 cDNA clone. 
Hybridizations were carried out according to the method of Church and 
Gilbert (Proc. Natl. Acad. Sci. USA 81: 1991-1995, 1984) using probes 
labeled by random priming. The washing conditions were as follows: two 
times (10 minutes each) in 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), and 
5.0% SDS at 63.degree. C.; then four times (5 minutes each) in 1 mM EDTA, 
40 mM NaHPO4 (pH 7.2), and 1% SDS at 63.degree. C. The condition for 
deprobing filters was as follows: two times (15 minutes each) in 2 mM Tris 
(pH 8.2), 2 mM EDTA (pH 8.0), and 0.1% SDS at 70.degree. C. for DNA blots 
and at 80.degree. C. for RNA blots. 
As shown in FIG. 4, digestion of two ecotypes (Columbia and Landsberg) of 
Arabidopsis DNA with the enzymes, Bgl II and Hind III, gave rise to two 
bands after the DNA blot was probed with a labelled AFT1 cDNA sequence. 
These data indicate that only one copy of AFT1 was present in both 
ecotypes of Arabidopsis, since there was one restriction site for Bgl II 
and one site for Hind III within the AFT1 cDNA, respectively. 
Developmental Expression Pattern of the AFT1 Gene in Arabidopsis 
The developmental and organ-specific expression of AFT1, as well as the 
light regulation of AFT1 expression, were studied by RNA (Northern blot) 
analysis. Total RNA was isolated according to the method of Logemann et 
al. (Anal. Biochem. 163: 16-20, 1987), separated by electrophoresis (15 
.mu.g per lane), blotted to a Biotrans.TM. Nylon membrane, and hybridized 
to the labeled AFT1 cDNA clone and the Arabidopsis Lhca2 cDNA clone. The 
conditions for hybridization and washing were the same as described in 
genomic Southern analysis supra. RNAs were extracted from Arabidopsis 
grown either in a greenhouse (16 hr light/8 hr dark at 
25.degree..+-.5.degree. C.) or on agarose plates in a tissue culture room 
(16 hr light/8 hr dark at 20.degree..+-.2.degree. C.). Greenhouse-grown 
plants were used for developmental expression analyses. Leaves were 
harvested weekly for RNA preparation. Greenhouse-grown plants were also 
used for light induction experiments. At four weeks, plants were moved to 
a dark chamber for three days, then shifted back to light. Leaves were 
then harvested every two hours. Tissue culture-grown plants were used for 
organ-specific expression analyses. Leaf, root, and stem mRNAs were 
isolated from plants grown for 35 days on agarose plate in MS media 
supplemented with 1% sucrose, and the flower and silique mRNAs were 
isolated from plants grown for 35 days in the greenhouse. The MS was 
purchased from Sigma (Cat# M-0153). As shown in FIG. 5, Panel A and Table 
I, when total RNAs isolated from leaves of one to five week-old plants 
were hybridized to a labelled AFT1 cDNA, the steady-state m/RNA level of 
AFT1 did not change significantly over a five week period. 
When RNAs isolated from different organs were analyzed, the steady-state 
mRNA level in silique was found to be about one fifth of that in flower, 
whereas the mRNA levels in leaves, roots, and stems were about the same 
(FIG. 5, Panel B; Table I). It should be noted that the mRNA levels from 
flowers and siliques are not directly comparable to those from leaves, 
roots, and stems (FIG. 5, Panel B), because they were from materials grown 
under different conditions (as described supra). However, the steady-state 
mRNA levels of flower and silique can be compared to that of five-week-old 
leaves shown in FIG. 5, Panel A. The quantitative data indicate that the 
AFT1 mRNA level in leaves is about two times higher than that in flowers 
and nine times higher than that in siliques (Table I, infra). The growth 
conditions can affect the steady-state mRNA level since greenhouse-grown 
plants contained three times more AFT1 mRNA than plate-grown plants (FIGS. 
5, Panels A and B; Table I, infra). These data indicate that although AFT1 
expression is probably required throughout much of the Arabidopsis life 
cycle, its steady-state mRNA level is still regulated organ-specifically. 
Furthermore, dark-adapted plants contain at least two times more 
steady-state mRNA than plants grown in light (FIG. 5, Panel C, Table I, 
infra), suggesting that light plays a role in the down-regulation of AFT1 
expression. 
The relative intensities of AFT1 mRNA derived from the data in FIG. 5 are 
shown below in Table I. The relative intensity data were collected from 
.beta.-scanning of RNA gel blots by a Blot Analyzer, and normalized using 
the intensity of the 18s RNA band. 
TABLE I 
__________________________________________________________________________ 
A. Developmental Expression.sup.a 
Time (in weeks): 
One Two Three 
Four 
Five 
Relative Intensity of AFT1: 
41 45 58 38 36 
B. Organ-specific Expression.sup.b 
Organs: Leaf 
Root 
Stem 
Flower 
Silique 
Relative Intensity of AFT1: 
11 11 12 19 4 
C. Light Regulation.sup.c 
Time (in hours): 
Zero 
Two Four 
Six Eight 
Ten 
Relative Intensity of Lhca2: 
0.2 0.24 
1.6 3.2 3.9 6.5 
Relative Intensity of AFT1: 
132 49 39 34 38 44 
__________________________________________________________________________ 
.sup.a and .sup.c : RNAs from greenhousegrown plants; 
.sup.b : RNAs of leaf, root, and stem from plategrown plants, RNAs of 
flower and silique from greenhousegrown plants. 
We have shown that the AFT1 gene of Arabidopsis encodes a novel protein 
which can activate transcription in yeast. Accordingly, we conclude that 
AFT1 functions as a transcriptional activator. 
Chimeric AFT1 Proteins as Targeted Transcriptional Activators 
Since plant gene expression varies in accordance with developmental stages 
of different cell types and in response to different environmental factors 
and hormonal cues, the proteins (including the gene regulatory sequences) 
of the present invention are most useful for applications aimed at 
improving or engineering plant varieties of agricultural or commercial 
interest. 
Accordingly, the invention, in general terms, also involves the 
construction of and use of novel chimeric AFT1 proteins capable of 
selectively activating transcription of a specified gene, e.g., a crucifer 
storage protein such as napin. Targeted transcription of a gene is 
acquired by imbuing the AFT1 transcriptional activator with the ability to 
selectively activate a specific gene by fusing it to a DNA-binding domain 
which is capable of binding to the 5' upstream regulatory region, e.g., in 
the vicinity of the transcription start site. Such chimeric proteins 
contain two parts: the AFT1 transcriptional activation region (described 
supra) and a DNA binding domain that is directed to or specific for the 
transcriptional initiation region of interest. For example, a chimeric 
AFT1 transcriptional activator protein may be produced by fusing a Gal4 
DNA binding region (see, e.g., Ma et al. Nature, 334: 631-633, 1988; Ma et 
al. Cell 48: 847-853, 1988) to the transcriptional activating portion of 
AFT1 according to methods known in the art (e.g., see Sadowski et al., 
Nature 335: 563-564, 1988). 
Importantly, the gene of interest, e.g., a napin storage protein gene, 
placed under the transcriptional control of an AFT1 chimeric activator 
must include the appropriate DNA recognition sequence in its 5' upstream 
region. For example, to activate napin gene expression with a Gal4-AFT1 
protein, the napin gene should contain a 5' GAL4 upstream activation 
sequence (UAS). Construction of such clones is well known in the art and 
is discussed infra. Moreover, those skilled in the art will easily 
recognize that the DNA binding domain component of the chimeric activator 
protein may be derived from any appropriate eukaryotic or prokaryotic 
source. Thus, fusion genes encoding chimeric AFT1 transcriptional 
activator proteins can be constructed which include virtually any DNA 
binding domain and the AFT1 transcriptional activator provided that the 
gene placed under the transcriptional control of the AFT1 chimeric 
activator contains the requisite DNA regulatory sequences which 
facilitates its binding. Such chimeric AFT1 transcriptional activator 
proteins are capable of activating transcription efficiently in transgenic 
plants (plasmid construction discussed infr). Furthermore, cells 
expressing such chimeric AFT1 transcriptional activator proteins, e.g., 
AFT1/Gal4, are capable of specifically activating and overexpressing the 
desired gene product. 
To identify effective chimeric AFT1 transcriptional activator proteins in 
vivo or in vitro, functional analyses are performed. Such assays may be 
carried out using transiently transformed plant cells or transgenic plants 
harboring the appropriate transgenes, e.g., an AFT1/Gal4 transcriptional 
activator and a storage protein promoter region containing the requisite 
Gal4 DNA binding sequences, according to standard methods (see, e.g., 
Gelvin et al., supra). 
To identify particularly useful combinations, i.e., chimeric AFT1 
activators and its cognate genes, plasmids are constructed and analyzed in 
either transient assays or in vivo in transgenic plants. Construction of 
chimeric transgenes is by standard methods (see, e.g., Ausubel et al, 
supra). The wild-type promoter of a specific gene, e.g., the crucifer 
napin storage protein, containing the regulatory region the appropriate 
DNA-binding sequence, e.g., Gal4, is fused to a reporter gene, for 
example, the .beta.-glucuronidase gene (GUS) (see, e.g., Jefferson, Plant. 
Mol. Biol. Rep. 316: 387, 1987) in a plant expression vector and 
introduced into a host by any established method (as described infra) 
along with the cognate AFT1 chimeric transcriptional activator expression 
construct. By "reporter gene" is meant a gene whose expression may be 
assayed; such genes include, without limitation, .beta.-glucuronidase 
(GUS), luciferase, chloramphenicol transacetylase (CAT), and 
.beta.-galactosidase. In one particular example, the expression vector is 
transformed into Agrobacterium followed by transformation of the plant 
material, e.g., leaf discs (see, e.g., Gelvin et al. infra). Regenerated 
shoots are selected on medium containing, e.g., kanamycin. After rooting, 
transgenic plantlets are transferred to soil and grown in a growth room. 
Primary transformants are then assayed for chimeric AFT1-induced GUS 
activity either by quantitating GUS activity or by histochemical staining 
as described below. Untransformed plants are taken as controls. 
Fluorometric analysis of GUS activity can be performed in any plant cell 
protoplast or transgenic plant according to standard methodologies. 
Alternatively, preparations of crude plant extracts can be assayed as 
described, e.g., by Jefferson (supra), using extracts standardized for 
protein concentration (see, e.g., Bradford, Anal. Biochem. 72: 248, 1976). 
GUS levels in different plant tissues are assayed by enzymatic conversion 
of 4-methylumbelliferyl glucuronide to 4-methylumbelliferone, which is 
quantified with a fluorimeter (e.g., Perkin-Elmer LS 2B, Norwalk, Conn.). 
Typically, the fluorimeter is set at 455 nm emission and 365 nm excitation 
wavelengths. GUS activity is generally expressed as picomoles per 
milligram of protein per minute (see, e.g., Jefferson supra). 
Alternatively, GUS activity can be assayed by in situ histochemical 
staining, e.g., as follows. Whole tissues and thin sections from 
transgenic plants and untransformed control plant tissue can be stained by 
incubation with 5-bromo-4-chloro-3-indoyl .beta.-D-glucuronic acid 
(X-gluc; Research Organics, Inc., Cleveland Ohio) as described by 
Jefferson et al (EMBO J 6: 3901, 1987) and Gallagher (GUS Protocols, 
1992). Tissue sections are incubated at 37.degree. C. in 2 mM X-gluc in 
0.1M NaPO.sub.4 (pH 7.0), and then sectioned. GUS activity in a 
transformed plant is easily identified by the presence of an indigo blue 
precipitate within the cells expressing the reporter gene. Stained 
material is optionally examined microscopically using bright-field and 
dark-field optics. AFT1 Interactinq Proteins 
Other properties of the AFT1 protein can be explored by modifying the 
interaction trap system described supra. For example, proteins which 
interact with AFT1 can be isolated and identified. To this end, we used a 
LexA and partial AFT1 fusion protein as a bait (LexA/AFT1 33-194, i.e., 
AFT1 residues 33-194 (SEQ ID NO: 29) fused to LexA) to search for proteins 
capable of interacting with AFT1. We identified five novel cDNAs showing 
sequence homology to several plant genes, including plant defense related 
gene products, e.g., 3-O-methyltransferase (see, e.g., Poeydomenge et al. 
Plant Physiol. 105: 749-750, 1994 and Jaek et al., Mol. Plant-Microbe 
Interactions 5: 294-300, 1992) and ascorbate peroxidase (see, e.g., 
Mittler et al., Plant J. 5: 397-405, 1994; Mehdy, Plant Physiol. 105: 
467-472, 1994), the proteasome gene product (see, e.g., Haffter et al., 
Nucleic Acids Res. 19: 5075, 1991), and an ankryin repeating protein gene 
product, AKR.sub.2. The nucleotide sequences for these cDNAs are shown in 
FIGS. 6 (SEQ ID NO: 17), 8 (SEQ ID NO: 19), 10 (SEQ ID NO: 21), 12 (SEQ ID 
NO: 23), and 14 (SEQ ID NO: 25). The deduced amino acid sequences coded 
for by these cDNAs are shown in FIGS. 7 (SEQ ID NO: 18), 9 (SEQ ID NO: 
20), 11 (SEQ ID NO: 22), 13 (SEQ ID NO: 24), and 15 (SEQ ID NO: 26). 
AFT1 Polypeptide Expression 
Polypeptides according to the invention may be produced by transformation 
of a suitable host cell with all or part of an AFT1 cDNA (e.g., the cDNA 
described above) in a suitable expression vehicle or with a plasmid 
construct designed to express the chimeric AFT1 transcriptional activator 
protein supra. 
Those skilled in the field of molecular biology will understand that any of 
a wide variety of expression systems may be used to provide the 
recombinant protein. The precise host cell used is not critical to the 
invention. The AFT1 protein or chimeric activator protein may be produced 
in a prokaryotic host, e.g., E. coli, or in a eukaryotic host, e.g., 
Saccharomyces cerevisiae, mammalian cells (e.g., COS 1 or NIH 3T3 cells), 
or any of a number of plant cells including, without limitation, algae, 
tree species, ornamental species, temperate fruit species, tropical fruit 
species, vegetable species, legume species, monocots, dicots, or in any 
plant of commercial or agricultural significance. Particular examples of 
suitable plant hosts include Chlamydomonas, Conifers, Petunia, Tomato, 
Potato, Tobacco, Arabidopsis, Lettuce, Sunflower, Oilseed rape, Flax, 
Cotton, Sugarbeet, Celery, Soybean, Alfalfa, Medicago, Lotus, Vigna, 
Cucumber, Carrot, Eggplant, Cauliflower, Horseradish, Morning Glory, 
Poplar, Walnut, Apple, Asparagus, Rice, Corn, Millet, Onion, Barley, 
Orchard grass, Oat, Rye, and Wheat. 
Such cells are available from a wide range of sources including: the 
American Type Culture Collection (Rockland, Md.); Chlamydomonas Culture 
Collection, (Duke University), Durham, N.C.; or from any of a number seed 
companies, e.g., W. Atlee Burpee Seed Co. (Warminster, Pa.), Park Seed Co. 
(Greenwood, S.C.), Johnny Seed Co. (Albion, Me.), or Northrup King Seeds 
(Harstville, S.C.). Descriptions and sources of useful host cells are also 
found in Vasil I.K., Cell Culture and Somatic Cell Genetics of Plants, Vol 
I, II, III Laboratory Procedures and Their Applications Academic Press, 
New York, 1984; Dixon, R. A., Plant Cell Culture-A Practical Approach, IRL 
Press, Oxford University, 1985; Green et al., Plant Tissue and Cell 
Culture, Academic Press, New York, 1987; Gasser and Fraley, Science 244: 
1293, 1989. 
For prokaryotic expression, DNA encoding an AFT1 polypeptide of the 
invention is carried on a vector operably linked to control signals 
capable of effecting expression in the prokaryotic host. If desired, the 
coding sequence may contain, at its 5' end, a sequence encoding any of the 
known signal sequences capable of effecting secretion of the expressed 
protein into the periplasmic space of the host cell, thereby facilitating 
recovery of the protein and subsequent purification. Prokaryotes most 
frequently used are various strains of E. coli; however, other microbial 
strains may also be used. Plasmid vectors are used which contain 
replication origins, selectable markers, and control sequences derived 
from a species compatible with the microbial host. Examples of such 
vectors may be found in Pouwels et al. (supra) or Ausubel et al. (supra). 
Commonly used prokaryotic control sequences (also referred to as 
"regulatory elements") are defined herein to include promoters for 
transcription initiation, optionally with an operator, along with ribosome 
binding site sequences. Promoters commonly used to direct protein 
expression include the beta-lactamase (penicillinase), the lactose (lac) 
(Chang et al., Nature 198: 1056, 1977), the tryptophan (Trp) (Goeddel et 
al., Nucl. Acids Res. 8: 4057, 1980) and the tac promoter systems as well 
as the lambda-derived P.sub.L promoter and N-gene ribosome binding site 
(Simatake et al., Nature 292: 128, 1981). 
For eukaryotic expression, the method of transformation or transfection and 
the choice of vehicle for expression of the AFT1 polypeptide or chimeric 
activator protein will depend on the host system selected. Transformation 
and transfection methods are described, e.g., in Ausubel et al. (supra); 
Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic 
Press, 1989; Gelvin et al., Plant Molecular Biology Manual, Kluwer 
Academic Publishers, 1990; Kindle, K., Proc. Natl. Acad. Sci., USA 87: 
1228, 1990; Potrykus, I., Annu. Rev. Plant Physiol. Plant Mol. Biology 42: 
205, 1991; and BioRad (Hercules, Calif.) Technical Bulletin #1687 
(Biolistic Particle Delivery Systems). Expression vehicles may be chosen 
from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. 
Pouwels et al., 1985, Supp. 1987); Gasser and Fraley (supra); Clontech 
Molecular Biology Catalog (Catalog 1992/93 Tools for the Molecular 
Biologist, Palo Alto, Calif.); and the references cited above. 
One preferred eukaryotic expression system is the mouse 3T3 fibroblast host 
cell transfected with a pMAMneo expression vector (Clontech, Palo Alto, 
Calif.). pMAMneo provides: an RSV-LTR enhancer linked to a 
dexamethasone-inducible MMTV-LTR promotor, an SV40 origin of replication 
which allows replication in mammalian systems, a selectable neomycin gene, 
and SV40 splicing and polyadenylation sites. DNA encoding an AFT1 
polypeptide would be inserted into the pMAMneo vector in an orientation 
designed to allow expression. The recombinant AFT1 protein would be 
isolated as described below. Other preferable host cells which may be used 
in conjunction with the pMAMneo expression vehicle include COS cells and 
CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively). 
Alternatively, an AFT1 polypeptide is produced by a stably-transfected 
mammalian cell line. A number of vectors suitable for stable transfection 
of mammalian cells are available to the public, e.g., see Pouwels et al. 
(supra); methods for constructing such cell lines are also publicly 
available, e.g., in Ausubel et al. (supra). In one example, cDNA encoding 
the AFT1 polypeptide is cloned into an expression vector which includes 
the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, 
therefore, the AFT1-encoding gene into the host cell chromosome is 
selected for by inclusion of 0.01-300 .mu.M methotrexate in the cell 
culture medium (as described in Ausubel et al., supra). This dominant 
selection can be accomplished in most cell types. Recombinant protein 
expression can be increased by DHFR-mediated amplification of the 
transfected gene. Methods for selecting cell lines bearing gene 
amplifications are described in Ausubel et al. (supra); such methods 
generally involve extended culture in medium containing gradually 
increasing levels of methotrexate. DHFR-containing expression vectors 
commonly used for this purpose include pCVSEII-DHRF and pAdD26SV(A) 
(described in Ausubel et al., supra). Any of the host cells described 
above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR.sup.+ 
cells, ATCC Accession No. CRL 9096) are among the host cells preferred 
for DHFR selection of a stably-transfected cell line or DHFR-mediated gene 
amplification. 
Most preferably, an AFT1 polypeptide or AFT1 chimeric transcriptional 
activator is produced by a stably-transfected plant cell line or by a 
transgenic plant. A number of vectors suitable for stable transfection of 
plant cells or for the establishment of transgenic plants are available to 
the public; such vectors are described in Pouwels et al. (supra), 
Weissbach and Weissbach (supra), and Gelvin et al. (supra). Methods for 
constructing such cell lines are described in, e.g., Weissbach and 
Weissbach (supra), and Gelvin et al. (Supra). Typically, plant expression 
vectors include (1) a cloned plant gene under the transcriptional control 
of 5' and 3' regulatory sequences and (2) a dominant selectable marker. 
Such plant expression vectors may also contain, if desired, a promoter 
regulatory region (e.g., one conferring inducible or constitutive, 
environmentally- or developmentally-regulated, or cell- or tissue-specific 
expression), a transcription initiation start site, a ribosome binding 
site, an RNA processing signal, a transcription termination site, and/or a 
polyadenylation signal. 
Once the desired AFT1 nucleic acid sequences is obtained it may be 
manipulated in a variety of ways known in the art. For example, where the 
sequence involves non-coding flanking regions, the flanking regions maybe 
subjected to mutagenesis. 
The AFT1 DNA sequence of the invention may, if desired, be combined with 
other DNA sequences in a variety of ways. The AFT1 DNA sequence of the 
invention may be employed with all or part of the gene sequences normally 
associated with the AFT1 protein. In its component parts a DNA sequence 
encoding an AFT1 protein is combined in the DNA construct having a 
transcription initiation control region capable of promoting transcription 
and translation in a host cell. 
In general, the constructs will involve regulatory regions functional in 
plants which provide for modified production of AFT1 protein or a chimeric 
AFT1 protein as discussed Supra. The open reading frame coding for the 
AFT1 protein or functional fragment thereof will be joined at its 5' end 
to a transcription initiation regulatory region such as the sequence 
naturally found in the 5' upstream region of the AFT1 structural gene. 
Numerous other transcription initiation regions are available which 
provide for constitutive or inducible regulation. 
For applications when developmental, hormonal or environmental expression 
is desired appropriate 5' upstream non-coding regions are obtained from 
other genes; for example, from genes regulated during seed development, 
embryo development, or leaf development. 
Regulatory transcript termination regions may be also be provided in DNA 
constructs of this invention as well. Transcript termination regions may 
be provided by the DNA sequence encoding the AFT1 protein or any 
convenient transcription termination region derived from a different gene 
source, especially the transcript termination region which is normally 
associated with the transcript initiation region. The transcript 
termination region will contain preferably at least 1 kb, preferably about 
3 kb of sequence 3' to the structurally gene from which the termination 
region is derived. Plant expression constructs having AFT1 as the DNA 
sequence of interest for expression thereof may be employed with a wide 
variety of plant life, particularly plant life involved in the production 
of seed storage proteins or storage lipids, useful for industrial and 
agricultural applications. Importantly, this invention is applicable to 
dicotyledons and monocotyledons, and will be readily applicable to any new 
or improved transformation or regeneration method. 
An example of a useful plant promoter according to the invention is a 
caulimovirus promoter, e.g., a cauliflower mosaic virus (CaMV) promoter. 
These promoters confer high levels of expression in most plant tissues, 
and the activity of these promoters is not dependent on virally encoded 
proteins. CaMV is a source for both the 35S and 19S promoters. In most 
tissues of transgenic plants, the CaMV 35S promoter is a strong promoter 
(see, e.g., Odell et al., Nature 313: 810, 1985). The CaMV promoter is 
also highly active in monocots (see, e.g., Dekeyser et al., Plant Cell 2: 
591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220: 389, 1990). 
Moreover, activity of this promoter can be further increased (i.e., 
between 2-10 fold) by duplication of the CaMV 35S promoter (see e.g., Kay 
et al., Science 236: 1299, 1987; Ow et al., Proc. Natl. Acad. Sci., USA 
84: 4870, 1987; and Fang et al., Plant Cell 1: 141, 1989). 
Other useful plant promoters include, without limitation, the nopaline 
synthase promoter (An et al., Plant Physiol. 88: 547, 1988) and the 
octopine synthase promoter (Fromm et al., Plant Cell 1: 977, 1989). 
For certain applications, it may be desirable to produce the AFT1 gene 
product in an appropriate tissue, at an appropriate level, or at an 
appropriate developmental time. Thus, there are an assortment of gene 
promoters, each with its own distinct characteristics embodied in its 
regulatory sequences, shown to be regulated in response to the 
environment, hormones, and/or developmental cues. These include gene 
promoters that are responsible for (1) heat-regulated gene expression 
(see, e.g., Callis et al., Plant Physiol. 88: 965, 1988), (2) 
light-regulated gene expression (e.g., the pea rbcS-3A described by 
Kuhlemeier et al., Plant Cell 1: 471, 1989; the maize rbcS promoter 
described by Schaffner and Sheen, Plant Cell 3: 997, 1991; or the 
cholorphyll a/b-binding protein gene found in pea described by Simpson et 
al., EMBO J. 4: 2723, 1985), (3) hormone-regulated gene expression (e.g., 
the abscisic acid responsive sequences from the Em gene of wheat described 
by Marcotte et al., Plant Cell 1: 969, 1989), (4) wound-induced gene 
expression (e.g., of wunI described by Siebertz et al., Plant Cell 1: 961, 
1989), or (5) organ-specific gene expression (e.g., of the tuber-specific 
storage protein gene described by Roshal et al., EMBO J. 6: 1155, 1987; 
the 23-kDa zein gene from maize described by Schernthaner et al., EMBO J. 
7: 1249, 1988; or the French bean .beta.-phaseolin gene described by 
Bustos et al., Plant Cell 1: 839, 1989). 
Plant expression vectors may also optionally include RNA processing 
signals, e.g, introns, which have been shown to be important for efficient 
RNA synthesis and accumulation (Callis et al., Genes and Dev. 1: 1183, 
1987). The location of the RNA splice sequences can dramatically influence 
the level of transgene expression in plants. In view of this fact, an 
intron may be positioned upstream or downstream of a AFT1 
polypeptide-encoding sequence in the transgene to modulate levels of gene 
expression. 
In addition to the aforementioned 5' regulatory control sequences, the 
expression vectors may also include regulatory control regions which are 
generally present in the 3' regions of plant genes (Thornburg et al., 
Proc. Natl. Acad. Sci. USA 84: 744, 1987; An et al., Plant Cell 1: 115, 
1989). For example, the 3' terminator region may be included in the 
expression vector to increase stability of the mRNA. One such terminator 
region may be derived from the PI-II terminator region of potato. In 
addition, other commonly used terminators are derived from the octopine or 
nopaline synthase signals. 
The plant expression vector also typically contains a dominant selectable 
marker gene used to identify those cells that have become transformed. 
Useful selectable genes for plant systems include genes encoding 
antibiotic resistance genes, for example, those encoding resistance to 
hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin. 
Genes required for photosynthesis may also be used as selectable markers 
in photosynthetic-deficient strains. Finally, genes encoding herbicide 
resistance may be used as selectable markers; useful herbicide resistance 
genes include the bar gene encoding the enzyme phosphinothricin 
acetyltransferase and conferring resistance to the broad spectrum 
herbicide Basta.RTM. (Hoechst AG, Frankfurt, Germany). 
Efficient use of selectable markers is facilitated by a determination of 
the susceptibility of a plant cell to a particular selectable agent and a 
determination of the concentration of this agent which effectively kills 
most, if not all, of the transformed cells. Some useful concentrations of 
antibiotics for tobacco transformation include, e.g., 75-100 .mu.g/ml 
(kanamycin), 20-50 .mu.g/ml (hygromycin), or 5-10 .mu.g/ml (bleomycin). A 
useful strategy for selection of transformants for herbicide resistance is 
described, e.g., by Vasil et al., supra. 
It should be readily apparent to one skilled in the art of molecular 
biology, especially in the field of plant molecular biology, that the 
level of gene expression is dependent, not only on the combination of 
promoters, RNA processing signals and terminator elements, but also on how 
these elements are used to increase the levels of selectable marker gene 
expression. 
Plant Transformation 
Upon construction of the plant expression vector, several standard methods 
are accessible for introduction of the recombinant genetic material into 
the host plant for the generation of a transgenic plant. These methods 
include (1) Agrobacterium-mediated transformation (A. tumefaciens or A. 
rhizogenes) (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, 
vol 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C. 
P., and Draper, J,. In: DNA Cloning, Vol II, D. M. Glover, ed, Oxford, IRI 
Press, 1985), (2) the particle delivery system (see, e.g., Gordon-Kamm et 
al., Plant Cell 2: 603, 1990; or BioRad Technical Bulletin 1687, supra), 
(3) microinjection protocols (see, e.g., Green et al., supra), (4) 
polyethylene glycol (PEG) procedures (see, e.g., Draper et al., Plant Cell 
Physiol. 23: 451, 1982; or e.g., Zhang and Wu, Theor. Appl. Genet. 76: 
835, 1988), (5) liposome-mediated DNA uptake (see, e.g., Freeman et al., 
Plant Cell Physiol. 25: 1353, 1984), (6) electroporation protocols (see, 
e.g., Gelvin et al., supra; Dekeyser et al., supra; or Fromm et al., 
Nature 319: 791, 1986), and (7) the vortexing method (see, e.g., Kindle 
supra). The method of transformation is not critical to the instant 
invention; various method of plant transformation are currently available 
(supra). As newer methods are available to transform crops or other host 
cells they may be directly applied. Accordingly, a wide variety of methods 
have been developed to insert a DNA sequence into the gene of a plant host 
to obtain the transcription or transcript and translation of the sequence 
to effect phenotypic changes in both dicots and monocots. Moreover, the 
manner in which the DNA construct is introduced into the plant host is not 
critical to the invention. Thus, any method which provides for efficient 
transformation maybe employed. 
The following is an example outlining an Agrobacterium-mediated plant 
transformation. The general process for manipulating genes to be 
transferred into the genome of plant cells is carried out in two phases. 
First, all the cloning and DNA modification steps are done in E. coli, and 
the plasmid containing the gene construct of interest is transferred by 
conjugation into Agrobacterium. Second, the resulting Agrobacterium strain 
is used to transform plant cells. Thus, for the generalized plant 
expression vector, the plasmid contains an origin of replication that 
allows it to replicate in Agrobacterium and a high copy number origin of 
replication functional in E. coli. This permits facile production and 
testing of transgenes in E. coli prior to transfer to Agrobacterium for 
subsequent introduction into plants. Resistance genes can be carried on 
the vector, one for selection in bacteria, e.g., streptomycin, and the 
other that will express in plants, e.g., a gene encoding for kanamycin 
resistance or an herbicide resistance gene. Also present are restriction 
endonuclease sites for the addition of one or more transgenes operably 
linked to appropriate regulatory sequences and directional T-DNA border 
sequences which, when recognized by the transfer functions of 
Agrobacterium, delimit the region that will be transferred to the plant. 
In another example, plants cells may be transformed by shooting into the 
cell tungsten microprojectiles on which cloned DNA is precipitated. In the 
Biolistic Apparatus (Bio-Rad, Hercules, Calif.) used for the shooting, a 
gunpowder charge (22 caliber Power Piston Tool Charge) or an air-driven 
blast drives a plastic macroprojectile through a gun barrel. An aliquot of 
a suspension of tungsten particles on which DNA has been precipitated is 
placed on the front of the plastic macroprojectile. The latter is fired at 
an acrylic stopping plate that has a hole through it that is too small for 
the macroprojectile to go through. As a result, the plastic 
macroprojectile smashes against the stopping plate and the tungsten 
microprojectiles continue toward their target through the hole in the 
plate. For the instant invention the target can be any plant cell, tissue, 
seed, or embryo. The DNA introduced into the cell on the microprojectiles 
becomes integrated into either the nucleus or the chloroplast. 
Transfer and expression of transgenes in plant cells is now routine 
practice to those skilled in the art. It has become a major tool to carry 
out gene expression studies and to attempt to obtain improved plant 
varieties of agricultural or commercial interest. 
Transgenic Plant Regeneration 
Plants cells transformed with a plant expression vector can be regenerated, 
e.g., from single cells, callus tissue or leaf discs according to standard 
plant tissue culture techniques. It is well known in the art that various 
cells, tissues, and organs from almost any plant can be successfully 
cultured to regenerate an entire plant; such techniques are described, 
e.g., in Vasil supra; Green et al., supra; Weissbach and Weissbach, supra; 
and Gelvin et al., supra. 
In one particular example, a cloned AFT1 polypeptide under the control of 
the 35S CaMV promoter and the nopaline synthase terminator and carrying a 
selectable marker (e.g., kanamycin resistance) is transformed into 
Agrobacterium. Transformation of leaf discs (e.g., of tobacco leaf discs), 
with vector-containing Agrobacterium is carried out as described by Horsch 
et al. (Science 227: 1229, 1985). Putative transformants are selected 
after a few weeks (e.g., 3 to 5 weeks) on plant tissue culture media 
containing kanamycin (e.g. 100 .mu.g/ml). Kanamycin-resistant shoots are 
then placed on plant tissue culture media without hormones for root 
initiation. Kanamycin-resistant plants are then selected for greenhouse 
growth. If desired, seeds from self-fertilized transgenic plants can then 
be sowed in a soil-less media and grown in a greenhouse. 
Kanamycin-resistant progeny are selected by sowing surfaced sterilized 
seeds on hormone-free kanamycin-containing media. Analysis for the 
integration of the transgene is accomplished by standard techniques (see, 
e.g., Ausubel et al. supra; Gelvin et al. supra). 
Transgenic plants expressing the selectable marker are then screened for 
transmission of the transgene DNA by standard immunoblot and DNA detection 
techniques. Each positive transgenic plant and its transgenic progeny are 
unique in comparison to other transgenic plants established with the same 
transgene. Integration of the transgene DNA into the plant genomic DNA is 
in most cases random and the site of integration can profoundly effect the 
levels, and the tissue and developmental patterns of transgene expression. 
Consequently, a number of transgenic lines are usually screened for each 
transgene to identify and select plants with the most appropriate 
expression profiles. 
Transgenic lines are evaluated on levels of transgene expression. 
Expression at the RNA level is determined initially to identify and 
quantitate expression-positive plants. Standard techniques for RNA 
analysis are employed and include PCR amplification assays using 
oligonucleotide primers designed to amplify only transgene RNA templates 
and solution hybridization assays using transgene-specific probes (see, 
e.g., Ausubel et al., supra). The RNA-positive plants are then analyzed 
for protein expression by Western immunoblot analysis using AFT1 specific 
antibodies (see, e.g., Ausubel et al., supra). In addition, in situ 
hybridization and immunocytochemistry according to standard protocols can 
be done using transgene-specific nucleotide probes and antibodies, 
respectively, to localize sites of expression within transgenic tissue. 
Once the recombinant AFT1 protein is expressed in any cell or in a 
transgenic plant (e.g., as described above), it may be isolated, e.g., 
using affinity chromatography. In one example, an anti-AFT1 antibody 
(e.g., produced as described in Ausubel et al., supra, or by any standard 
technique) may be attached to a column and used to isolate the 
polypeptide. Lysis and fractionation of AFT1-producing cells prior to 
affinity chromatography may be performed by standard methods (see, e.g., 
Ausubel et al., supra). Once isolated, the recombinant protein can, if 
desired, be further purified, e.g., by high performance liquid 
chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry 
And Molecular Biology, eds., Work and Burdon, Elsevier, 1980). 
These general techniques of polypeptide expression and purification can 
also be used to produce and isolate useful AFT1 fragments or analogs. 
In other applications, however, expression of the transgene in the plant 
cell or the transgenic plant may be the desired result. These include 
applications such as AFT1 controlled regulation of modulating plant 
defense related proteins, e.g., 3-O-methyltransferase or ascorbate 
peroxidase, or altering the normal development of the plant. 
Use 
Introduction of AFT1 or a chimeric AFT1 transcriptional activator into a 
transformed plant cell facilitates the manipulation of developmental 
events. For example, transgenic plants of the instant invention expressing 
AFT1 or an AFT1 chimeric transcriptional activator might be used to alter, 
simply and inexpensively, or regulate plant gene expression, e.g., plant 
defense mechanism, expression of plant storage components, or any number 
of other plant developmental events. 
Other Embodiments 
The invention also includes any biologically active fragment or analog of a 
crucifer AFT1 protein. By "biologically active" is meant possessing any in 
vivo or in vitro activity which is characteristic of the crucifer AFT1 
polypeptide shown in FIG. 1 (SEQ ID NO: 2). Because crucifer AFT1 protein 
exhibits a range of physiological properties and because such properties 
may be attributable to different portions of the crucifer AFT1 protein 
molecule, a useful AFT1 fragment or analog is one which exhibits a 
biological activity in any biological assay for AFT1 transcriptional 
activation or binding activity, for example, those assays described supra. 
Such fragment or analog may function in accordance with developmental 
stages of different cell types and in response to different environmental 
factors and hormonal cues, or in response to a particular signal 
transduction pathway. 
Preferred analogs include AFT1 proteins (or biologically active fragments 
thereof) whose sequences differ from the wild-type sequence only by 
conservative amino acid substitutions, for example, substitution of one 
amino acid for another with similar characteristics (e.g., valine for 
glycine, arginine for lysine, etc.) or by one or more non-conservative 
amino acid substitutions, deletions, or insertions which do not abolish 
the polypeptide's biological activity. 
Analogs can differ from naturally occurring AFT1 protein in amino acid 
sequence or can be modified in ways that do not involve sequence, or both. 
Analogs of the invention will generally exhibit at least 70%, preferably 
80%, more preferably 90%, and most preferably 95% or even 99%, homology 
with a segment of 20 amino acid residues, preferably 40 amino acid 
residues, or more preferably the entire sequence of a naturally occurring 
AFT1 polypeptide sequence. 
Alterations in primary sequence include genetic variants, both natural and 
induced. Also included are analogs that include residues other than 
naturally occurring L-amino acids, e.g., D-amino acids or non-naturally 
occurring or synthetic amino acids, e.g., .beta. or .gamma. amino acids. 
Alternatively, increased stability may be conferred by cyclizing the 
peptide molecule. Also included in the invention are crucifer AFT1 
proteins modified by in vivo or in vitro chemical derivatization of 
polypeptides, including acetylation, methylation, phosphorylation, 
carboxylation, or glycosylation. 
In addition to substantially full-length polypeptides, the invention also 
includes biologically active fragments of the polypeptides. As used 
herein, the term "fragment", as applied to a polypeptide, will ordinarily 
be at least 20 residues, more typically at least 40 residues, and 
preferably at least 60 residues in length. Fragments of crucifer AFT1 
proteins can be generated by methods known to those skilled in the art. 
The ability of a candidate fragment to exhibit a biological activity of 
crucifer AFT1 protein can be assessed by those methods described herein. 
Also included in the invention are crucifer AFT1 proteins containing 
residues that are not required for biological activity of the peptide, 
e.g., those added by alternative mRNA splicing or alternative protein 
processing events. 
Other embodiments are within the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 29 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 845 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
AAAAAAAAATCAAATCTCTCTCTTTCTCTCTCTAATGGCGGCGACATTAGGCAGAGACCA60 
GTATGTGTACATGGCGAAGCTCGCCGAGCAGGCGGAGCGTTACGAAGAGATGGTTCAATT120 
CATGGAACAGCTCGTTACAGGCGCTACTCCAGCGGAAGAGCTCACCGTTGAAGAGAGGAA180 
TCTCCTCTCTGTTGCTTACAAGAACGTGATCGGATCTCTACGCGCCGCCTGGAGGATCGT240 
GTCTTCGATTGAGCAGAAGGAAGAGAGTAGGAAGAACGACGAGCACGTGTCGCTTGTCAA300 
GGATTACAGATCTAAAGTTGAGTCTGAGCTTTCTTCTGTTTGCTCTGGAATCCTTAAGCT360 
CCTTGACTCGCATCTGATCCCATCTGCTGGAGCGAGTGAGTCTAAGGTCTTTTACTTGAA420 
GATGAAAGGTGATTATCATCGGTACATGGCTGAGTTTAAGTCTGGTGATGAGAGGAAAAC480 
TGCTGCTGAAGATACCATGCTCGCTTACAAAGCAGCTCAGGATATCGCAGCTGCGGATAT540 
GGCACCTACTCATCCGATAAGGCTTGGTCTGGCCCTGAATTTCTCAGTGTTCTACTATGA600 
GATTCTCAATTCTTCAGACAAAGCTTGTAACATGGCCAAACAGGCTTTTGAGGAGGCCAT660 
AGCTGAGCTTGACACTCTGGGAGAGGAATCCTACAAAGACAGCACTCTCATAATGCAGTT720 
GCTGAGGGACAATTTAACCCTTTGGACCTCCGATATGCAGGAGCAGATGGACGAGGCCTG780 
AGGATCTAGATGAAGGGGGGGAGGGTTGTTACGCGATGTTTCTGCCACCAAATCGATCTC840 
AAAAT845 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 248 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
MetAlaAlaThrLeuGlyArgAspGlnTyrValTyrMetAlaLysLeu 
151015 
AlaGluGlnAlaGluArgTyrGluGluMetValGlnPheMetGluGln 
202530 
LeuValThrGlyAlaThrProAlaGluGluLeuThrValGluGluArg 
354045 
AsnLeuLeuSerValAlaTyrLysAsnValIleGlySerLeuArgAla 
505560 
AlaTrpArgIleValSerSerIleGluGlnLysGluGluSerArgLys 
65707580 
AsnAspGluHisValSerLeuValLysAspTyrArgSerLysValGlu 
859095 
SerGluLeuSerSerValCysSerGlyIleLeuLysLeuLeuAspSer 
100105110 
HisLeuIleProSerAlaGlyAlaSerGluSerLysValPheTyrLeu 
115120125 
LysMetLysGlyAspTyrHisArgTyrMetAlaGluPheLysSerGly 
130135140 
AspGluArgLysThrAlaAlaGluAspThrMetLeuAlaTyrLysAla 
145150155160 
AlaGlnAspIleAlaAlaAlaAspMetAlaProThrHisProIleArg 
165170175 
LeuGlyLeuAlaLeuAsnPheSerValPheTyrTyrGluIleLeuAsn 
180185190 
SerSerAspLysAlaCysAsnMetAlaLysGlnAlaPheGluGluAla 
195200205 
IleAlaGluLeuAspThrLeuGlyGluGluSerTyrLysAspSerThr 
210215220 
LeuIleMetGlnLeuLeuArgAspAsnLeuThrLeuTrpThrSerAsp 
225230235240 
MetGlnGluGlnMetAspGluAla 
245 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
GCGGAATTCATGAGGCCCATTAAAATT27 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
GTAGGATCCGGTCGGATTTCTTGTCGC27 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
CGCGAATTCAATAGCGACAAGTACGAT27 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
GTAGGATCCGTCTCTCTTCCAAGGTAGA28 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
GATCCTAGAATTCAAGAAGAATCGGCGTGGC31 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
CTGACTGAATTCATGGCGGCGACATTAGG29 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
GACTGAGTCGACCCTTCATCTAGATCCTC29 
(2) INFORMATION FOR SEQ ID NO: 10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 
GACTGACTCGAGCCTTCATCTAGATCCTCA30 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 
CTGACTGAATTCGAGTCTAAGGTCTTTAC29 
(2) INFORMATION FOR SEQ ID NO: 12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 
GACTGACTCGAGACTCGCTCCAGCAGATGG30 
(2) INFORMATION FOR SEQ ID NO: 13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
GACTGACTCGAGTGAAGAATTGAGAATCTC30 
(2) INFORMATION FOR SEQ ID NO: 14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 
GACTGAGTCGACACTCGCTCCAGCAGATGG30 
(2) INFORMATION FOR SEQ ID NO: 15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 
GACTGAGTCGACTGAAGAATTGAGAATCTC30 
(2) INFORMATION FOR SEQ ID NO: 16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 
CTGACTGAATTCGTTACAGGCGCTACTCCAG31 
(2) INFORMATION FOR SEQ ID NO: 17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 557 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 
TCACCCAGAGAGGTCAGGCTTTGATGGACCATGGACCCAAGAGCCGCTGAAGTTTGACAA60 
CTCCTACTTCGTGGAACTGCTGAAAGGAGAATCAGAGGGCTTGTTGAAACTTCCAACTGA120 
CAAGACCTTATTGGAAGACCCGGAGTTCCGTCGTCTTGTTGAGCTTTATGCAAAGGATGA180 
AGATGCATTCTTCAGAGACTACGCGGAATCGCACAAGAAACTCTCTGAGCTTGGTTTCAA240 
CCCAAACTCCTCAGCAGGCAAAGCAGTTGCAGACAGCACGATTCTGGCACAGAGTGCGTT300 
CGGGGTTGCAGTTGCTGCTGCGGTTGTGGCATTTGGTTACTTTTACGAGATTCGGAAGAG360 
GATGAAGTAAACGAAATAGGAAGGAAAACACGAAGCAACGATGCTCTTATTTGGGTATTA420 
AAGAAACTATTAATCGTCTATCGAATCTATTTTGCTGCTACAAGATTCTAAACTCTTTGA480 
ATCCACGATTCCACTGTTTAGTAGTAAAAAAGTTAAAAAGTCAATATTTTGGGTCCGTGA540 
TTCATTTTTGCGATAAA557 
(2) INFORMATION FOR SEQ ID NO: 18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 122 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 
HisProGluArgSerGlyPheAspGlyProTrpThrGlnGluProLeu 
151015 
LysPheAspAsnSerTyrPheValGluLeuLeuLysGlyGluSerGlu 
202530 
GlyLeuLeuLysLeuProThrAspLysThrLeuLeuGluAspProGlu 
354045 
PheArgArgLeuValGluLeuTyrAlaLysAspGluAspAlaPhePhe 
505560 
ArgAspTyrAlaGluSerHisLysLysLeuSerGluLeuGlyPheAsn 
65707580 
ProAsnSerSerAlaGlyLysAlaValAlaAspSerThrIleLeuAla 
859095 
GlnSerAlaPheGlyValAlaValAlaAlaAlaValValAlaPheGly 
100105110 
TyrPheTyrGluIleArgLysArgMetLys 
115120 
(2) INFORMATION FOR SEQ ID NO: 19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 478 
(B) TYPE: nucliec acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 
GAGTGACGAACATTGCGTGAAATTCTTGAAGAACTGCTACGAGTCACTTCCAGAGGATGG60 
AAAAGTGATATTAGCAGAGTGTATTCTTCCAGAGACACCAGACTCAAGCCTCTCAACCAA120 
ACAAGTAGTCCATGTCGATTGCATTATGTTGGCTCACAATCCCGGAGGCAAAGAACGAAC180 
CGAGAAAGAGTTTGAGGCATTAGCCAAAGCATCAGGCTTCAAGGGCATCAAAGTTGTCTG240 
CGACGCTTTTGGTGTTAACCTTATTGAGTTACTCAAGAAGCTCTAAAAACAAACAATGTT300 
CCTATGAAGATGATTTATATGTAAACATTATCTCATATCTCCTTCCACGGTTCCAAAACT360 
ATGCTGTTTAATAATGGTTTTTACAAGAATTTGATTATGAGTTTGTATTTTTGTTTGTTT420 
GGAACAAAATTATGTGATTATAGGGAAAAATAAAATGAGCTATTATTGAAGAAAAAAA478 
(2) INFORMATION FOR SEQ ID NO: 20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 94 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 
SerAspGluHisCysValLysPheLeuLysAsnCysTyrGluSerLeu 
151015 
ProGluAspGlyLysValIleLeuAlaGluCysIleLeuProGluThr 
202530 
ProAspSerSerLeuSerThrLysGlnValValHisValAspCysIle 
354045 
MetLeuAlaHisAsnProGlyGlyLysGluArgThrGluLysGluPhe 
505560 
GluAlaLeuAlaLysAlaSerGlyPheLysGlyIleLysValValCys 
65707580 
AspAlaPheGlyValAsnLeuIleGluLeuLeuLysLysLeu 
8590 
(2) INFORMATION FOR SEQ ID NO: 21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1357 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 
CCAGATTATCCCTCCCCCGAATTCGGCACGAGGAAAAATCCTCTTCTTTCAGATGAGAAA60 
CCCAAATCGACGGAGGAGAATAAGAGTTCTAAGCCGGAATCAGCTTCTGGGAGTTCAACT120 
TCATCAGCTATGCCTGGCTTGAATTTCAATGCTTTTGATTTCTCTAATATGGCTAGTATT180 
CTCAACGATCCTAGCATCAGAGAAATGGCTGAGCAAATAGCTAAAGATCCTGCCTTTAAC240 
CAATTGGCTGAGCAGCTTCAGAGATCTATTCCTAACGCTGGCCAGGAAGGTGGTTTCCCT300 
AACTTTGATCCTCAACAGTATGTCAATACAATGCAACAGGTTATGCATAACCCTGAGTTT360 
AAGACAATGGCCGAGAAACTTGGTACCGCCTTAGTTCAGGATCCACAAATGTCTCCTTTT420 
TTGGATGCTTTCTCGAATCCTGAAACAGCAGAACACTTTACTGAGCGTATGGCGCGGATG480 
AAAGAAGATCCAGAGTTGAAACCTATACTAGATGAGATTGATGCTGGTGGTCCTTCTGCC540 
ATGATGAAGTACTGGAATGATCCAGAAGTGCTGAAAAAGCTGGGTGAAGCAATGGGTATG600 
CCTGTTGCTGGCTTACCAGACCAGACTGTTTCAGCTGAACCTGAGGTAGCAGAAGAAGGT660 
GAAGAAGAAGAGTCTATTGTTCACCAAACTGCCAGTCTTGGTGATGTTGAGGGTTTGAAA720 
GCTGCCTTGGCATCTGGTGGTAACAAAGATGAAGAAGATTCTGAAGGAAGGACAGCATTG780 
CATTTTGCTTGTGGATACGGCGAGTTGAAATGTGCTCAAGTTCTTATCGATGCTGGAGCA840 
AGTGTTAATGCGGTTGACAAAAACAAGAACACACCTCTGCATTATGCTGCTGGTTACGGG900 
AGGAAAGAGAGTGTAAGCCTTCTCCTGGAGAATGGTGCTGCAGTCACTCTGCAAAACCTA960 
GACGAGAAGACGCCAATTGATGTAGCGAAGCTCAACAGCCAGCTGGAGGTGGTGAAGCTG1020 
CTTGAGAAGGATGCTTTCCTTTGAGCTCTGCTGGTTAAAGGAAAGCTCTAAGCTCATATT1080 
GTCTTTGAGGCATTTGTCTTGTGTGTGTCCTGAACCAGTTTCACAGGCTTTTTGTGTACA1140 
CTTTTTATTAGTTCCTCTCTTCTTCTAAATTTGTCTCTTATGTTGTTTTAAAAGTCAATA1200 
AAGAAAGAAATAGCAATCAATGATTTAATTTATGATTATATTCTTTATTTCGTCGACCTC1260 
TACAGAATGATTCAATTTGGAAGAATCATTCTGGTTTGGAGGATATGTAAGAAAAACTAC1320 
TTGATCTCCAAGTTATTCCATTCTTCTGTTGAAAAAA1357 
(2) INFORMATION FOR SEQ ID NO: 22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 339 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 
GlyThrArgLysAsnProLeuLeuSerAspGluLysProLysSerThr 
151015 
GluGluAsnLysSerSerLysProGluSerAlaSerGlySerSerThr 
202530 
SerSerAlaMetProGlyLeuAsnPheAsnAlaPheAspPheSerAsn 
354045 
MetAlaSerIleLeuAsnAspProSerIleArgGluMetAlaGluGln 
505560 
IleAlaLysAspProAlaPheAsnGlnLeuAlaGluGlnLeuGlnArg 
65707580 
SerIleProAsnAlaGlyGlnGluGlyGlyPheProAsnPheAspPro 
859095 
GlnGlnTyrValAsnThrMetGlnGlnValMetHisAsnProGluPhe 
100105110 
LysThrMetAlaGluLysLeuGlyThrAlaLeuValGlnAspProGln 
115120125 
MetSerProPheLeuAspAlaPheSerAsnProGluThrAlaGluHis 
130135140 
PheThrGluArgMetAlaArgMetLysGluAspProGluLeuLysPro 
145150155160 
IleLeuAspGluIleAspAlaGlyGlyProSerAlaMetMetLysTyr 
165170175 
TrpAsnAspProGluValLeuLysLysLeuGlyGluAlaMetGlyMet 
180185190 
ProValAlaGlyLeuProAspGlnThrValSerAlaGluProGluVal 
195200205 
AlaGluGluGlyGluGluGluGluSerIleValHisGlnThrAlaSer 
210215220 
LeuGlyAspValGluGlyLeuLysAlaAlaLeuAlaSerGlyGlyAsn 
225230235240 
LysAspGluGluAspSerGluGlyArgThrAlaLeuHisPheAlaCys 
245250255 
GlyTyrGlyGluLeuLysCysAlaGlnValLeuIleAspAlaGlyAla 
260265270 
SerValAsnAlaValAspLysAsnLysAsnThrProLeuHisTyrAla 
275280285 
AlaGlyTyrGlyArgLysGluSerValSerLeuLeuLeuGluAsnGly 
290295300 
AlaAlaValThrLeuGlnAsnLeuAspGluLysThrProIleAspVal 
305310315320 
AlaLysLeuAsnSerGlnLeuGluValValLysLeuLeuGluLysAsp 
325330335 
AlaPheLeu 
(2) INFORMATION FOR SEQ ID NO: 23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 663 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 
TTTTAAAAAATTTTGCCATCAACCGTAGATGTTCCGCCAAAGGGTGGGTTTAGCTTCGAT60 
CTGTGTAAGAGAAATGATATTCTTACACAAAAGGGTCTTAAAGCTCCGTCTTTTTTGAAG120 
ACTGGAACAACCATTGTTGGTTTGATTTTCAAGGATGGTGTGATACAAGGGGCAGATACC180 
CGAGCAACTGAGGGGCCAATTGTTGCTGATAAGAACTGTGAGAAGATTCACTATATGGCA240 
CCAAACATATATTGCTGTGGTGCAGGAACTCGGGCTGATACTGAAGCAGTCACTGATATG300 
GTCAGCTCACAGCTGCGATTGCATCGTTACCAGACTGGTCGAGACTCTCGGGTCATTACT360 
GCTTTGACCCTTCTCAAAAAACATTTTTTCAGCTACCAAGGTCATGTCTCTGCTGCTCTT420 
GTACTCGGTGGAGTTGATATCACTGGTCCACATCTGCATACTATATACCCACACGGTTCA480 
ACTGACACTCTTCCATTCGCCACAATGGGTTCGGGTTCTCTTGCTGCTATGTCTGTGTTT540 
GAGGCAAAGTATAAAGAAGGCCTAACTAGGGATGAAGGAATTAAGCTGGTCGCTGAATCC600 
ATATGCTCGGGTATATCCAATGACCTGGGTAGTGGTAGCAACGTGGACATCTGCGTGATC660 
ACA663 
(2) INFORMATION FOR SEQ ID NO: 24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 219 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 
LysIleLeuProSerThrValAspValProProLysGlyGlyPheSer 
151015 
PheAspLeuCysLysArgAsnAspIleLeuThrGlnLysGlyLeuLys 
202530 
AlaProSerPheLeuLysThrGlyThrThrIleValGlyLeuIlePhe 
354045 
LysAspGlyValIleGlnGlyAlaAspThrArgAlaThrGluGlyPro 
505560 
IleValAlaAspLysAsnCysGluLysIleHisTyrMetAlaProAsn 
65707580 
IleTyrCysCysGlyAlaGlyThrArgAlaAspThrGluAlaValThr 
859095 
AspMetValSerSerGlnLeuArgLeuHisArgTyrGlnThrGlyArg 
100105110 
AspSerArgValIleThrAlaLeuThrLeuLeuLysLysHisPhePhe 
115120125 
SerTyrGlnGlyHisValSerAlaAlaLeuValLeuGlyGlyValAsp 
130135140 
IleThrGlyProHisLeuHisThrIleTyrProHisGlySerThrAsp 
145150155160 
ThrLeuProPheAlaThrMetGlySerGlySerLeuAlaAlaMetSer 
165170175 
ValPheGluAlaLysTyrLysGluGlyLeuThrArgAspGluGlyIle 
180185190 
LysLeuValAlaGluSerIleCysSerGlyIleSerAsnAspLeuGly 
195200205 
SerGlySerAsnValAspIleCysValIleThr 
210215 
(2) INFORMATION FOR SEQ ID NO: 25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 976 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 
ACGAGAGGCCCTGAGACGCGGCAGATATCAGGTCCTGCGACTTCAACACAGATCAGGAAC60 
TTCACATTATGTCAGCATCTGCAAGGAATCCACACACATATCTCATCCATGGTAGCGGAC120 
CTTCCCAGTATTGCTACTGATGTATTGTCTCCTTATCTGGCTGCAATCTATAATGCGGCA180 
TGTGAGCCAGTTACACCTTTGTTTAAAGCAATGCGAGACAAGCTCGAGTCATGCATTCTT240 
CAAATCCATGATCAAAACTTTGGTGCTGATGACGCTGACATGGACAACAACGCTTCCTCA300 
TACATGGAGGAGTTGCAGAGATCGATTCTTCACTTCCGCAAGGAGTTCCTATCTAGACTA360 
TTGCCTTCCGCAGCAAATGCTAACACTGCAGGAACAGAATCGATCTGCACAAGACTCACA420 
AGACAAATGGCGTCAAGGGTTTTGATCTTCTACATCAGACATGCATCCCTTGTGCGACCA480 
CTTTCAGAATGGGGAAAACTCAGAATGGCCAAAGACATGGCCGAGCTGGAACTAGCAGTG540 
GGACAGAATCTATTTCCCGTGGAACAACTCGGAGCACCGTACAGAGCTCTTAGAGCGTTT600 
AGGCCTTTGGTTTTCCTGGAAACATCTCAAATGGGATCATCTCCTCTCATCAATGATCTA660 
CCACCGAGCATCGTCCTACATCATCTCTACACAAGAGGCCCAGACGAGTTAGAGTCACCG720 
ATGCAGAAGAACAGACTAAGTCCTAAACAGTACTCACTGTGGCTTGATAACCAAAGAGAG780 
GATCAGATCTGGAAAGGGATAAAAGCAACTTTGGATGATTATGCAGTGAAGATCAGATCG840 
AGAGGGGACAAAGAGTTTAGTCCAGGTTATCCTCTAATGCTTCAAATTGGTTCATCTTTA900 
ACACAAGAAAACTTATAAGCTGTGCTTTGTTACCGAATCAATATTCTTCTATTGCGAACT960 
TTTTTGTCTCAAAAAA976 
(2) INFORMATION FOR SEQ ID NO: 26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 305 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 
ThrArgGlyProGluThrArgGlnIleSerGlyProAlaThrSerThr 
151015 
GlnIleArgAsnPheThrLeuCysGlnHisLeuGlnGlyIleHisThr 
202530 
HisIleSerSerMetValAlaAspLeuProSerIleAlaThrAspVal 
354045 
LeuSerProTyrLeuAlaAlaIleTyrAsnAlaAlaCysGluProVal 
505560 
ThrProLeuPheLysAlaMetArgAspLysLeuGluSerCysIleLeu 
65707580 
GlnIleHisAspGlnAsnPheGlyAlaAspAspAlaAspMetAspAsn 
859095 
AsnAlaSerSerTyrMetGluGluLeuGlnArgSerIleLeuHisPhe 
100105110 
ArgLysGluPheLeuSerArgLeuLeuProSerAlaAlaAsnAlaAsn 
115120125 
ThrAlaGlyThrGluSerIleCysThrArgLeuThrArgGlnMetAla 
130135140 
SerArgValLeuIlePheTyrIleArgHisAlaSerLeuValArgPro 
145150155160 
LeuSerGluTrpGlyLysLeuArgMetAlaLysAspMetAlaGluLeu 
165170175 
GluLeuAlaValGlyGlnAsnLeuPheProValGluGlnLeuGlyAla 
180185190 
ProTyrArgAlaLeuArgAlaPheArgProLeuValPheLeuGluThr 
195200205 
SerGlnMetGlySerSerProLeuIleAsnAspLeuProProSerIle 
210215220 
ValLeuHisHisLeuTyrThrArgGlyProAspGluLeuGluSerPro 
225230235240 
MetGlnLysAsnArgLeuSerProLysGlnTyrSerLeuTrpLeuAsp 
245250255 
AsnGlnArgGluAspGlnIleTrpLysGlyIleLysAlaThrLeuAsp 
260265270 
AspTyrAlaValLysIleArgSerArgGlyAspLysGluPheSerPro 
275280285 
GlyTyrProLeuMetLeuGlnIleGlySerSerLeuThrGlnGluAsn 
290295300 
Leu 
305 
(2) INFORMATION FOR SEQ ID NO: 27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 215 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 
ValThrGlyAlaThrProAlaGluGluLeuThrValGluGluArgAsn 
151015 
LeuLeuSerValAlaTyrLysAsnValIleGlySerLeuArgAlaAla 
202530 
TrpArgIleValSerSerIleGluGlnLysGluGluSerArgLysAsn 
354045 
AspGluHisValSerLeuValLysAspTyrArgSerLysValGluSer 
505560 
GluLeuSerSerValCysSerGlyIleLeuLysLeuLeuAspSerHis 
65707580 
LeuIleProSerAlaGlyAlaSerGluSerLysValPheTyrLeuLys 
859095 
MetLysGlyAspTyrHisArgTyrMetAlaGluPheLysSerGlyAsp 
100105110 
GluArgLysThrAlaAlaGluAspThrMetLeuAlaTyrLysAlaAla 
115120125 
GlnAspIleAlaAlaAlaAspMetAlaProThrHisProIleArgLeu 
130135140 
GlyLeuAlaLeuAsnPheSerValPheTyrTyrGluIleLeuAsnSer 
145150155160 
SerAspLysAlaCysAsnMetAlaLysGlnAlaPheGluGluAlaIle 
165170175 
AlaGluLeuAspThrLeuGlyGluGluSerTyrLysAspSerThrLeu 
180185190 
IleMetGlnLeuLeuArgAspAsnLeuThrLeuTrpThrSerAspMet 
195200205 
GlnGluGlnMetAspGluAla 
210215 
(2) INFORMATION FOR SEQ ID NO: 28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 127 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 
GluSerLysValPheTyrLeuLysMetLysGlyAspTyrHisArgTyr 
151015 
MetAlaGluPheLysSerGlyAspGluArgLysThrAlaAlaGluAsp 
202530 
ThrMetLeuAlaTyrLysAlaAlaGlnAspIleAlaAlaAlaAspMet 
354045 
AlaProThrHisProIleArgLeuGlyLeuAlaLeuAsnPheSerVal 
505560 
PheTyrTyrGluIleLeuAsnSerSerAspLysAlaCysAsnMetAla 
65707580 
LysGlnAlaPheGluGluAlaIleAlaGluLeuAspThrLeuGlyGlu 
859095 
GluSerTyrLysAspSerThrLeuIleMetGlnLeuLeuArgAspAsn 
100105110 
LeuThrLeuTrpThrSerAspMetGlnGluGlnMetAspGluAla 
115120125 
(2) INFORMATION FOR SEQ ID NO: 29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 162 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 
LeuValThrGlyAlaThrProAlaGluGluLeuThrValGluGluArg 
151015 
AsnLeuLeuSerValAlaTyrLysAsnValIleGlySerLeuArgAla 
202530 
AlaTrpArgIleValSerSerIleGluGlnLysGluGluSerArgLys 
354045 
AsnAspGluHisValSerLeuValLysAspTyrArgSerLysValGlu 
505560 
SerGluLeuSerSerValCysSerGlyIleLeuLysLeuLeuAspSer 
65707580 
HisLeuIleProSerAlaGlyAlaSerGluSerLysValPheTyrLeu 
859095 
LysMetLysGlyAspTyrHisArgTyrMetAlaGluPheLysSerGly 
100105110 
AspGluArgLysThrAlaAlaGluAspThrMetLeuAlaTyrLysAla 
115120125 
AlaGlnAspIleAlaAlaAlaAspMetAlaProThrHisProIleArg 
130135140 
LeuGlyLeuAlaLeuAsnPheSerValPheTyrTyrGluIleLeuAsn 
145150155160 
SerSer 
__________________________________________________________________________