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Matched Legal Cases: ['Application No. 08', 'Application No. 08', 'Application No. 08', 'Application No. 08', 'Application No. 08', 'application No. 08', 'application No. 08', 'application No. 08', 'application No. 08', 'application No. 08']

Patent US5955573 - Ubiquitin-lytic peptide fusion gene constructs, protein products deriving ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsStabilized ubiquitin-lytic peptide fusion polypeptides and a method of making the same by sub-cloning nucleic acid sequences coding for lytic peptides into a plasmid vector comprising a promoter and ubiquitin polypeptide coding sequence, wherein the ubiquitin polypeptide sequence is linked to the 5'...http://www.google.com/patents/US5955573?utm_source=gb-gplus-sharePatent US5955573 - Ubiquitin-lytic peptide fusion gene constructs, protein products deriving therefrom, and methods of making and using sameAdvanced Patent SearchPublication numberUS5955573 APublication typeGrantApplication numberUS 08/505,486Publication dateSep 21, 1999Filing dateJul 21, 1995Priority dateJun 4, 1993Fee statusPaidAlso published asUS6018102, US6084156Publication number08505486, 505486, US 5955573 A, US 5955573A, US-A-5955573, US5955573 A, US5955573AInventorsJoan Garbarino, Jesse Jaynes, William BelknapOriginal AssigneeDemegen, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (6), Non-Patent Citations (6), Referenced by (1), Classifications (50), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetUbiquitin-lytic peptide fusion gene constructs, protein products deriving therefrom, and methods of making and using same
US 5955573 AAbstract
1. A lytic peptide comprising the amino acid sequence of SEQ ID NO. 97 .
2. A lytic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS. 48, 55-65.
3. A lytic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS. 66-80.
4. A lytic peptide of claim 2 wherein the peptide has the amino acid sequence of SEQ ID NO. 57.
5. A lytic peptide of claim 3 wherein the peptide has the amino acid sequence of SEQ ID NO. 66.
6. A lytic peptide of claim 2 wherein the peptide has the amino acid sequence of SEQ ID NO. 48.
This is a divisional of application Ser. No. 08/279,472, filed Jul. 22, 1994, now abandoned, which is a continuation-in-part of Application No. 08/231,730, filed April 20, 1994, now abandoned, in the names of Jesse M. Jaynes and Gordon R. Julian, which in turn is a continuation-in-part of Application No. 08/225,476, filed Apr. 8, 1994, now abandoned, in the names of Jesse M. Jaynes and Gordon R. Julian, which is in turn a continuation-in-part of Application No. 08/148,491, filed Nov. 8, 1993, now abandoned, and Application No. 08/148,889, filed Nov. 8, 1993, now abandoned, both filed in the name of Gordon R. Julian, which are in turn continuation-in-part of Application No. 08/039,620, filed Jun. 4, 1993, now abandoned, in the name of Jesse M. Jaynes and Gordon R. Julian.
Naturally occurring lytic peptides play an important if not critical role as immunological agents in insects and have some, albeit secondary, defense functions in a range of other animals. The function of these peptides is to destroy procaryotic and other non-host cells by disrupting the cell membrane and promoting cell lysis. Common features of these naturally occurring lytic peptides include an overall basic charge, a small size (23-39 amino acid residues), and the ability to form amphipathic α-helices or β-pleated sheets. Several types of lytic peptides have been identified: cecropins (described in U.S. Pat. Nos. 4,355,104 and 4,520,016 to Hultmark et al.), defensins, sarcotoxins, melittin, and magainins (described in U.S. Patent No. 4,810,777 to Zasloff). Each of these peptide types is distinguished by sequence and secondary structure characteristics.
Several hypotheses have been suggested for the mechanism of action of the lytic peptides: disruption of the membrane lipid bilayer by the anphipathic α-helix portion of the lytic peptide; lytic peptide formation of ion channels, which results in osmotically induced cytolysis; lytic peptide promotion of protein aggregation, which results in ion channel formation; and lytic peptide-induced release of phospholipids. Whatever the mechanism of lytic peptide-induced membrane damage, an ordered secondary conformation such as an amphipathic α-helix and positive charge density are features that appear to participate in the function of the lytic peptides.
Active synthetic analogs of naturally occurring lytic peptides have been produced and tested in vitro against a variety of procaryotic and eukaryotic cell types (see for example Arrowood, M. J., et al., J. Protozool. 38: 161s 199!; Jaynes, J. M., et al., FASEB J. 2: 2878 1988!), including: gram positive and gram negative bacteria, fungi, yeast, protozoa, envelope viruses, virus-infected eukaryotic cells, and neoplastic or transformed mammalian cells. The results from these studies indicate that many of the synthetic lytic peptide analogs have similar or higher levels of lytic activity for many different types of cells, compared to the naturally occurring forms. In addition, the peptide concentration required to lyse microbial pathogens such as protozoans, yeast, and bacteria does not lyse normal mammalian cells. However, because previous work demonstrates that absolute sequence is not important as long as positive charge and amphipathy are preserved, the level of activity for a given synthetic peptide is difficult to predict.
Synthetic lytic peptide analogs can also act as agents of eukaryotic cell proliferation. Peptides that promote lysis of transformed cells will, at lower concentrations, promote cell proliferation in some cell types. This stimulatory activity is thought to depend on the channel-forming capability of the peptides, which somehow stimulates nutrient uptake, calcium influx or metabolite release, thereby stimulating cell proliferation (see Jaynes, J. M., Drug News & Perspectives 3: 69 1990!; and Reed, W. A. et al., Molecular Reproduction and Development 31: 106 1992!). Thus, at a given concentration, these peptides stimulate or create channels that can be beneficial to the normal mammalian cell in a benign environment where it is not important to exclude toxic compounds.
For example, in an attempt to sub-clone into a standard plasmid vector a gene coding for frog magainin, a natural lytic peptide, bacterial transformants contained deletion mutations in the magainin coding region. Another attempt was made to sub-clone a synthetic lytic peptide (SEQ ID NO. 98) into a standard plasmid vector (pUC19) containing the Cauliflower Mosaic virus 35S promoter. The resulting transformants were screened by polymerase chain reaction (PCR). However, out of 30 colonies, only 2 sub-clones gave faint positive signals. These two sub-clones were sequenced. The sequence showed that one clone had a point mutation that introduced a stop codon 3/4 of the way through the lytic peptide, and the other clone had a point mutation that changed the start codon from methionine to isoleucine. Both mutations would prevent the biosynthesis of the protein. Four more clones were analyzed, and of these four, one was sub-cloned an the wrong orientation, and three others had mutations introduced into the sequence. One of these sub-clones was selected for further analysis, but it inhibited the growth of its E. coli host. Thus, the production of recombinant DNA molecules coding for lytic peptides is difficult due to the uncertainty in obtaining the correct sub-clone.
Ubiquitin is a small, highly conserved protein present in all eukaryotes. Ubiquitins are encoded by gene families that are characterized by two types of basic structures. Polyubiquitin genes contain several direct repeats of ubiquitin, and ubiquitin-ribosomal fusion genes encode a single ubiquitin unit fused to the coding region for a small ribosomal associated protein. Both of these gene types are translated as polyproteins and then are processed By an endogenous ubiquitin hydrolase present in eukaryotes to release multiple ubiquitin proteins or ubiquitin and the ribosomal associated protein. A number of ubiquitin cDNAs or genomic clones have been isolated, including plant ubiquitin cDNAs and genomic clones from the potato (Garbarino, J. and Belknap, W., Plant Molecular Biology 24: 119 (1994); Garbarino. J. et al., Plant Molecular Biology 20: 235 (1992)).
The forgoing facts suggest that although lytic peptides as a class may include species that are efficacious in destroying bacteria, neoplastic cells, fungi, virus-infected cells, and protozoa, this lytic characteristic also decreases the stability of sub-cloned lytic peptides in host cells. This decreased stability hinders efforts to develop a more economical and efficient means of synthesizing lytic peptides.
It is yet another object of this invention to provide expression vectors with procaryotic promoters that express ubiquitin-lytic peptide fusion genes in procaryotic hosts, the products of which can be cleaved in vitro by ubiquitin hydrolases.
FIG. 1 is a map of a recombinant nucleic acid expression vector pUCUbi3-LP98 containing a 920 bp ubiquitin-ribosomal fusion gene promoter region linked to a 228 bp coding region for a ubiquitin polypeptide with a six bp BamHI site at the 3' end (SEQ ID NO. 93) that is fused at its 3' end to a gene coding for a lytic peptide (D5D*, SEQ ID NO. 98). The Ubi3 ubiquitin-lytic peptide nucleotide sequence corresponds to SEQ ID NO. 92. A nopaline synthase polyadenylation signal is located at the 3' end of the lytic peptide gene.
FIG. 2 is a map of a recombinant nucleic acid expression vector pUCUbi7-LP98 containing a 1220 bp polyubiquitin promoter region and 568 bp intron linked to a 228 bp coding region for a ubiquitin polypeptide with a six bp BamHI site at the 3' end (SEQ ID NO. 96) that is fused at its 3' end to a gene coding for a lytic peptide (D5D*, SEQ ID NO. 98). The Ubi7 ubiquitin-lytic peptide nucleotide sequence corresponds to SEQ ID NO. 95. A nopaline synthase polyadenylation signal is located at the 3' end of the lytic peptide gene.
The disclosures of prior co-pending U.S. patent application No. 08/039,620 filed Jun. 4, 1993 in the names of Jesse M. Jaynes and Gordon R. Julian, U.S. patent application No. 08/148,889 filed Nov. 8, 1993 in the name of Gordon R. Julian, U.S. patent application No. 08/148,491 filed Nov. 8, 1993 in the name of Gordon R. Julian, U.S. patent application No. 08/225,476 filed Apr. 8, 1994 in the names of Jesse M. Jaynes and Gordon R.
Julian, and U.S. patent application No. 08/231,730 filed Apr. 20, 1994 in the names of Jesse M. Jaynes and Gordon R. Julian, are all hereby incorporated herein by reference in their entirety.
The term "amphipathic" as used herein refers to the distribution of hydrophobic and hydrophilic amino acid residues along opposing faces of an α-helix structure or other secondary conformation, which results in one face of the α-helix structure being predominantly hydrophobic and the other ace being predominantly hydrophilic. The degree of amphipathy of a peptide can be assessed by plotting the sequential amino acid residues on an Edmunson helical wheel (see also Kamtekar, S. et al., Science 262: 1680 (1993). The terms "peptide" and "polypeptide" as used herein refer to a molecule composed of a chain of amino acid residues and is intended to be construed as inclusive of polypeptides and peptides per se having molecular weights of up to 10,000 daltons, as well as proteins having molecular weights of greater that about 10,000 daltons, wherein the molecular weights are number average molecular weights. The term is also intended to be construed as inclusive of functional equivalents thereof when used in reference to a specific peptide coding sequence in the specification and claims herein. Functional equivalents of peptides and polypeptides include but are not limited to deletions, additions, and substitutions of amino acids in the polypeptide or peptide chain that do not adversely affect the overall function of the resulting peptide or polypeptide.
The term "plasmid" as used herein refers to a DNA molecule that is capable of autonomous replication within a host cell, either extrachromosomaily or as part of the host cell chromosome(s). The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids as disclosed herein and/or in accordance wish published procedures. In certain instances, as will be apparent to the ordinarily skilled artisan, other plasmids known in the art may be used interchangeable with plasmids described herein.
The term "ligation" as used herein refers to the process of forming phosphodiester bonds between two double-stranded DNA fragments. Unless otherwise specified, ligation is accomplished using standard procedures known to one skilled in the art.
The term "polymerase chain reaction," or "PCR" as used herein refers to a method for amplification of a desired nucleotide sequence in vitro, as described in U.S. Pat. No. 4,683,195, herein incorporated by reference in its entirety.
The term "nucleic acid" as used herein refers to deoxyribonucleic acid molecules (DNA) composed of a chain of deoxyribonucleotides and ribonucleic acid molecules (RNA) composed of a chain of ribonucleotides. The term "nucleic acid" as used herein is to be construed as including functional equivalents thereof when used in reference to a specific nucleotide sequence in the specification and claims herein. Functional equivalents of nucleic acid molecules include synonymous coding sequences with one or more codon substitutions and deletions or additions that do not effect the overall function of the resulting nucleic acid molecule. The degeneracy of the genetic code is well known to the art; therefore, synonymous coding sequences with one or more codon substitutions can be readily determined by one of ordinary skill in the art. Synonymous nucleotide coding sequences vary from the exemplified coding sequences but encode proteins of the same amino acid sequences as those specifically provided herein or proteins with similar function and are therefore also regarded as functional equivalents thereof.
The term "promoter" as used herein refers to an untranslated (i.e. one that does not result in a peptide or protein product) sequence upstream of the polypeptide coding region of a nucleotide sequence that controls transcription of a gene. Promoters typically fall into two classes, constitutive and inducible. Inducible promoters initiate high levels of transcription of the nucleic acid under their control in response to external stimuli. Constitutive promoters maintain a relatively constant level of transcription in a given cell. Suitable promoters for use in the present may include both procaryotic and eukaryotic promoters, with all ubiquitin promoters being preferred, solanaceous plant ubiquitin promoters being highly preferred, and potato ubiquitin promoters being most preferred. Additional control sequences such as ribosomal binding sites and enhancers may be included as control sequences when necessary.
The term "polyadenylation" sites as used herein refers to a control sequence located on the 3' end of a gene construct that provides a signal for cleavage and polyadenylation of the transcription unit expressed from the promoter. These control sequences are known to one skilled in the art
The term "expression" as used herein refers to transcription and or translation of a nucleic acid sequence coding for a protein or peptide.
In one embodiment, the present invention is directed to an isolated nucleotide sequence comprising a gene coding for a ubiquitin polypeptide and functional equivalents thereof, linked to a ubiquitin promoter and functional equivalents thereof. Suitable ubiquitin promoters for use in the present invention include, but are not limited to, ubiquitin promoters from solanaceous plants. Preferably, the ubiquitin promoter is a potato plant ubiquitin promoter and most preferably it is the potato Ubi3 or Ubi7 promoter. In embodiments wherein the isolated nucleotide sequence codes for the potato Ubi3 promoter linked to a gene coding for a ubiquitin polypeptide it has a nucleotide sequence according to SEQ ID NO. 93. The Ubi3 promoter alone also has utility as constitutive promoter in eukaryotes.
In embodiments wherein the isolated nucleotide sequence codes for the potato Ubi7 promoter linked to a gene coding for a ubiquitin polypeptide it has a nucleotide sequence according to SEQ ID NO. 96. The Ubi7 nucleotide sequence according to SEQ ID NO. 96 includes an intron that is part of the ubiquitin transcription unit. The intron is not required for gene expression from the Ubi7 promoter, thus the Ubi7 promoter region without the intron can be considered as a specific functional equivalent of the Ubi7 promoter. The Ubi7 promoter alone, with or without the intron, has utility as a wound inducible promoter in eukaryotes.
Preferably, the nucleotide sequence comprising the isolated ubiquitin promoter and gene coding for a ubiquitin polypeptide further comprises a gene coding for a lytic peptide ligated to the 3' end of the gene coding for a ubiquitin polypeptide. Suitable genes coding for a lytic peptide have a nucleotide sequence coding for any one of the amino acid sequences according to SEQ ID NO. 1-91 and 97-98.
In one preferred embodiment, the present invention is directed to an isolated nucleotide sequence comprising a gene coding for a lytic peptide ligated to the 3' end of the gene coding for a ubiquitin polypeptide linked to the Ubi3 ubiquitin promoter having a nucleotide sequence according to SEQ ID NO. 92. In an alternative of this embodiment, the present invention is directed to an isolated nucleotide sequence comprising a gene coding for a lytic peptide ligated to the 3' end of the gene coding for a ubiquitin polypeptide linked to a Ubi7 ubiquitin promoter having a nucleotide sequence according to SEQ ID NO. 95.
In another embodiment, the present invention is directed to a recombinant nucleic acid expression vector. The vector is characterized in that it comprises a nucleotide sequence wherein a gene coding for a ubiquitin polypeptide is linked to a ubiquitin promoter. Preferably, the present invention is directed to a recombinant nucleic acid expression vector characterized in that it further comprises a nucleotide sequence wherein a gene coding for a lytic peptide is ligated to the 3' end of the gene coding for a ubiquitin polypeptide linked to a ubiquitin promoter. Suitable vectors for use in this invention include any eukaryotic or procaryotic expression vectors known in the art. Preferable vectors for use in this invention are pUC19 and pCGN1547.
In another embodiment, the present invention is directed to a host cell that is transformed by a recombinant DNA expression vector comprising a gene coding for a ubiquitin polypeptide linked to a ubiquitin promoter. Suitable host cells for transformation in the present invention include all known bacterial host cells, with all strains of Escherichia coli and Agrobacterium tumefaciens being preferred. Preferably, the present invention is directed to a host cell the recombinant DNA expression vector further comprises a gene coding for a lytic peptide ligated to the 3' end of the gene coding for a ubiquitin polypeptide linked to a ubiquitin promoter. Suitable genes coding for a lytic peptide have a nucleotide sequence coding for any one of the amino acid sequences according :to SEQ ID NO. 1-91 and 97-98.
In another embodiment, the present invention is directed to a method of sub-cloning nucleotide sequences coding for lytic peptides and expressing such sequences in cells. The method comprises a first step wherein a recombinant nucleic acid containing a gene coding for a lytic peptide ligated to a gene coding for a ubiquitin polypeptide linked to a ubiquitin promoter is produced in a first host cell. Suitable first host cells include any known bacterial host cells. Preferably, the first host cell is either an Escherichia coli cell or an Agrobacterium tumefaciens cell.
If the peptides are sub-cloned using such a ubiquitin-fusion expression vector, the following advantage results: the lytic peptide gene constructs have increased stability in the bacterial host. While not wishing to be bound by any one theory, the present inventors believe that the stability is due to the ubiquitin protein coding nucleic acid region fused to the 5' end of the lytic peptide nucleic acid sequence. Bacteria do not contain the endogenous hydrolase necessary for cleavage of the ubiquitin fusion protein, so the gene constructs are not toxic to bacteria, since active lytic peptide cannot released. Thus functional equivalents of the ubiquitin fusion polypeptide include any ubiquitin molecule that is capable of deceiving the host cell into viewing the gene construct and its products as non-toxic.
The present invention is also directed to a method of sub-cloning nucleotide sequences coding for lytic peptides and expressing such sequences in cells. The method comprises producing in a host cell a recombinant nucleic acid expression vector comprising a gene coding for a lytic peptide ligated to the 3' end of a gene coding for a ubiquitin promoter linked to a procaryotic promoter sequence. Suitable procaryotic promoters include those known to one skilled in the art to be active in prokaryotes and used in plasmid vectors for bacterial gene expression.
Set out in Table 1 below as illustrative examples of lytic peptides are the amino acid sequences of families of related lytic peptides. These lytic peptides are designated for ease of reference as SEQ ID NO. 1-91 and 97-98. Nucleic acid sequences coding for these lytic peptides and functional equivalents thereof represent examples of lytic peptide nucleic acid sequences that are sub-cloned to make ubiquitin-lytic peptide fusion gene constructs and polypeptides. The ubiquitin polypeptide, designated for ease of reference as SEQ ID NO. 94, and functional equivalents thereof, represents an example of the 5' fusion ubiquitin polypeptide.
TABLE 1__________________________________________________________________________LYTIC PEPTIDE SEQUENCES__________________________________________________________________________SEQ ID NO. 1Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys Lys Val Lys 1               5                  10                  15Lys Ala Val Lys Lys Ala Val Lys Lys Lys Lys       20                   25SEQ ID NO. 2Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val Lys Lys Ala 1               5                  10                  15Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala Val Lys Lys Lys Lys        20                  25                  30SEQ ID NO. 3Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val Ala Val Lys 1               5                  10                  15Ala Val Lys Lys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala       20                   25                 30Val Lys Lys Lys Lys   35SEQ ID NO. 4Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys Lys Val Lys 1               5                  10                  15Lys Ala Val Lys Lys Ala Val       20SEQ ID NO. 5Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val Lys Lys Ala 1               5                  10                  15Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala Val       20                  25SEQ ID NO. 6Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val Ala Val Lys 1               5                  10                  15Ala Val Lys Lys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala       20                   25                 30ValSEQ ID NO. 7Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Ala Leu Lys Lys Leu Arg 1               5                  10                  15Arg Gly Val Arg Lys Val Ala Lys Arg Lys Arg       20                  25SEQ ID NO. 8Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Ala Leu Lys Lys Leu Arg 1               5                  10                  15Arg Gly Val Arg Lys Val Ala       20SEQ ID NO. 9Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu 1               5                  10                  15Ala Arg Lys Ile Ala Arg Leu Gly Val Ala Phe       20                   25SEQ ID NO. 10Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu Ala Arg Lys Ile 1               5                  10                  15Ala Arg Leu Gly Val Ala Phe       20SEQ ID NO. 11Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Ala Leu Lys Lys Leu Arg 1               5                  10                  15Arg Gly Val Arg Lys Val Ala Lys Arg Lys Arg Lys Lys Asp Leu       20                   25                 30SEQ ID NO. 12Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Ala Leu Lys Lys Leu Arg 1               5                  10                  15Arg Gly Val Arg Lys Val Ala Lys Asp Leu       20                   25SEQ ID NO. 13Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu 1               5                  10                  15Ala Arg Lys Ile Ala Arg Leu Gly Val Ala Phe Lys Asp Leu       20                  25                  30SEQ ID NO. 14Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu Ala Arg Lys Ile 1               5                  10                  15Ala Arg Leu Gly Val Ala Phe Lys Asp Leu       20                  25SEQ ID NO. 15Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 1               5                  10                  15Ala Lys Lys Val Ala Lys Val Ala Val Ala Val       20                  25SEQ ID NO. 16Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 1               5                  10                  15Ala Lys Lys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Val       20                   25                 30SEQ ID NO. 17Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 1               5                  10                  15Ala Lys Lys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Lys       20                   25                 30Val Ala Val Ala Val   35SEQ ID NO. 18Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala Lys Lys Val 1               5                  10                  15Ala Lys Val Ala Val Ala Val       20SEQ ID NO. 19Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala Lys Lys Val 1               5                  10                  15Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Val       20                  25SEQ ID NO. 20Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala Lys Lys Val 1               5                  10                  15Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala       20                  25                  30ValSEQ ID NO. 21Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val 1               5                  10                  15Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val       20                  25SEQ ID NO. 22Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val 1               5                  10                  15Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala       20                  25                  30SEQ ID NO. 23Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val 1               5                  10                  15Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala       20                  25                  30Lys Val Ala Lys Lys   35SEQ ID NO. 24Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1               5                  10                  15Lys Lys Val Ala Lys Lys Val       20SEQ ID NO. 25Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1               5                  10                  15Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala       20                  25SEQ ID NO. 26Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1               5                  10                  15Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Val Ala Lys       20                  25                  30LysSEQ ID NO. 27Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1               5                  10                  15Lys Lys Val Ala Lys Lys Val Lys Lys Lys Lys       20                  25SEQ ID NO. 28Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1               5                  10                  15Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Lys Lys       20                  25                  30SEQ ID NO. 29Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1               5                  10                  15Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Val Ala Lys       20                  25                  30Lys Lys Lys Lys Lys   35SEQ ID NO. 30Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Lys Lys Lys Lys 1               5                  10                  15SEQ ID NO. 31Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15Ala Lys Lys Lys Lys       20SEQ ID NO. 32Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15Ala Lys Val Lys Ala Lys Val Lys Lys Lys Lys       20                  25SEQ ID NO. 33Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10SEQ ID NO. 34Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15AlaSEQ ID NO. 35Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15Ala Lys Val Lys Ala Lys Val       20SEQ ID NO. 36Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15SEQ ID NO. 37Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15Ala Lys Val Lys Ala       20SEQ ID NO. 38Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1               5                  10                  15Ala Lys Val Lys Ala Lys Val Lys Ala Lys Val       20                  25SEQ ID NO. 39Phe Lys Lys Val Lys Lys Val Ala Lys Lys Val Cys Lys Cys Val Lys 1               5                  10                  15Lys Ala Val Lys Lys Val`Lys Lys Phe       20                  25SEQ ID NO. 40Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys Lys Val Lys 1               5                  10                  15Lys Ala Val Lys Lys Ala Val Cys Cys Cys Cys       20                  25SEQ ID NO. 41Cys Cys Cys Cys Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 1               5                  10                  15Ala Lys Lys Val Ala Lys Val Ala Val Ala Val       20                  25SEQ ID NO. 42Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys Lys Val Lys 1               5                  10                  15Lys Ala Val Lys Lys Ala Val Ser Ser Ser Ser       20                  25SEQ ID NO. 43Ser Ser Ser Ser Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 1               5                  10                  15Ala Lys Lys Val Ala Lys Val Ala Val Ala Val       20                  25SEQ ID NO. 44Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Ala Leu Lys Lys Leu Lys 1               5                  10                  15Lys Ala Leu Lys Lys Ala Leu       20SEQ ID NO. 45Leu Ala Lys Lys Leu Ala Lys Lys Leu Lys Lys Leu Ala Lys Lys Leu 1               5                  10                  15Ala Lys Leu Ala Leu Ala Phe       20SEQ ID NO. 46Phe Ala Phe Ala Phe Lys Ala Phe Lys Lys Ala Phe Lys Lys Phe Lys 1               5                  10                  15Lys Ala Phe Lys Lys Ala Phe       20SEQ ID NO. 47Phe Ala Ile Ala Ile Lys Ala Ile Lys Lys Ala Ile Lys Lys Ile Lys 1               5                  10                  15Lys Ala Ile Lys Lys Ala Ile       20SEQ ID NO. 48Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe 1               5                  10                  15Ala Lys Phe Ala Phe Ala Phe       20SEQ ID NO. 49Phe Lys Arg Leu Ala Lys Ile Lys Val Leu Arg Leu Ala Lys Ile Lys 1               5                  10                  15ArgSEQ ID NO. 50Lys Leu Lys Leu Ala Val Lys Leu Val Gly Leu Leu Arg Lys Lys Arg 1               5                  10                  15Ala Leu Lys Ile Ala Leu Arg Gly Val Ala Lys Arg Ala Gly Arg Leu       20                  25                  30Ala Val Arg Lys Phe   35SEQ ID NO. 51Phe Ala Arg Ala Arg Lys Ala Arg Lys Lys Ala Arg Lys Lys Arg Lys 1               5                  10                  15Lys Ala Arg Lys Lys Ala Arg Lys Asp Arg       20                  25SEQ ID NO. 52Phe Ala Val Ala Val Cys Ala Val Cys Cys Ala Val Cys Cys Val Cys 1               5                  10                  15Cys Ala Val Cys Cys Ala Val       20SEQ ID NO. 53Phe Ala Val Ala Val Ser Ala Val Ser Ser Ala Val Ser Ser Val Ser 1               5                  10                  15Ser Ala Val Ser Ser Ala Val       20SEQ ID NO. 54Phe Ala Val Ala Val Ser Ala Val Ser Ser Ala Val Ser Ser Val Ser 1               5                  10                  15Ser Ala Val Ser Ser Ala Val Ser Ser Ser Ser       20                  25SEQ ID NO. 55Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe 1               5                  10                  15Ala Lys Phe Ala Phe Ala Phe Lys Lys Lys Lys       20                  25SEQ ID NO. 56Lys Lys Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe 1               5                  10                  15Ala Lys Lys Phe Ala Lys Phe Ala Phe Ala Phe       20                  25SEQ ID NO. 57Phe Ala Arg Lys Phe Leu Lys Arg Phe Lys Lys Phe Val Arg Lys Phe 1               5                  10                  15Ile Arg Phe Ala Phe Leu Phe       20SEQ ID NO. 58Phe Ala Arg Lys Phe Leu Lys Arg Phe Lys Lys Phe Val Arg Lys Phe 1               5                  10                  15Ile Arg Phe Ala Phe Leu Phe Lys Arg Lys Arg       20                  25SEQ ID NO. 59Lys Arg Lys Arg Phe Ala Arg Lys Phe Leu Lys Arg Phe Lys Lys Phe 1               5                  10                  15Val Arg Lys Phe Ile Arg Phe Ala Phe Leu Phe       20                  25SEQ ID NO. 60Ile Ala Lys Lys Ile Ala Lys Lys Ile Lys Lys Ile Ala Lys Lys Ile 1               5                  10                  15Ala Lys Ile Ala Ile Ala Ile       20SEQ ID NO. 61Ile Ala Lys Lys Ile Ala Lys Lys Ile Lys Lys Ile Ala Lys Lys Ile 1               5                  10                  15Ala Lys Ile Ala Ile Ala Ile Lys Lys Lys Lys       20                  25SEQ ID NO. 62Lys Lys Lys Lys Ile Ala Lys Lys Ile Ala Lys Lys Ile Lys Lys Ile 1               5                  10                  15Ala Lys Lys Ile Ala Lys Ile Ala Ile Ala Ile       20                  25SEQ ID NO. 63Ile Ala Arg Lys Ile Leu Lys Arg Ile Lys Lys Ile Val Arg Lys Phe 1               5                  10                  15Ile Arg Ile Ala Ile Leu Ile       20SEQ ID NO. 64Ile Ala Arg Lys Ile Leu Lys Arg Ile Lys Lys Ile Val Arg Lys Phe 1               5                  10                  15Ile Arg Ile Ala Ile Leu Ile Lys Arg Lys Arg       20                  25SEQ ID NO. 65Lys Arg Lys Arg Ile Ala Arg Lys Ile Leu Lys Arg Ile Lys Lys Ile 1               5                  10                  15Val Arg Lys Phe Ile Arg Ile Ala Ile Leu Ile       20                  25SEQ ID NO. 66Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15LeuSEQ ID NO. 67Lys Arg Lys Arg Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg 1               5                  10                  15Ala Lys Ile Lys Leu       20SEQ ID NO. 68Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Lys Arg Lys Arg       20SEQ ID NO. 69Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Arg Val Lys Leu Lys Ile       20SEQ ID NO. 70Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Arg Val Lys Leu Lys Ile Lys Arg Lys Arg       20                  25SEQ ID NO. 71Lys Arg Lys Arg Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg 1               5                  10                  15Ala Lys Ile Lys Leu Arg Val Lys Leu Lys Ile       20                  25SEQ ID NO. 72Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Arg Val Lys Leu Lys Ile Arg Ala Arg Ile Lys Leu       20                  25SEQ ID NO. 73Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Arg Val Lys Leu Lys Ile Arg Ala Arg Ile Lys Leu Lys Arg Lys       20                  25                  30ArgSEQ ID NO. 74Lys Arg Lys Arg Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg 1               5                  10                  15Ala Lys Ile Lys Leu Arg Val Lys Leu Lys Ile Arg Ala Arg Ile Lys       20                  25                  30LeuSEQ ID NO. 75Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Val Phe Ala Ile Leu Leu       20SEQ ID NO. 76Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg Ala Lys Ile Lys 1               5                  10                  15Leu Val Phe Ala Ile Leu Leu Lys Arg Lys Arg       20                  25SEQ ID NO. 77Lys Arg Lys Arg Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Leu Arg 1               5                  10                  15Ala Lys Ile Lys Leu Val Phe Ala Ile Leu Leu       20                  25SEQ ID NO. 78Val Phe Ala Ile Leu Leu Phe Lys Leu Arg Ala Lys Ile Lys Val Arg 1               5                  10                  15Leu Arg Ala Lys Ile Lys Leu       20SEQ ID NO. 79Val Phe Ala Ile Leu Leu Phe Lys Leu Arg Ala Lys Ile Lys Val Arg 1               5                  10                  15Leu Arg Ala Lys Ile Lys Leu Arg Lys Arg       20                  25SEQ ID NO. 80Lys Arg Lys Arg Val Phe Ala Ile Leu Leu Phe Lys Leu Arg Ala Lys 1               5                  10                  15Ile Lys Val Arg Leu Arg Ala Lys Ile Lys Leu       20                  25SEQ ID NO. 81Val Gly Glu Cys Val Arg Gly Arg Cys Pro Ser Gly Met Cys Cys Ser 1               5                  10                  15Gln Phe Gly Tyr Cys Gly Lys Gly Pro Lys Tyr Cys Gly       20                  25SEQ ID NO. 82Val Gly Glu Cys Val Arg Gly Arg Cys Pro Ser Gly Met Cys Cys Ser 1               5                  10                  15Gln Phe Gly Tyr Cys Gly Lys Gly Pro Lys Tyr Cys Gly Arg       20                  25                  30SEQ ID NO. 83Leu Gly Asp Cys Leu Lys Gly Lys Cys Pro Ser Gly Met Cys Cys Ser 1               5                  10                  15Asn Tyr Gly Phe Cys Gly Arg Gly Pro Arg Phe Cys Gly Lys       20                  25                  30SEQ ID NO. 84Gln Cys Ile Gly Asn Gly Gly Arg Cys Asn Glu Asn Val Gly Pro Pro 1               5                  10                  15Tyr Cys Cys Ser Gly Phe Cys Leu Arg Gln Pro Gly Gln Gly Tyr Gly       20                  25                  30Tyr Cys Lys Asn Arg   35SEQ ID NO. 85Cys Ile Gly Asn Gly Gly Arg Cys Asn Glu Asn Val Gly Pro Pro Tyr 1               5                  10                  15Cys Cys Ser Gly Phe Cys Leu Arg Gln Pro Asn Gln Gly Tyr Gly Val       20                  25                  30Cys Arg Asn Arg   35SEQ ID NO. 86Cys Ile Gly Gln Gly Gly Lys Cys Gln Asp Gln Leu Gly Pro Pro Phe 1               5                  10                  15Cys Cys Ser Gly Tyr Cys Val Lys Asn Pro Gln Asn Gly Phe Gly Leu       20                  25                  30Cys Lys Gln Lys   35SEQ ID NO. 87Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1               5                  10                  15Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys Ala Arg       20                  25                  30His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys   35                  40SEQ ID NO. 88Gln Arg Val Cys Asp Lys Pro Ser Gly Thr Trp Ser Gly Leu Cys Gly 1               5                  10                  15Asn Asn Asn Ala Cys Arg Gln Asn Cys Ile Gln Val Asp Arg Ala Lys       20                  25                  30Lys Gly Ser Cys Gln Phe Leu Tyr Pro Ala Lys Lys   35                  40SEQ ID NO. 89Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1               5                  10                  15Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg       20                  25                  30His Gly Ser Cys   35SEQ ID NO. 90Gln Arg Val Cys Asn Lys Pro Ser Gly Thr Trp Ser Gly Leu Cys Gly 1               5                  10                  15Asn Asn Asn Ala Cys Arg Gln Asn Cys Ile Lys Val Asp Arg Ala Lys       20                  25                  30Lys Gly Ser Cys   35SEQ ID NO. 91Met Leu Glu Glu Leu Phe Glu Glu Met Thr Glu Phe Ile Glu Glu Val 1               5                  10                  15Ile Glu Thr Met       20SEQ ID NO. 94Met Gln Ile Phe Val Lys Thr Leu 1                5Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser Asp Thr    10                  15                  20Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile   25                  30                  35Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu       40                   45                  50Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu           55                  60Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Gly Ser65                  70                  75SEQ ID NO. 97Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu 1               5                  10                  15Ala Arg Lys Ile Ala Arg Leu Gly Val Ala Lys Leu Ala Gly Leu Arg       20                  25                  30Ala Val Leu Lys Phe   35SEQ ID NO. 98Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu Asp Arg Lys Ile 1               5                  10                  15Asp Arg Leu Gly Val Asp Phe       20__________________________________________________________________________
To obtain the genomic clone containing a ubiquitin-ribosomal fusion promoter and ubiquitin polypeptide coding region, a αFIXII potato genomic library is first prescreened using PCR. The PCR primers are homologous to regions of the ubiquitin-ribosomal fusion cDNA (see Garbarino J., et al., Plant Molecular Biology 20: 235(1992); Garbarino J. and Belknap W., Plant Molecular Biology 30 24: 119 (1994); both of which are hereby incorporated by reference herein in their entirety). A primer 5' to the beginning ATG of ubiquitin and a primer complementary to a sequence near the 5' end of the ribosomal protein are used.
The library is plated in 22 aliquots containing approximately 0.5�106 pfu (plaque forming units) each on an E. coli lawn. A plug is taken from each of the 22 resulting plaques and the eluant from each is subjected to PCR under standard conditions. The PCR products are run on agarose gels. The gels are then blotted and probed with the ubiquitin coding region of the ubiquitin-ribosomal fusion cDNA according to standard conditions. Two of the plugs produce PCR products that hybridize to the cDNA probe., Both of these are the correct size for the predicted ubiquitin-ribosomal fusion genomic fragment.
The eluants from these two plugs are then plated and screened with the ubiquitin coding region of the ubiquitin-ribosomal fusion cDNA according to standard conditions. For verification, the positive plaques from the initial screen are replated and screened with a probe containing both the ribosomal protein-coding region and the 3' end of the potato ubiquitin-ribosomal fusion cDNA.
The genomic clones are sequenced using Sequenase version 2.0 15 (United States Biochemical Corporation) or Promega fmol DNA Sequencing System using standard conditions. A genomic clone containing both the ubiquitin-ribosomal fusion promoter region and the ubiquitin-ribosomal fusion coding region is identified.
A chimeric gene is then constructed with a portion of the potato ubiquitin-ribosomal fusion genomic clone ligated to a lytic peptide gene. PCR is used to generate the Ubi3 promoter and ubiquitin portion of the chimeric gene. The Ubi3 promoter region includes the 920 bp promoter region upstream of the ubiquitin ATG, and the ubiquitin polypeptide coding region is 228 bp plus 6 bp of a BamHI restriction site at the 3' end (SEQ ID NO. 93). The primers contain BamHI restriction sites and are homologous to the 5' end of the Ubi3 promoter and to the 3' end of the ubiquitin polypeptide coding region. The ubiquitin-ribosomal fusion genomic clone is used as the amplification template. This insert is first sub-cloned into she plasmid pCGN1547, as described in Garbarino et al., Plant Molecular Biology 24: 119 (1994). The Ubi3 insert is then isolated from pCGN1547 using the BamHI sites and ligated into pUCl9 under standard conditions. Transformation of E. coli is done according to standard conditions and correct sub-clones are confirmed by mini-prep or PCR DNA analysis. This plasmid is designated pUCUbi3.
A nucleotide fragment coding for the lytic peptide (corresponding to the amino acid sequence SEQ ID NO. 98) is synthesized using a nucleic acid synthesizer, adding a stop codon to the 3' end, and used as a PCR template. The 5' PCR primer homologous to the lytic peptide nucleotide sequence contains a BamHI site, and the 3' primer contains an XbaI site. These sites are used to sub-clone the PCR generated insert into pUC19. A nopaline synthase polyadenylation signal (NOS3' ) is then cloned 3' to the lytic peptide sequence. Following sequence analysis, the BamHI insert containing the Ubi3 promoter and ubiquitin coding region (SEQ ID NO. 93) is cloned 5' to the lytic peptide.
The entire Ubi3 ubiquitin-lytic peptide fusion gene construct, including the polyadenylation site, was isolated from pUC19 as an Asp718/HindIII restriction fragment and sub-cloned into the pCGN1547 Agrobacterium vector for use in plant transformation (see McBride, et al., Plant Molecular Biology 14:269 (1990). This plasmid is designated as pCGNUbi3-LP98.
TABLE 2__________________________________________________________________________NUCLEOTIDE SEQUENCE OF POTATO UBIQUITIN-RIBOSOMALFUSION PROMOTER (UBI3) AND UBIQUITIN CODING REGIONINSERT, AND UBIQUITIN-LYTIC PEPTIDE FUSION GENE CONSTRUCT__________________________________________________________________________SEQ ID NO. 92CCAAAGCACA TACTTATCGA TTTAAATTTC ATCGAAGAGA TTAATATCGA                               50ATAATCATAT ACATACTTTA AATACATAAC AAATTTTAAA TACATATATC                               100TGGTATATAA TTAATTTTTT AAAGTCATGA AGTATGTATC AAATACACAT                               150ATGGAAAAAA TTAACTATTC ATAATTTAAA AAATAGAAAA GATACATCTA                               200GTGAAATTAG GTGCATGTAT CAAATACATT AGGAAAAGGG CATATATCTT                               250GATCTAGATA ATTAACGATT TTGATTTATG TATAATTTCC AAATGAAGGT                               300TTATATCTAC TTCAGAAATA ACAATATACT TTTATCAGAA CATTCAACAA                               350AGCAACAACC AACTAGAGTG AAAAATACAC ATTGTTCTCT AGACATACAA                               400AATTGAGAAA AGAATCTCAA AATTTAGAGA AACAAATCTG AATTTCTAGA                               450AGAAAAAAAT AATTATGCAC TTTGCTATTG CTCGAAAAAT AAATGAAAGA                               500AATTAGACTT TTTTAAAAGA TGTTAGACTA GATATACTCA AAAGCTATTA                               550AAGGAGTAAT ATTCTTCTTA CATTAAGTAT TTTAGTTACA GTCCTGTAAT                               600TAAAGACACA TTTTAGATTG TATCTAAACT TAAATGTATC TAGAATACAT                               650ATATTTGAAT GCATCATATA CATGTATCCG ACACACCAAT TCTCATAAAA                               700AACGTAATAT CCTAAACTAA TTTATCCTTC AAGTCAACTT AAGCCCAATA                               750TACATTTTCA TCTCTAAAGG CCCAAGTGGC ACAAAATGTC AGGCCCAATT                               800ACGAAGAAAA GGGCTTGTAA AACCCTAATA AAGTGGCACT GGCAGAGCTT                               850ACACTCTCAT TCCATCAACA AAGAAACCCT AAAAGCCGCA GCGCCACTGA                               900TTTCTCTCCT CCAGGCGAAG ATG CAG ATC TTC GTG AAG ACC TTA                               944                Met Gln Ile Phe Val Lys Thr Leu                1                5ACG GGG AAG ACG ATC ACC CTA GAG GTT GAG TCT TCC GAC ACC                               986Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser Asp Thr    10                  15                  20ATC GAC AAT GTC AAA GCC AAG ATC CAG GAC AAG GAA GGG ATT                               1028Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile   25                  30                   35CCC CCA GAC CAG CAG CGT TTG ATT TTC GCC GGA AAG CAG CTT                               1070Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu       40                  45                  50GAG GAT GGT CGT ACT CTT GCC GAC TAC AAC ATC�CAG AAG GAG                               1112Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu           55                  60TCA ACT CTC CAT CTC GTG CTC CGT CTC CGT GGT GGT                               1148Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly65                  70                  75GGA TCC GCT GTT AAA AGA GTG GGT CGT AGG TTG AAA AAG TTG                               1190Gly Ser Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu       80                  85                  90GAC CGT AAG ATT GAT AGG TTA GGA GTT GAT TTT TGATC                               1228Asp Arg Lys Ile Asp Arg Leu Gly Val Asp Phe           95                  100SEQ ID NO. 93CCAAAGCACA TACTTATCGA TTTAAATTTC ATCGAAGAGA TTAATATCGA                               50ATAATCATAT ACATACTTTA AATACATAAC AAATTTTAAA TACATATATC                               100TGGTATATAA TTAATTTTTT AAAGTCATGA AGTATGTATC AAATACACAT                               150ATGGAAAAAA TTAACTATTC ATAATTTAAA AAATAGAAAA GATACATCTA                               200GTGAAATTAG GTGCATGTAT CAAATACATT AGGAAAAGGG CATATATCTT                               250GATCTAGATA ATTAACGATT TTGATTTATG TATAATTTCC AAATGAAGGT                               300TTATATCTAC TTCAGAAATA ACAATATACT TTTATCAGAA CATTCAACAA                               350AGCAACAACC AACTAGAGTG AAAAATACAC ATTGTTCTCT AGACATACAA                               400AATTGAGAAA AGAATCTCAA AATTTAGAGA AACAAATCTG AATTTCTAGA                               450AGAAAAAAAT AATTATGCAC TTTGCTATTG CTCGAAAAAT AAATGAAAGA                               500AATTAGACTT TTTTAAAAGA TGTTAGACTA GATATACTCA AAAGCTATTA                               550AAGGAGTAAT ATTCTTCTTA CATTAAGTAT TTTAGTTACA GTCCTGTAAT                               600TAAAGACACA TTTTAGATTG TATCTAAACT TAAATGTATC TAGAATACAT                               650ATATTTGAAT GCATCATATA CATGTATCCG ACACACCAAT TCTCATAAAA                               700AACGTAATAT CCTAAACTAA TTTATCCTTC AAGTCAACTT AAGCCCAATA                               750TACATTTTCA TCTCTAAAGG CCCAAGTGGC ACAAAATGTC AGGCCCAATT                               800ACGAAGAAAA GGGCTTGTAA AACCCTAATA AAGTGGCACT GGCAGAGCTT                               850ACACTCTCAT TCCATCAACA AAGAAACCCT AAAAGCCGCA GCGCCACTGA                               900TTTCTCTCCT CCAGGCGAAG ATG CAG ATC TTC GTG AAG ACC TTA                               944                 Met Gln Ile Phe Val Lys Thr Leu                 1               5ACG GGG AAG ACG ATC ACC CTA GAG GTT GAG TCT TCC GAC ACC                               986Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser Asp Thr    10                  15                  20ATC GAC AAT GTC AAA GCC AAG ATC CAG GAC AAG GAA GGG ATT                               1028Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile   25                  30                   35CCC CCA GAC CAG CAG CGT TTG ATT TTC GCC GGA AAG CAG CTT                               1070Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu       40                  45                  50GAG GAT GGT CGT ACT CTT GCC GAC TAC AAC ATC CAG AAG GAG                               1112Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu       40                  45                  50TCA ACT CTC CAT CTC GTG CTC CGT CTC CGT GGT GGT GGA TCC                               1154Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Gly Ser65                  70                  75__________________________________________________________________________
To obtain the genomic clone containing a polyubiquitin promoter, intron and ubiquitin polypeptide coding region, a λFIXII potato genomic library was first prescreened using PCR as described in Example 2 above. The PCR primers are homologous to regions of the polyubiquitin cDNA (see Garbarino J., et al., Plant Molecular Biology 20: 235(1992)). A primer homologous to the 5' untranslated region of ubiquitin in the polyubiquitin cDNA and a primer complementary to the amino terminus of the ubiquitin coding region in the polyubiquitin cDNA are used. A genomic clone containing both the polyubiquitin promoter region, intron, and the polyubiquitin coding region was identified.
A chimeric gene is then constructed with a portion of the potato polyubiquitin genomic clone ligated to a lytic peptide gene, as described in Example 2. PCR is used to generate the Ubi7-ubiquitin portion of the chimeric gene. The Ubi7 promoter region includes the 1220 bp promoter and 568 bp intron upstream of the ubiquitin ATG, and the ubiquitin polypeptide coding region is 328 bp plus 6 bp of a BamHI restriction site (SEQ ID NO. 96). This plasmid is designated pUCUbi7.
The entire Ubi7 ubiquitin-lytic peptide fusion gene construct, including the polyadenylation site, is isolated from pUC19 as an Asp78/partial HindIII: restriction fragment (the intron has an internal HindIII site) and sub-cloned into the pCGN1547 Agrobacterium vector for use in plant transformation. This plasmid is designated pCGNUbi7-LP98.
TABLE 3__________________________________________________________________________NUCLEOTIDE SEQUENCE OF POTATO POLYUBIQUITIN PROMOTERREGION (UB17) AND UBIQUITIN CODING REGION INSERT,AND UBIQUITIN-LYTIC PRPTIDE FUSION GENE CONSTRUCT__________________________________________________________________________SEQ ID NO. 95TTTATCAATC AGATTTGAAC ATATAAATAA ATATAAATTG TCTCAATAAT                               50TCTACATTAA ACTAATATTT GAAATCTCAA TTTTATGATT TTTTAAATTC                               100ACTTTATATC CAAGACAATT TNCANCTTCA AAAAGTTTTA TTAAANATTT                               150ACATTAGTTT TGTTGATGAG GATGACAAGA TNTTGGTCAT CAATTACATA                               200TACCCAAATT GAATAGTAAG CAACTTCAAT CTTTTTCATA ATGATAATGA                               250CAGACACAAN NNAAACCCAT TTATTATTCA CATTGATTGA GTTTTATATG                               300CAATATAGTA ATAATAATAA TATTTCTTAT AAAGCAAGAG GTCAATTTTT                               350TTTTAATTAT ACCACGTCAC TAAATTATAT TTGATAATGT AAAACAATTC                               400AAATTTTACT TAAATATCAT GAAATAAACT ATTTTTATAA CCAAATTACT                               450AAATTTTTCC AATAAAAAAA AGTCATTAAG AAGACATAAA ATAAATTTGA                               500GGTAAANGAG TGAAGTCGAC TGACTTTTTT TTTTTTTATC ATAAGAAAAT                               550AAATTATTAA CTTTAACCTA ATAAAACACT AATATAATTT CATGGAATCT                               600AATACTTACC TCTTAGAAAT AAGAAAAAGT GTTTCTAATA GACCCTCAAT                               650TTACATTAAA TATTTTCAAT CAAATTTAAA TAACAAATAT CAATATGAGG                               700TCAATAACAA TATCAAAATA ATATGAAAAA AGAGCAATAC ATAATATAAG                               750GGACGATTTA AGTGCGATTA TCAAGGTAGT ATTATATCCT AATTTGCTAA                               800TATTTGNGCT CTTATATTTA AGGTCATGTT CATGATAAAC TTGAAATGCG                               850CTATATTAGA GCATATATTA AAATAAAAAA ATACCTAAAA TAAAATTAAG                               900TTATTTTTAG TATATATTTT TTTACATGAC CTACATTTTT CTGGGTTTTT                               950CTAAAGGAGC GTGTAAGTGT CGACCTCATT CTCCTAATTT TCCCCACCAC                               1000ATAAAAATTA AAAAGGAAAG GTAGCTTTTG CGTGTTGTTT TGGTACACTA                               1050CACCTCATTA TTACACGTGT CCTCATATAA TTGGTTAACC CTATGAGGCG                               1100GTTTCGTCTA GAGTCGGCCA TGCCATCTAT AAAATGAAGC TTTCTGCACC                               1150TCATTTTTTT CATCTTCTAT CTGATTTCTA TTATAATTTC TCTCAATTGC                               1200CTTCAAATTT CTCTTTAAGG TTAGAATCTT CTCTATTTTT                               1240GGTTTTTGTA TGTTTAGATT CTCGAATTAG CTAATCAGGC GCTGTTATAG                               1290CCCTTCCTTT TGAGTCTCTC CTCGGTTGTC TTGATGGAAA AGGCCTAACA                               1340TTTGAGTTTT TTTACGTCTG GTTTGATGGA AAAGGCCTAC AATTGGCCGT                               1390TTTCCCCGTT CGTTTTGATG AAAAAGCCCC TAGTTTGAGA TTTTTTTTCT                               1440GTCGTTCGTT CTAAAGGTTT AAAATTAGAG TTTTTACATT TGTTTGATGA                               1490AAAAGGCCTT AAATTTGAGT TTTTCCGGTT GATTTGATGA AAAAGCCCTA                               1540GAATTTGTGT TTTTCCGTCG GTTTGATTCT GAAGGCCTAA AATTTGAGTT                               1590TCTCCGGCTG TTTTGATGAA AAAGCCCTAA ATTTGAGTTT CTCCGGCTGT                               1640TTTGATGAAA AAGCCCTAAA TTTGAAGTTT TTTCCCCGTG TTTTAGATTG                               1690TTTAGGTTTT AATTCTCGAA TCAGCTAATC AGGGAGTGTG AAAGCCCTAA                               1740ATTGAAGTTT TTTTCGTTGT TCTGATTGTT GTTTTTATGA ATTTGCAG                               1788ATG CAG ATC TTT GTG AAA ACT CTC ACC GGA AAG ACT ATC ACC                               1830Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr 1               5                  10CTA GAG GTG GAA AGT TCT GAT ACA ATC GAC AAC GTT AAG GCT                               1872Leu Glu Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala15                  20                  25AAG ATC CAG GAT AAG GAA GGA ATT CCC CCG GAT CAG CAA AGG                               1914Lys Ile Glu Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg    30                  35                  40CTT ATC TTC GCC GGA AAG CAG TTG GAG GAC GGA CGT ACT CTA                               1956Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu   45                  50                  55GCT GAT TAC AAC ATC CAG AAG GAG TCT ACC CTC CAT TTG GTG                               1998Ala Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu ValCTC CGT CTA CGT GGA GGT GGA TCC GCT GTT AAA AGA GTG GGT -                               2040Leu Arg Leu Arg Gly Gly Gly Ser Ala Val Lys Arg Val Gly           75                    80CGT AGG TTG AAA AAG TTG GAC CGT AAG ATT GAT AGG TTA GGA                               2082Arg Arg Leu Lys Lys Leu Asp Arg Lys Ile Asp Arg Leu Gly85                  90                  95GTT GAT TTT TGATCTAGAG CCCCGAATTT CCCCGA                               2127Val Asp Phe    100SEQ ID NO 96TTTATCAATC AGATTTGAAC ATATAAATAA ATATAAATTG TCTCAATAAT                               50TCTACATTAA ACTAATATTT GAAATCTCAA TTTTATGATT TTTTAAATTC                               100ACTTTATATC CAAGACAATT TNCANCTTCA AAAAGTTTTA TTAAANATTT                               150ACATTAGTTT TGTTGATGAG GATGACAAGA TNTTGGTCAT CAATTACATA                               200TACCCAAATT GAATAGTAAG CAACTTCAAT GTTTTTCATA ATGATAATGA                               250CAGACACAAN NNAAACCCAT TTATTATTCA CATTGATTGA GTTTTATATG                               300CAATATAGTA ATAATAATAA TATTTCTTAT AAAGCAAGAG GTCAATTTTT                               350TTTTAATTAT ACCACGTCAC TAAATTATAT TTGATAATGT AAAACAATTC                               400AAATTTTACT TAAATATCAT GAAATAAACT ATTTTTATAA CCAAATTACT                               450AAATTTTTCC AATAAAAAAA AGTCATTAAG AAGACATAAA ATAAATTTGA                               500GGTAAANGAG TGAAGTCGAC TGACTTTTTT TTTTTTTATC ATAAGAAAAT                               550AAATTATTAA CTTTAACCTA ATAAAACACT AATATAATTT CATGGAATCT                               600AATACTTACC TCTTAGAAAT AAGAAAAAGT GTTTCTAATA GACCCTCAAT                               650TTACATTAAA TATTTTCAAT CAAATTTAAA TAACAAATAT CAATATGAGG                               700TCAATAACAA TATCAAAATA ATATGAAAAA AGAGCAATAC ATAATATAAG                               750GGACGATTTA AGTGCGATTA TCAAGGTAGT ATTATATCCT AATTTGCTAA                               800TATTTGNGCT CTTATATTTA AGGTCATGTT CATGATAAAC TTGAAATGCG                               850CTATATTAGA GCATATATTA AAATAAAAAA ATACCTAAAA TAAAATTAAG                               900TTATTTTTAG TATATATTTT TTTACATGAC CTACATTTTT CTGGGTTTTT                               950CTAAAGGAGC GTGTAAGTGT CGACCTCATT CTCCTAATTT TCCCCACCAC                               1000ATAAAAATTA AAAAGGAAAG GTAGCTTTTG CGTGTTGTTT TGGTACACTA                               1050CACCTCATTA TTACACGTGT CCTCATATAA TTGGTTAACC CTATGAGGCG                               1100GTTTCGTCTA GAGTCGGCCA TGCCATCTAT AAAATGAAGC TTTCTGCACC                               1150TCATTTTTTT CATCTTCTAT CTGATTTCTA TTATAATTTC TCTCAATTGC                               1200CTTCAAATTT CTCTTTAAGG TTAGAATCTT CTCTATTTTT                               1240GGTTTTTGTA TGTTTAGATT CTCGAATTAG CTAATCAGGC GCTGTTATAG                               1290CCCTTCCTTT TGAGTCTCTC CTCGGTTGTC TTGATGGAAA AGGCCTAACA                               1340TTTGAGTTTT TTTACGTCTG GTTTGATGGA AAAGGCCTAC AATTGGCCGT                               1390TTTCCCCGTT CGTTTTGATG AAAAAGCCCC TAGTTTGAGA TTTTTTTTCT                               1440GTCGTTCGTT CTAAAGGTTT AAAATTAGAG TTTTTACATT TGTTTGATGA                               1490AAAAGGCCTT AAATTTGAGT TTTTCCGGTT GATTTGATGA AAAAGCCCTA                               1540GAATTTGTGT TTTTCCGTCG GTTTGATTCT GAAGGCCTAA AATTTGAGTT                               1590TCTCCGGCTG TTTTGATGAA AAAGCCCTAA ATTTGAGTTT CTCCGGCTGT                               1640TTTGATGAAA AAGCCCTAAA TTTGAAGTTT TTTCCCCGTG TTTTAGATTG                               1690TTTAGGTTTT AATTCTCGAA TCAGCTAATC AGGGAGTGTG AAAGCCCTAA                               1740ATTGAAGTTT TTTTCGTTGT TCTGATTGTT GTTTTTATGA ATTTGCAG                               1788ATG CAG ATC TTT GTG AAA ACT CTC ACC GGA AAG ACT ATC ACC                               1830Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr 1               5                  10CTA GAG GTG GAA AGT TCT GAT ACA ATC GAC AAC GTT AAG GCT                               1872Leu Glu Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala15                  20                  25AAG ATC CAG GAT AAG GAA GGA ATT CCC CCG GAT CAG CAA AGG                               1914Lys Ile Glu Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg    30                  35                  40CTT ATC TTC GCC GGA AAG CAG TTG GAG GAC GGA CGT ACT CTA                               1956Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu   45                  50                  55GCT GAT TAC AAC ATC CAG AAG GAG TCT ACC CTC CAT TTG GTG                               1998Ala Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val       60                  65                  70CTC CGT CTA CGT GGA GGT GGA TCC     2022Leu Arg Leu Arg Gly Gly Gly Ser           75__________________________________________________________________________
pUC19 and pCGN1547 plasmid vectors containing a potato (Solanum tuberosum) Ubi7 promoter and intron, a region coding for a ubiquitin polypeptide, and a gene coding for a lytic peptide are constructed according to Example 3. Each plasmid respectively contains one lytic peptide nucleotide sequence coding for an amino acid sequence corresponding to SEQ ID NO. 1, 7, 15, 21, 30, 39, 43, 52, 83, 86, 88, 90, and 91. The resultant pUC19 Ubi7 ubiquitin-lytic peptide recombinant plasmids are designated as follows: pUCUbi7-LP1, pUCUbi7-LP7, pUCUbi7-LP15, pUCUbi7-LP21, pUCUbi7-LP30, pUCUbi7-P39, pUCUbi7-LP43, pUCUbi7-LP52, pUCUbi7-LP83, pUCUbi7-LP86, pUCUbi7-LP88, pUCUbi7-LP90, and pUCUbi7-LP91. The resultant pCGN1547 Ubi7 ubiquitin-lytic peptide recombinant plasmids are designated as follows: pCGNUbi7-LP1, pCGNUbi7-LP7, pCGNUbi7-LP15, pCGNUbi7,-LP21, pCGNUbi7-LP30, pCGNUbi7-LP39, pCGNUbi7-LP43, pCGNUbi7-LP52, pCGNUbi7-LP83, pCGNUbi7-LP86, pCGNUbi7-LP88, pCGNUbi7-LP90, and pCGNUbi7-LP91.
Two chimeric genes containing a β-glucuronidase (GUS) reporter gene and the Ubi3 promoter were constructed in-pCGN1547 plasmid vectors according to Garbarino, J., and Belknap, W., Plant Molecular Biology 24: 119 (1994), hereby incorporated by reference in its entirety. The first vector contains the 920 bp Ubi3 promoter ligated to the GUS gene, and expresses the GUS protein. This plasmid is designated pCGNUbi3-GUS. The second vector contains the 920 bp Ubi3 promoter and 228 bp ubiquitin coding region ligated in frame to the GUS gene. This plasmid expresses a ubiquitin-GUS fusion polypeptide. This plasmid is designated pCGNUbi3-GUSf.
Two chimeric genes containing a β-glucuronidase (GUS) reporter gene and the Ubi7 promoter minus the intron region were constructed in pCGN1547 plasmid vectors using PCR, as described in Example 3 and in Garbarino, J., and Belknap, W., Plant Molecular Biology 24: 119 (1994). The first vector contains a 1156 bp Ubi7 promoter region insert, including she 5' untranslated region of ubiquitin, ligated to the GUS gene. This plasmid does not contain the Ubi7 intron and expresses the GUS protein. This plasmid is designated pCGNUbi7-GUS. The second vector contains the 1156 Ubi7 ubiquitin promoter from pCGNUbi7-GUS and the 228 bp ubiquitin coding region fused in frame to the GUS reporter gene. This plasmid expresses a ubiquitin-GUS fusion polypeptide and is designated pCGNUbi7-GUSf.
The chimeric plasmids pCGNUbi3-GUS, pCGNUbi3-GUSf, pCGNUbi7-GUS, and pCGNUbi7-GUSf from Example 5 are introduced into the potato (Solanum tuberosum) using Agrobacterium mediated transformation according to Garbarino, J., and Belknap, W. Plant Molecular Biology 24:119 (1994). The strain of Agrobacterium tumefaciens used for transformation (PC2760, see An, G., et al., EMBO J. 4: 277 (1985)) harbors the disarmed Ti plasmid pAL4404 (see Hoekema, A., et al., Nature 303: 179 (1983). Plant transformation is carried out as previously described in Synder, G. W., et al., Plant Cell Rep 12:324 (1993), except that 1 mg/l silver thiosulfate is added to the stage II transformation medium (see Chang, H. H., et al., Bot Bull Acad Sci 32: 63 (1991).
Expression of the ubiquitin-GUS fusion polypeptide and MRNA products and the GUS protein alone is examined by northern and western analysis, as described in Garbarino J., and Belknap, W., Plant Molecular Biology 24: 119 (1994). GUS protein expression is examined in the transgenic plants using western analysis. Although there is a wide range of activity among individual clones, the ubiquitin-GUS fusion polypeptide containing plants generally give 5-10 fold higher expression than the plants containing GUS protein alone. This higher level of protein expression Corresponds to similarly elevated mRNA transcription levels for the ubiquitin-GUS fusion constructs, as shown by northern analysis (described in Garbarino et al., Plant Molecular Biology 24: 119 (1994)). Western analysis also shows that the ubiquitin-GUS fusion polypeptide was appropriately processed by endogenous ubiquitin hydrolases co produce free GUS protein.
GUS protein activity is measured as described by Jefferson, R. A., et al., EMBO J. 6: 3901 (1987). Table 4 below shows a comparison of the GUS activities in plants transformed with pCGNUbi3-GUS (ubi-) and plants transformed with pCGNUbi3-GUSf (ubi+). The activity is measured in nmoles methyl umbelliferon (MU) per minute per milligram of protein. Methyl umbelliferon is the fluorescent product of the GUS enzymatic reaction.
TABLE 4______________________________________COMPARISON OF GUS PROTEIN ACTIVITY IN PLANTSTRANSFORMED WITH THE UBI3 PROMOTER WITH (+UBI)AND WITHOUT (-UBI) UBIQUITIN POLYPEPTIDE FUSIONGUS Activity (nmoles MU/min/mg protein)   Leaf                     SenescentConstruct   Meristem 2nd Leaf 5th Leaf                            Leaf   Tuber______________________________________3.2 - ubi   6.31 �            2.51 �                     1.79 �                             5.42 �                                   3.26 �   0.74     0.52     0.22    1.24  0.278.1 - ubi   25.8 �            9.98 �                     6.34 �                            19.20 �                                   14.2 �   2.08     2.10     1.00    6.11   1.63.5 + ubi   94.8 �            60.3 �                     32.7 �                             50.1 �                                   37.6 �   12.6     25.1     8.71    11.6  10.49.8 + ubi   33.3 �            18.9 �                     9.74 �                             22.7 �                                   20.7 �    0.5     2.75     0.99    3.57  3.45______________________________________
The chimeric plasmids pCGNUbi3-LP98 from Example 2 and pCGNUbi7-LP98 from Example 3 are introduced into the potato (Solanum tuberosum) using Agrobacterium mediated transformation according to Garbarino, J., and Belknap, W. Plant Molecular Biology 24:119 (1994). The strain of Agrobacterium tumefaciens used for transformation (PC2760, see An, G., et al., 3EMBO J. 4: 277 (1985)) harbors the disarmed Ti plasmid pAL4404 (see Hoekema, A., et al., Nature 303: 179 (1983). Plant transformation is carried out as previously described in Synder, G. W., et al., Plant Cell Rep 12:324 (1993), except that 1 mg/l silver thiosulfate is added to the stage II transformation medium (see Chang, H. H., et al., Bot Bull Acad Sci 32: 63 (1991).
Expression of the ubiquitin-lytic peptide fusion polypeptide and mRNA products is examined by northern and western analysis, in Garbarino J., and Belknap, W., Plant Molecular Biology 24: 119 (1994). Northern analysis shows that ubiquitin-lytic peptide mRNA is transcribed from the gene construct in the transgenic plants. Western analysis shows that the ubiquitin-lytic peptide fusion polypeptide is appropriately processed by endogenous ubiquitin in hydrolases to produce free lytic peptide.
mRNA expression from the cloned Ubi3 promoter was examined before and after wounding to determine if the cloned Ubi3 promoter is wound inducible in transformed plants (see Garbarino, J. and Belknap, W., Plant Molecular Biology 24:119 (1994)). Northern analysis comparing endogenous Ubi3 mRNA expression levels to pCGNUbi3-GUS and pCGNUbi3-GUSf mRNA expression levels in transformed plants (see Example 5) shows that while the endogenous Ubi3 mRNA transcription increases upon wounding, transcription from the recombinant Ubi3 plasmids does not. Thus the recombinant Ubi3 promoter does not have the wound inducible characteristic of the endogenous Ubi3 promoter. This result suggests that the 920 bp of upstream sequence cloned in the Ubi3 genomic clone is not sufficient to obtain wound-dependent activation of the promoter. The promoter instead is constitutive, however, it still demonstrates developmental regulation, as shown in Table 4 above.
In contrast, the cloned TUi-b promoter retains its wound-dependent induction. Northern analysis comparing the endogenous Ubi7 mRNA expression levels to the expression levels from pCGNUbi7-GUS and pCGNUbi7-GUSf in transformed plants (see Example 5) shows that both the endogenous and the cloned Ubi7 promoter have wound-dependent activation.
E. coli cultures, each respectively transformed with pUCUbi7-LP98 (Local Accession No. PBT-0273), pUCUbi3-LP98 (Local Accession No. PBT-0276), pUCUbi7 (Local Accession No. PBT-0277), and pUCUbi3 (Local Accession No. PBT-0234) were deposited in the Agricultural Research Service Culture Collection (NRRL). The depository is located at located at 1815 North University Street, Peoria, Ill.,
__________________________________________________________________________#             SEQUENCE LISTING- (1) GENERAL INFORMATION:-    (iii) NUMBER OF SEQUENCES: 98- (2) INFORMATION FOR SEQ ID NO: 1:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Ala Val Lys Ala Val Lys Lys Al - #a Val Lys Lys Val Lys#                15- Lys Ala Val Lys Lys Ala Val Lys Lys Lys Ly - #s#            25- (2) INFORMATION FOR SEQ ID NO: 2:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Ala Val Lys Ala Val Ala Val Ly - #s Ala Val Lys Lys Ala#                15- Val Lys Lys Val Lys Lys Ala Val Lys Lys Al - #a Val Lys Lys Lys Lys#            30- (2) INFORMATION FOR SEQ ID NO: 3:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 37     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Ala Val Lys Ala Val Ala Val Ly - #s Ala Val Ala Val Lys#                15- Ala Val Lys Lys Ala Val Lys Lys Val Lys Ly - #s Ala Val Lys Lys Ala#            305- Val Lys Lys Lys Lys   35- (2) INFORMATION FOR SEQ ID NO: 4:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Ala Val Lys Ala Val Lys Lys Al - #a Val Lys Lys Val Lys#                15- Lys Ala Val Lys Lys Ala Val       20- (2) INFORMATION FOR SEQ ID NO: 5:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Ala Val Lys Ala Val Ala Val Ly - #s Ala Val Lys Lys Ala#                15- Val Lys Lys Val Lys Lys Ala Val Lys Lys Al - #a Val#            25- (2) INFORMATION FOR SEQ ID NO: 6:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 33     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Ala Val Lys Ala Val Ala Val Ly - #s Ala Val Ala Val Lys#                15- Ala Val Lys Lys Ala Val Lys Lys Val Lys Ly - #s Ala Val Lys Lys Ala#            305- Val- (2) INFORMATION FOR SEQ ID NO: 7:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Al - #a Leu Lys Lys Leu Arg#                15- Arg Gly Val Arg Lys Val Ala Lys Arg Lys Ar - #g#            25- (2) INFORMATION FOR SEQ ID NO: 8:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Al - #a Leu Lys Lys Leu Arg#                15- Arg Gly Val Arg Lys Val Ala       20- (2) INFORMATION FOR SEQ ID NO: 9:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Arg Lys Arg Ala Val Lys Arg Val Gly Ar - #g Arg Leu Lys Lys Leu#                15- Ala Arg Lys Ile Ala Arg Leu Gly Val Ala Ph - #e#             25- (2) INFORMATION FOR SEQ ID NO: 10:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Ala Val Lys Arg Val Gly Arg Arg Leu Lys Ly - #s Leu Ala Arg Lys Ile#                15- Ala Arg Leu Gly Val Ala Phe       20- (2) INFORMATION FOR SEQ ID NO: 11:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 31     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Al - #a Leu Lys Lys Leu Arg#                15- Arg Gly Val Arg Lys Val Ala Lys Arg Lys Ar - #g Lys Lys Asp Leu#            305- (2) INFORMATION FOR SEQ ID NO: 12:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Ala Val Gly Leu Arg Ala Ile Lys Arg Al - #a Leu Lys Lys Leu Arg#                15- Arg Gly Val Arg Lys Val Ala Lys Asp Leu#            25- (2) INFORMATION FOR SEQ ID NO: 13:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Arg Lys Arg Ala Val Lys Arg Val Gly Ar - #g Arg Leu Lys Lys Leu#                15- Ala Arg Lys Ile Ala Arg Leu Gly Val Ala Ph - #e Lys Asp Leu#            30- (2) INFORMATION FOR SEQ ID NO: 14:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Ala Val Lys Arg Val Gly Arg Arg Leu Lys Ly - #s Leu Ala Arg Lys Ile#                15- Ala Arg Leu Gly Val Ala Phe Lys Asp Leu#            25- (2) INFORMATION FOR SEQ ID NO: 15:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Val Lys Lys Val Ala Ly - #s Lys Val Lys Lys Val#                15- Ala Lys Lys Val Ala Lys Val Ala Val Ala Va - #l#            25- (2) INFORMATION FOR SEQ ID NO: 16:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Val Lys Lys Val Ala Ly - #s Lys Val Lys Lys Val#                15- Ala Lys Lys Val Ala Lys Val Ala Val Ala Ly - #s Val Ala Val Ala Val#            305- (2) INFORMATION FOR SEQ ID NO: 17:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 37     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Val Lys Lys Val Ala Ly - #s Lys Val Lys Lys Val#                15- Ala Lys Lys Val Ala Lys Val Ala Val Ala Ly - #s Val Ala Val Ala Lys#            30- Val Ala Val Ala Val   35- (2) INFORMATION FOR SEQ ID NO: 18:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Val Lys Lys Val Ala Lys Lys Val Lys Ly - #s Val Ala Lys Lys Val#                15- Ala Lys Val Ala Val Ala Val       20- (2) INFORMATION FOR SEQ ID NO: 19:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Val Lys Lys Val Ala Lys Lys Val Lys Ly - #s Val Ala Lys Lys Val#                15- Ala Lys Val Ala Val Ala Lys Val Ala Val Al - #a Val#            25- (2) INFORMATION FOR SEQ ID NO: 20:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 33     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Val Lys Lys Val Ala Lys Lys Val Lys Ly - #s Val Ala Lys Lys Val#                15- Ala Lys Val Ala Val Ala Lys Val Ala Val Al - #a Lys Val Ala Val Ala#            30- Val- (2) INFORMATION FOR SEQ ID NO: 21:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Val Lys Lys Val Ala Ly - #s Val Ala Lys Lys Val#                15- Ala Lys Val Ala Lys Lys Val Ala Lys Lys Va - #l#            25- (2) INFORMATION FOR SEQ ID NO: 22:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Val Lys Lys Val Ala Ly - #s Val Ala Lys Lys Val#                15- Ala Lys Val Ala Lys Lys Val Ala Lys Lys Va - #l Ala Lys Lys Val Ala#            30- (2) INFORMATION FOR SEQ ID NO: 23:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 37     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Val Lys Lys Val Ala Ly - #s Val Ala Lys Lys Val#                15- Ala Lys Val Ala Lys Lys Val Ala Lys Lys Va - #l Ala Lys Lys Val Ala#            305- Lys Val Ala Lys Lys   35- (2) INFORMATION FOR SEQ ID NO: 24:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - 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#SLY PUBLISHED#36:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Va - #l Lys Ala Lys Val Lys#                15- (2) INFORMATION FOR SEQ ID NO: 37:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#37:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Va - #l Lys Ala Lys Val Lys#                15- Ala Lys Val Lys Ala       20- (2) INFORMATION FOR SEQ ID NO: 38:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#38:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Va - #l Lys Ala Lys Val Lys#                15- Ala Lys Val Lys Ala Lys Val Lys Ala Lys Va - #l#            25- (2) INFORMATION FOR SEQ ID NO: 39:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#39:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Lys Lys Val Lys Lys Val Ala Lys Lys Va - #l Cys Lys Cys Val Lys#                15- Lys Ala Val Lys Lys Val Lys Lys Phe#            25- (2) INFORMATION FOR SEQ ID NO: 40:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - 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#SLY PUBLISHED#74:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Arg Lys Arg Phe Lys Leu Arg Ala Lys Il - #e Lys Val Arg Leu Arg#                150- Ala Lys Ile Lys Leu Arg Val Lys Leu Lys Il - #e Arg Ala Arg Ile Lys#            30- Leu- (2) INFORMATION FOR SEQ ID NO: 75:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#75:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Le - #u Arg Ala Lys Ile Lys#                150- Leu Val Phe Ala Ile Leu Leu       20- (2) INFORMATION FOR SEQ ID NO: 76:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#76:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Phe Lys Leu Arg Ala Lys Ile Lys Val Arg Le - #u Arg Ala Lys Ile Lys#                150- Leu Val Phe Ala Ile Leu Leu Lys Arg Lys Ar - #g#            25- (2) INFORMATION FOR SEQ ID NO: 77:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#77:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Arg Lys Arg Phe Lys Leu Arg Ala Lys Il - #e Lys Val Arg Leu Arg#                150- Ala Lys Ile Lys Leu Val Phe Ala Ile Leu Le - #u#            25- (2) INFORMATION FOR SEQ ID NO: 78:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#78:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Val Phe Ala Ile Leu Leu Phe Lys Leu Arg Al - #a Lys Ile Lys Val Arg#                150- Leu Arg Ala Lys Ile Lys Leu       20- (2) INFORMATION FOR SEQ ID NO: 79:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#79:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Val Phe Ala Ile Leu Leu Phe Lys Leu Arg Al - #a Lys Ile Lys Val Arg#                150- Leu Arg Ala Lys Ile Lys Leu Lys Arg Lys Ar - #g#            25- (2) INFORMATION FOR SEQ ID NO: 80:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#80:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Arg Lys Arg Val Phe Ala Ile Leu Leu Ph - #e Lys Leu Arg Ala Lys#                150- Ile Lys Val Arg Leu Arg Ala Lys Ile Lys Le - #u#            25- (2) INFORMATION FOR SEQ ID NO: 81:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 29     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#81:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Val Gly Glu Cys Val Arg Gly Arg Cys Pro Se - #r Gly Met Cys Cys Ser#                150- Gln Phe Gly Tyr Cys Gly Lys Gly Pro Lys Ty - #r Cys Gly#            25- (2) INFORMATION FOR SEQ ID NO: 82:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#82:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Val Gly Glu Cys Val Arg Gly Arg Cys Pro Se - #r Gly Met Cys Cys Ser#                150- Gln Phe Gly Tyr Cys Gly Lys Gly Pro Lys Ty - #r Cys Gly Arg#            30- (2) INFORMATION FOR SEQ ID NO: 83:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#83:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Leu Gly Asp Cys Leu Lys Gly Lys Cys Pro Se - #r Gly Met Cys Cys Ser#                150- Asn Tyr Gly Phe Cys Gly Arg Gly Pro Arg Ph - #e Cys Gly Lys#            30- (2) INFORMATION FOR SEQ ID NO: 84:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 37     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#84:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Gln Cys Ile Gly Asn Gly Gly Arg Cys Asn Gl - #u Asn Val Gly Pro Pro#                150- Tyr Cys Cys Ser Gly Phe Cys Leu Arg Gln Pr - #o Gly Gln Gly Tyr Gly#            30- Tyr Cys Lys Asn Arg   35- (2) INFORMATION FOR SEQ ID NO: 85:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#85:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Ile Gly Asn Gly Gly Arg Cys Asn Glu As - #n Val Gly Pro Pro Tyr#                150- Cys Cys Ser Gly Phe Cys Leu Arg Gln Pro As - #n Gln Gly Tyr Gly Val#            30- Cys Arg Asn Arg   35- (2) INFORMATION FOR SEQ ID NO: 86:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#86:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Ile Gly Gln Gly Gly Lys Cys Gln Asp Gl - #n Leu Gly Pro Pro Phe#                150- Cys Cys Ser Gly Tyr Cys Val Lys Asn Pro Gl - #n Asn Gly Phe Gly Leu#            30- Cys Lys Gln Lys   35- (2) INFORMATION FOR SEQ ID NO: 87:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 44     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#87:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Tr - #p Ser Gly Val Cys Gly#                150- Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile As - #n Leu Glu Lys Ala Arg#            30- His Gly Ser Cys Asn Tyr Val Phe Pro Ala Hi - #s Lys#        40- (2) INFORMATION FOR SEQ ID NO: 88:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 44     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#88:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Gln Arg Val Cys Asp Lys Pro Ser Gly Thr Tr - #p Ser Gly Leu Cys Gly#                150- Asn Asn Asn Ala Cys Arg Gln Asn Cys Ile Gl - #n Val Asp Arg Ala Lys#            30- Lys Gly Ser Cys Gln Phe Leu Tyr Pro Ala Ly - #s Lys#        40- (2) INFORMATION FOR SEQ ID NO: 89:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#89:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Tr - #p Ser Gly Val Cys Gly#                150- Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Ar - #g Leu Glu Lys Ala Arg#            30- His Gly Ser Cys   35- (2) INFORMATION FOR SEQ ID NO: 90:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#90:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Gln Arg Val Cys Asn Lys Pro Ser Gly Thr Tr - #p Ser Gly Leu Cys Gly#                150- Asn Asn Asn Ala Cys Arg Gln Asn Cys Ile Ly - #s Val Asp Arg Ala Lys#            30- Lys Gly Ser Cys   35- (2) INFORMATION FOR SEQ ID NO: 91:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#91:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Leu Glu Glu Leu Phe Glu Glu Met Thr Gl - #u Phe Ile Glu Glu Val#                150- Ile Glu Thr Met       20- (2) INFORMATION FOR SEQ ID NO: 92:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1228     (B) TYPE: NUCLEIC ACID#STRANDED (C) STRANDEDNESS: DOUBLE     (D) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:#DNA AND OTHER NUCLEIC ACIDGENOMIC#92:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#              50TATCGA TTTAAATTTC ATCGAAGAGA TTAATATCGA#             100ACTTTA AATACATAAC AAATTTTAAA TACATATATC#             150TTTTTT AAAGTCATGA AGTATGTATC AAATACACAT#             200CTATTC ATAATTTAAA AAATAGAAAA GATACATCTA#             250ATGTAT CAAATACATT AGGAAAAGGG CATATATCTT#             300ACGATT TTGATTTATG TATAATTTCC AAATGAAGGT#             350GAAATA ACAATATACT TTTATCAGAA CATTCAACAA#             400AGAGTG AAAAATACAC ATTGTTCTCT AGACATACAA#             450TCTCAA AATTTAGAGA AACAAATCTG AATTTCTAGA#             500ATGCAC TTTGCTATTG CTCGAAAAAT AAATGAAAGA#             550AAAAGA TGTTAGACTA GATATACTCA AAAGCTATTA#             600TTCTTA CATTAAGTAT TTTAGTTACA GTCCTGTAAT#             650AGATTG TATCTAAACT TAAATGTATC TAGAATACAT#             700CATATA CATGTATCCG ACACACCAAT TCTCATAAAA#             750AACTAA TTTATCCTTC AAGTCAACTT AAGCCCAATA#             800TAAAGG CCCAAGTGGC ACAAAATGTC AGGCCCAATT#             850TTGTAA AACCCTAATA AAGTGGCACT GGCAGAGCTT#             900TCAACA AAGAAACCCT AAAAGCCGCA GCGCCACTGA- TTTCTCTCCT CCAGGCGAAG ATG CAG ATC TTC GTG AAG AC - #C TTA#944#    Met Gln Ile Phe Val Lys Thr Leu#  5 1- ACG GGG AAG ACG ATC ACC CTA GAG GTT GAG TC - #T TCC GAC ACC# 986Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Se - #r Ser Asp Thr#    20- ATC GAC AAT GTC AAA GCC AAG ATC CAG GAC AA - #G GAA GGG ATT#1028Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Ly - #s Glu Gly Ile#         35- CCC CCA GAC CAG CAG CGT TTG ATT TTC GCC GG - #A AAG CAG CTT#1070Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gl - #y Lys Gln Leu#            50- GAG GAT GGT CGT ACT CTT GCC GAC TAC AAC AT - #C CAG AAG GAG#1112Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Il - #e Gln Lys Glu#                60#     1148CTC CAT CTC GTG CTC CGT CTC CGT GG - #T GGTSer Thr Leu His Leu Val Leu Arg Leu Arg Gl - #y Gly#75- GGA TCC GCT GTT AAA AGA GTG GGT CGT AGG TT - #G AAA AAG TTG#1190Gly Ser Ala Val Lys Arg Val Gly Arg Arg Le - #u Lys Lys Leu#            90#   1228T AAG ATT GAT AGG TTA GGA GTT GAT TT - #T TGATCAsp Arg Lys Ile Asp Arg Leu Gly Val Asp Ph - #e#                100- (2) INFORMATION FOR SEQ ID NO: 93:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1154     (B) TYPE: NUCLEIC ACID#STRANDED (C) STRANDEDNESS: DOUBLE     (D) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:#DNA      (A) DESCRIPTION: GENOMIC#93:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#              50TATCGA TTTAAATTTC ATCGAAGAGA TTAATATCGA#             100ACTTTA AATACATAAC AAATTTTAAA TACATATATC#             150TTTTTT AAAGTCATGA AGTATGTATC AAATACACAT#             200CTATTC ATAATTTAAA AAATAGAAAA GATACATCTA#             250ATGTAT CAAATACATT AGGAAAAGGG CATATATCTT#             300ACGATT TTGATTTATG TATAATTTCC AAATGAAGGT#             350GAAATA ACAATATACT TTTATCAGAA CATTCAACAA#             400AGAGTG AAAAATACAC ATTGTTCTCT AGACATACAA#             450TCTCAA AATTTAGAGA AACAAATCTG AATTTCTAGA#             500ATGCAC TTTGCTATTG CTCGAAAAAT AAATGAAAGA#             550AAAAGA TGTTAGACTA GATATACTCA AAAGCTATTA#             600TTCTTA CATTAAGTAT TTTAGTTACA GTCCTGTAAT#             650AGATTG TATCTAAACT TAAATGTATC TAGAATACAT#             700CATATA CATGTATCCG ACACACCAAT TCTCATAAAA#             750AACTAA TTTATCCTTC AAGTCAACTT AAGCCCAATA#             800TAAAGG CCCAAGTGGC ACAAAATGTC AGGCCCAATT#             850TTGTAA AACCCTAATA AAGTGGCACT GGCAGAGCTT#             900TCAACA AAGAAACCCT AAAAGCCGCA GCGCCACTGA- TTTCTCTCCT CCAGGCGAAG ATG CAG ATC TTC GTG AAG AC - #C TTA#944#    Met Gln Ile Phe Val Lys Thr Leu#  5 1- ACG GGG AAG ACG ATC ACC CTA GAG GTT GAG TC - #T TCC GAC ACC# 986Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Se - #r Ser Asp Thr#    20- ATC GAC AAT GTC AAA GCC AAG ATC CAG GAC AA - #G GAA GGG ATT#1028Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Ly - #s Glu Gly Ile#         35- CCC CCA GAC CAG CAG CGT TTG ATT TTC GCC GG - #A AAG CAG CTT#1070Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gl - #y Lys Gln Leu#            50- GAG GAT GGT CGT ACT CTT GCC GAC TAC AAC AT - #C CAG AAG GAG#1112Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Il - #e Gln Lys Glu#                60- TCA ACT CTC CAT CTC GTG CTC CGT CTC CGT GG - #T GGT GGA TCC#1154Ser Thr Leu His Leu Val Leu Arg Leu Arg Gl - #y Gly Gly Ser#75- (2) INFORMATION FOR SEQ ID NO: 94:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 78     (B) TYPE: AMINO ACID     (D) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE#94:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Gln Ile Phe Val Lys Thr Leu 1                5- Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Se - #r Ser Asp Thr#    20- Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Ly - #s Glu Gly Ile#         35- Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gl - #y Lys Gln Leu#            50- Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Il - #e Gln Lys Glu#                60- Ser Thr Leu His Leu Val Leu Arg Leu Arg Gl - #y Gly Gly Ser#75- (2) INFORMATION FOR SEQ ID NO: 95:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2127     (B) TYPE: NUCLEIC ACID#STRANDED (C) STRANDEDNESS: DOUBLE     (D) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:#DNA AND OTHER DNARIPTION: GENOMIC#95:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#              50TTGAAC ATATAAATAA ATATAAATTG TCTCAATAAT#             100ATATTT GAAATCTCAA TTTTATGATT TTTTAAATTC#             150ACAATT TNCANCTTCA AAAAGTTTTA TTAAANATTT#             200GATGAG GATGACAAGA TNTTGGTCAT CAATTACATA#             250AGTAAG CAACTTCAAT GTTTTTCATA ATGATAATGA#             300ACCCAT TTATTATTCA CATTGATTGA GTTTTATATG#             350TAATAA TATTTCTTAT AAAGCAAGAG GTCAATTTTT#             400CGTCAC TAAATTATAT TTGATAATGT AAAACAATTC#             450TATCAT GAAATAAACT ATTTTTATAA CCAAATTACT#             500AAAAAA AGTCATTAAG AAGACATAAA ATAAATTTGA#             550GTCGAC TGACTTTTTT TTTTTTTATC ATAAGAAAAT#             600AACCTA ATAAAACACT AATATAATTT CATGGAATCT#             650AGAAAT AAGAAAAAGT GTTTCTAATA GACCCTCAAT#             700TTCAAT CAAATTTAAA TAACAAATAT CAATATGAGG#             750AAAATA ATATGAAAAA AGAGCAATAC ATAATATAAG#             800CGATTA TCAAGGTAGT ATTATATCCT AATTTGCTAA#             850TATTTA AGGTCATGTT CATGATAAAC TTGAAATGCG#             900ATATTA AAATAAAAAA ATACCTAAAA TAAAATTAAG#             950TATTTT TTTACATGAC CTACATTTTT CTGGGTTTTT#            1000AAGTGT CGACCTCATT CTCCTAATTT TCCCCACCAC#            1050GGAAAG GTAGCTTTTG CGTGTTGTTT TGGTACACTA#            1100ACGTGT CCTCATATAA TTGGTTAACC CTATGAGGCG#            1150CGGCCA TGCCATCTAT AAAATGAAGC TTTCTGCACC#            1200TTCTAT CTGATTTCTA TTATAATTTC TCTCAATTGC#  1240            AAGG TTAGAATCTT CTCTATTTTT#            1290TAGATT CTCGAATTAG CTAATCAGGC GCTGTTATAG#            1340TCTCTC CTCGGTTGTC TTGATGGAAA AGGCCTAACA#            1390CGTCTG GTTTGATGGA AAAGGCCTAC AATTGGCCGT#            1440TTGATG AAAAAGCCCC TAGTTTGAGA TTTTTTTTCT#            1490AGGTTT AAAATTAGAG TTTTTACATT TGTTTGATGA#            1540TTGAGT TTTTCCGGTT GATTTGATGA AAAAGCCCTA#            1590CCGTCG GTTTGATTCT GAAGGCCTAA AATTTGAGTT#            1640GATGAA AAAGCCCTAA ATTTGAGTTT CTCCGGCTGT#            1690CCTAAA TTTGAAGTTT TTTCCCCGTG TTTTAGATTG#            1740CTCGAA TCAGCTAATC AGGGAGTGTG AAAGCCCTAA#              1788TTGT TCTGATTGTT GTTTTTATGA ATTTGCAG- ATG CAG ATC TTT GTG AAA ACT CTC ACC GGA AA - #G ACT ATC ACC#1830Met Gln Ile Phe Val Lys Thr Leu Thr Gly Ly - #s Thr Ile Thr#                10- CTA GAG GTG GAA AGT TCT GAT ACA ATC GAC AA - #C GTT AAG GCT#1872Leu Glu Val Glu Ser Ser Asp Thr Ile Asp As - #n Val Lys Ala#25- AAG ATC CAG GAT AAG GAA GGA ATT CCC CCG GA - #T CAG CAA AGG#1914Lys Ile Glu Asp Lys Glu Gly Ile Pro Pro As - #p Gln Gln Arg#    40- CTT ATC TTC GCC GGA AAG CAG TTG GAG GAC GG - #A CGT ACT CTA#1956Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gl - #y Arg Thr Leu#         55- GCT GAT TAC AAC ATC CAG AAG GAG TCT ACC CT - #C CAT TTG GTG#1998Ala Asp Tyr Asn Ile Gln Lys Glu Ser Thr Le - #u His Leu Val#            70- CTC CGT CTA CGT GGA GGT GGA TCC GCT GTT AA - #A AGA GTG GGT#2040Leu Arg Leu Arg Gly Gly Gly Ser Ala Val Ly - #s Arg Val Gly#                80- CGT AGG TTG AAA AAG TTG GAC CGT AAG ATT GA - #T AGG TTA GGA#2082Arg Arg Leu Lys Lys Leu Asp Arg Lys Ile As - #p Arg Leu Gly#95#                2127GAG TCGACCGATC CCCCGAATTT CCCCGAVal Asp Phe    100- (2) INFORMATION FOR SEQ ID NO: 96:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2022     (B) TYPE: NUCLEIC ACID#STRANDED (C) STRANDEDNESS: DOUBLE     (D) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:#DNA      (A) DESCRIPTION: GENOMIC#96:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#              50TTGAAC ATATAAATAA ATATAAATTG TCTCAATAAT#             100ATATTT GAAATCTCAA TTTTATGATT TTTTAAATTC#             150ACAATT TNCANCTTCA AAAAGTTTTA TTAAANATTT#             200GATGAG GATGACAAGA TNTTGGTCAT CAATTACATA#             250AGTAAG CAACTTCAAT GTTTTTCATA ATGATAATGA#             300ACCCAT TTATTATTCA CATTGATTGA GTTTTATATG#             350TAATAA TATTTCTTAT AAAGCAAGAG GTCAATTTTT#             400CGTCAC TAAATTATAT TTGATAATGT AAAACAATTC#             450TATCAT GAAATAAACT ATTTTTATAA CCAAATTACT#             500AAAAAA AGTCATTAAG AAGACATAAA ATAAATTTGA#             550GTCGAC TGACTTTTTT TTTTTTTATC ATAAGAAAAT#             600AACCTA ATAAAACACT AATATAATTT CATGGAATCT#             650AGAAAT AAGAAAAAGT GTTTCTAATA GACCCTCAAT#             700TTCAAT CAAATTTAAA TAACAAATAT CAATATGAGG#             750AAAATA ATATGAAAAA AGAGCAATAC ATAATATAAG#             800CGATTA TCAAGGTAGT ATTATATCCT AATTTGCTAA#             850TATTTA AGGTCATGTT CATGATAAAC TTGAAATGCG#             900ATATTA AAATAAAAAA ATACCTAAAA TAAAATTAAG#             950TATTTT TTTACATGAC CTACATTTTT CTGGGTTTTT#            1000AAGTGT CGACCTCATT CTCCTAATTT TCCCCACCAC#            1050GGAAAG GTAGCTTTTG CGTGTTGTTT TGGTACACTA#            1100ACGTGT CCTCATATAA TTGGTTAACC CTATGAGGCG#            1150CGGCCA TGCCATCTAT AAAATGAAGC TTTCTGCACC#            1200TTCTAT CTGATTTCTA TTATAATTTC TCTCAATTGC#  1240            AAGG TTAGAATCTT CTCTATTTTT#            1290TAGATT CTCGAATTAG CTAATCAGGC GCTGTTATAG#            1340TCTCTC CTCGGTTGTC TTGATGGAAA AGGCCTAACA#            1390CGTCTG GTTTGATGGA AAAGGCCTAC AATTGGCCGT#            1440TTGATG AAAAAGCCCC TAGTTTGAGA TTTTTTTTCT#            1490AGGTTT AAAATTAGAG TTTTTACATT TGTTTGATGA#            1540TTGAGT TTTTCCGGTT GATTTGATGA AAAAGCCCTA#            1590CCGTCG GTTTGATTCT GAAGGCCTAA AATTTGAGTT#            1640GATGAA AAAGCCCTAA ATTTGAGTTT CTCCGGCTGT#            1690CCTAAA TTTGAAGTTT TTTCCCCGTG TTTTAGATTG#            1740CTCGAA TCAGCTAATC AGGGAGTGTG AAAGCCCTAA#              1788TTGT TCTGATTGTT GTTTTTATGA ATTTGCAG- ATG CAG ATC TTT GTG AAA ACT CTC ACC GGA AA - #G ACT ATC ACC#1830Met Gln Ile Phe Val Lys Thr Leu Thr Gly Ly - #s Thr Ile Thr#                10- CTA GAG GTG GAA AGT TCT GAT ACA ATC GAC AA - #C GTT AAG GCT#1872Leu Glu Val Glu Ser Ser Asp Thr Ile Asp As - #n Val Lys Ala#25- AAG ATC CAG GAT AAG GAA GGA ATT CCC CCG GA - #T CAG CAA AGG#1914Lys Ile Glu Asp Lys Glu Gly Ile Pro Pro As - #p Gln Gln Arg#    40- CTT ATC TTC GCC GGA AAG CAG TTG GAG GAC GG - #A CGT ACT CTA#1956Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gl - #y Arg Thr Leu#         55- GCT GAT TAC AAC ATC CAG AAG GAG TCT ACC CT - #C CAT TTG GTG#1998Ala Asp Tyr Asn Ile Gln Lys Glu Ser Thr Le - #u His Leu Val#            70#              2022GA GGT GGA TCCLeu Arg Leu Arg Gly Gly Gly Ser           75- (2) INFORMATION FOR SEQ ID NO: 97:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 37     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#97:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Arg Lys Arg Ala Val Lys Arg Val Gly Ar - #g Arg Leu Lys Lys Leu#                15- Ala Arg Lys Ile Ala Arg Leu Gly Val Ala Ly - #s Leu Ala Gly Leu Arg#            305- Ala Val Leu Lys Phe   35- (2) INFORMATION FOR SEQ ID NO: 98:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23     (B) TYPE: AMINO ACID     (C) TOPOLOGY: LINEAR-     (ii) MOLECULE TYPE:     (A) DESCRIPTION: PEPTIDE-    (iii) HYPOTHETICAL: NO-      (v) FRAGMENT TYPE: COMPLETE PEPTIDE-     (vi) ORIGINAL SOURCE: SYNTHETIC-    (vii) IMMEDIATE SOURCE: SYNTHETIC-      (x) PUBLICATION INFORMATION: NOT PREVIOU - #SLY PUBLISHED#98:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Ala Val Lys Arg Val Gly Arg Arg Leu Lys Ly - #s Leu Asp Arg Lys Ile#                 15- Asp Arg Leu Gly Val Asp Phe       20__________________________________________________________________________
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS5294605 *Jul 8, 1991Mar 15, 1994The Scripps Research InstituteAmphiphilic peptide compositions and analogues thereofUS5357044 *Feb 11, 1993Oct 18, 1994Xoma CorporationCecropins fusion proteinsEP0342926A2 *May 16, 1989Nov 23, 1989Mycogen Plant Science, Inc.Plant ubiquitin promoter systemEP0552559A2 *Dec 21, 1992Jul 28, 1993Unilever PlcTransgenic plants resistant to microbian infectionWO1990012866A1 *Apr 10, 1990Nov 1, 1990Univ Louisiana StateLytic peptides, use for growth, infection and cancerWO1994016076A1 *Jan 5, 1994Jul 21, 1994Zeneca LtdAntimicrobial-protein-producing endosymbiotic microorganisms* Cited by examinerNon-Patent CitationsReference1J.E. Garbarino et al., "Isolation of a Polyubiquitin . . . Plants", Plant Physiol. 109, pp. 1-8, 1995.2J.E. Garbarino et al., "Isolation of a ubiquitin-ribosomal . . . plants", Plant Molecular Biology 24, pp. 119-127, 1994.3 *J.E. Garbarino et al., Isolation of a Polyubiquitin . . . Plants , Plant Physiol. 109, pp. 1 8, 1995.4 *J.E. Garbarino et al., Isolation of a ubiquitin ribosomal . . . plants , Plant Molecular Biology 24, pp. 119 127, 1994.5N.E. Hoffman et al., "Isolation and characterization of . . . ubi3", Plant Molecular Biology 17, pp. 1189-1201, 1991.6 *N.E. Hoffman et al., Isolation and characterization of . . . ubi3 , Plant Molecular Biology 17, pp. 1189 1201, 1991.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS7288622Sep 19, 2006Oct 30, 2007Issar Pharmaceuticals Pvt LtdComposition for treatment of burns and woundsClassifications U.S. Classification530/324, 530/858International ClassificationC07K14/435, C07K14/47, C12N15/82, A61K38/17, C07K14/00, A61K38/10, A61K38/00, C07K7/08, C07K14/46, C07K14/415Cooperative ClassificationY10S530/858, A61K38/1709, A61K38/1703, C07K14/43563, C07K14/46, C07K14/415, C12N15/8239, A61K38/17, A61K38/10, C12N15/8279, C12N15/8216, C07K14/00, C07K14/435, C07K14/001, C07K14/4723, C07K2319/00, C07K7/08, C12N15/8257, C07K14/43572, C12N15/8222European ClassificationC07K7/08, C12N15/82C8B6, C07K14/415, C12N15/82B20, C07K14/47A14, C12N15/82B, C07K14/00B, C07K14/435, C12N15/82C4D, A61K38/17, C07K14/46, C07K14/435A4, A61K38/17A, C07K14/00, A61K38/17A2, C12N15/82B24D, C07K14/435A4C, A61K38/10Legal EventsDateCodeEventDescriptionOct 24, 2000CCCertificate of correctionApr 9, 2003REMIMaintenance fee reminder mailedSep 22, 2003SULPSurcharge for late paymentSep 22, 2003FPAYFee paymentYear of fee payment: 4Mar 20, 2007FPAYFee paymentYear of fee payment: 8Jan 27, 2011FPAYFee paymentYear of fee payment: 12RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services