Papaya ringspot virus replicase gene

FIELD OF THE INVENTION
This invention relates to a replicase gene derived from papaya ringspot virus. More specifically, the invention relates to the genetic engineering of plants and to a method for conferring viral resistance to a plant using an expression cassette encoding papaya ringspot virus PRV FLA.83 W replicase.
BACKGROUND OF THE INVENTION
Many agriculturally important crops are susceptible to infection by plant viruses, particularly papaya ringspot virus, which can seriously damage a crop, reduce its economic value to the grower, and increase its cost to the consumer. Attempts to control or prevent infection of a crop by a plant virus such as papaya ringspot virus have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the genetic material which provides the protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. "Resistant" is the opposite of "susceptible," and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
Potyviruses are a distinct group of plant viruses which are pathogenic to various crops, and which demonstrate cross-infectivity between plant members of different families. Generally, a potyvirus is a single-stranded RNA virus that is surrounded by a repeating protein monomer, which is termed the coat protein (CP). The majority of the potyviruses are transmitted in a nonpersistent manner by aphids. As can be seen from the wide range of crops affected by potyviruses, the host range includes such diverse families of plants as Solanaceae, Chenopodiaceae, Gramineae, Compositae, Leguminosae, Dioscroeaceae, Cucurbitaceae, and Caricaceae. Potyviruses include watermelon mosaic virus II (WMVII); zucchini yellow mosaic virus (ZYMV), potato virus Y, tobacco etch and many others.
Another potyvirus of economic significance is papaya ringspot virus (PRV). Two groups of PRV have been identified: the "P" or "papaya ringspot" type infects papayas; and the "W" or "watermelon" type infects cucurbits, e.g., squash, but it is unable to infect papaya. Thus, these two groups can be distinguished by host range differences.
The potyviruses consist of flexous, filamentous particles of dimensions approximately 780.times.12 nanometers. The viral particles contain a single-stranded positive polarity RNA genome containing about 10,000 nucleotides. Translation of the RNA genome of potyviruses shows that the RNA encodes a single large polyprotein of about 330 kD. This polyprotein contains several proteins; these include the coat protein, nuclear inclusion proteins NIa and NIb, cytoplasmic inclusion protein (CI), and other proteases and movement proteins. These proteins are found in the infected plant cell and form the necessary components for viral replication. One of the proteins contained in the polyprotein is a 35 kD capsid or coat protein which coats and protects the viral RNA from degradation. One of the nuclear inclusion proteins, NIb, is an RNA replicase component and is thought to have polymerase activity. CI, a second inclusion protein, is believed to participate in the replicase complex and have a helicase activity. NIa, a third inclusion protein, has a protease activity. In the course of potyvirus infection, NIa and NIb are translationally transported across the nuclear membrane into the nucleus of the infected plant cell at the later stages of infection and accumulate to high levels.
The location of the protease gene appears to be conserved in these viruses. In the tobacco etch virus, the protease cleavage site has been determined to be the dipeptide Gln-Ser, Gln-Gly, or Gln-Ala. Conservation of these dipeptides at the cleavage sites in these viral polyproteins is apparent from the sequences of the above-listed potyviruses.
Expression of the coat protein genes from tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus. For reviews, see Fitchen et al., Annu. Rev. Microbiol., 47, 739 (1993) and Wilson, Proc. Natl. Acad. Sci. USA 90, 3134 (1993). For papaya ringspot virus, Ling et al. (Bio/Technology, 9, 752 (1991)) found that transgenic tobacco plants expressing the PRV coat protein gene isolated from the PRV strain HA 5-1 (mild) showed delayed symptom development and attenuation of symptoms after infection by a number of potyviruses, including tobacco etch (TEV), potato virus Y (PVY), and pepper mottle virus (PeMV). PRV does not infect tobacco, however. Thus, PRV CP transgenic tobacco plants cannot be used to evaluate protection against PRV. Fitch et al. (Bio/Technology, 10, 1466 (1992)), Gonsalve (American J. of Bot., 79, 88 (1992)), and Lius et al (91st Annual Meeting of the American Society for Horticultural Science Hortscience, 29, 483 (1994)) reported that four R.sub.o papaya plants made transgenic for a PRV coat protein gene taken from strain HA 5-1 (mild) displayed varying degrees of resistance against PRV infection, and one line (S55-1) appeared completely resistant to PRV. This appears to be the only papaya line that shows complete resistance to PRV infection.
Even though coat protein-mediated viral resistance has proven to be useful in variety of situations, it may not always be the most effective or the most desirable means for providing viral resistance. In such instances, it would be advantageous to have other methods for conferring viral resistance to plants. Interference with plant viral RNA polymerase activity is an approach to inhibit viral RNA replication and inhibit viral symptoms in plants.
A fragment of the putative replicase gene from tobacco mosaic virus (TMV) recently has been found to provide resistance against TMV when expressed in plants (Golemboski et al., Proc. Natl. Acad. Sci. USA, 87, 6311(1990); Carr et al., Molec Plant-Microbe Interactions, 4, 579 (1991); Carr et al., Mol. Plant-Microbe Interactions, 5, 397 (1992); and Zaitlin et al., PCT publication WO 91/1354)). In addition, the following viral polymerase genes also confer resistance: a defective replicase gene from cucmber mosaic virus (Anderson et al., Proc. Natl. Acad. Sci. USA, 89, 8759 (1992)), a region of the 201-kDa replicase gene from pea early browning virus (MacFarlane et al., Proc. Natl. Acad. Sci USA, 89, 5829 (1992)), AL1 antisense gene of tomato golden mosaic virus (Day et al., Proc. Natl. Acad. Sci. USA, 88, 6721 (1991); Bejarano et al., TIBTECH, 10, 383 (1992)), a modified component of the putative potato virus X replicase (Longstaff et al., EMBO Journal 12, 379 (1993)), and a defective 126-kDa protein of tobacco mosaic virus (Donson, Phytopathology, 82, 1071 (1992).
Thus, there is a continuing need for the transgenic expression of genes derived from potyviruses at levels which confer resistance to infection by these viruses.
SUMMARY OF THE INVENTION
This invention provides an isolated and purified DNA molecule that encodes the replicase for the FLA.83 W-type strain of papaya ringspot virus (PRV). The invention also provides a chimeric expression cassette comprising this DNA molecule, a promoter which functions in plant cells to cause the production of an RNA molecule, and at least one polyadenylation signal comprising 3' nontranslated DNA which functions in plant cells to cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3' end of the transcribed mRNA sequences, wherein the promoter is operably linked to the DNA molecule, and the DNA molecule is operably linked to the polyadenylation signal. Another embodiment of the invention is exemplified by the insertion of multiple virus gene expression cassettes into one purified DNA molecule, e.g., a plasmid. Preferably, these cassettes include the promoter of the 35S gene of cauliflower mosaic virus and the polyadenylation signal of the cauliflower mosaic virus 35S gene.
Also provided are bacterial cells, and transformed plant cells, containing the chimeric expression cassettes comprising the replicase gene derived from the FLA.83 W-type strain of papaya ringspot virus (referred to herein as PRV FLA83 W), and preferably the 35S promoter of cauliflower mosaic virus and the polyadenylation signal of the cauliflower mosaic virus 35S gene. Plants are also provided, wherein the plants comprise a plurality of transformed cells transformed with an expression cassettes comprising the replicase gene derived from the PRV FLA83 W strain, and preferably the cauliflower mosaic virus 35S promoter and the polyadenylation signal of the cauliflower mosaic virus gene. Transformed plants of this invention include tobacco, corn, cucumber, peppers, potatoes, soybean, squash, and tomatoes. Especially preferred are members of the Cucurbitaceae (e.g., squash and cucumber) family.
Another aspect of the present invention is a method of preparing a PRV-resistant plant, such as a dicot, comprising: transforming plant cells with a chimeric expression cassette comprising a promoter functional in plant cells operably liked to a DNA molecule that encodes a replicase as described above; regenerating the plant cells to provide a differentiated plant; and identifying a transformed plant that expresses the PRV replicase at a level sufficient to render the plant resistant to infection by the specific strain of PRV disclosed herein.
As used herein, with respect to a DNA molecule or "gene," the phrase "isolated and purified" is defined to mean that the molecule is either extracted from its context in the viral genome by chemical means and purified and/or modified to the extent that it can be introduced into the present vectors in the appropriate orientation, i.e., sense or antisense. As used herein, the term "chimeric" refers to the linkage of two or more DNA molecules which are derived from different sources, strains or species (e.g., from bacteria and plants), or the linkage of two or more DNA molecules, which are derived from the same species and which are linked in a way that does not occur in the native genome. As used herein, the term "expression" is defined to mean transcription or transcription followed by translation of a particular DNA molecule.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1(a-c). The nucleotide sequence and deduced amino acid sequence of the nuclear inclusion body B (NIb) replicase gene of PRV FLA83 W [SEQ ID NO:1 and SEQ ID NO:2, respectively]. The amino acid sequence of the encoded open reading frame is shown below the nucleotide sequence.
FIGS. 2(a-d). The alignment of the nucleotide sequences of the nuclear inclusion body B (NIb) coding sequences from PRV isolates: P99-HA-P (Quemada et al., J. Gen. Virol., 71, 203 (1990)) [SEQ ID NO:5]; Prv-Ha-P (Yeh et al., J. Gen. Virol., 73, 2531 (1992)) [SEQ ID NO:6]; and Prv-W (Quemada et al., J. Gen. Virol., 71, 203 (1990)) [SEQ ID NO:7]. The position of primers RMM335 [SEQ ID NO:3] and RMM36 [SEQ ID NO:4] relative to the NIb sequence is shown. The sequences in RMM35 and RMM36 are homologous to sequences in PRV HA (attenuated) USA P (Quemada et al., J. Gen. Virol., 71, 203 (1990)). In addition, RMM335 has novel restriction endonuclease cleavage sites for EcoRI and NcoI while RMM36 has novel restriction endonuclease cleavage sites for BamHI and NcoI. The dots represent either the lack of sequence information at the ends of the NIb gene or gaps in homology in sequences relative to others in the alignment. Sequence alignments were generated using the UWGCG program Pileup.
FIGS. 3(a-b). The alignment of the amino acid sequences from papaya ringspot virus isolates described in FIG. 2 (Prv-W is identified as SEQ ID NO:8, P99-Ha-P is identified as SEQ ID NO:9, and Prv-Ha-P is identified as SEQ ID NO: 10). Sequence differences between virus strains are underlined. The dots represent either the lack of sequence information at the ends of the NIb gene or gaps in homology in sequences relative to others in the alignment. Alignments were generated using the UWGCG Pileup program.
FIGS. 4(a-c). The alignment of PRV FLA83 W NIb and PVY (Strain N, "Pvyaaa" in the Figure [SEQ ID NO:11]) NIb (Robaglia et al., J. Gen. Virol., 70, 935 (1989)) nucleotide sequences. The dots represent either the lack of sequence information at the ends of the NIb gene or gaps in homology in sequences relative to others in the alignment. Alignments were generated using the UWGCG Pileup program.
FIG. 5. The alignment of PRV FLA83 W NIb and PVY (Strain N) NIb (Robaglia et al., J. Gen. Virol., 70, 935 (1989)) [SEQ ID NO:12] amino acid sequences. The dots represent either the lack of sequence information at the ends of the NIb gene or gaps in homology in sequences relative to others in the alignment. Alignments were generated using the UWGCG Pileup program.
FIGS. 6(a-b). Schematic representation of the assembly of the papaya ringspot virus FLA83 NIb expression cassette vectors. Installation of cassettes into binary vectors is described in Table 1. (A) Assembly of the papaya ringspot virus FLA83 NIb gene expression cassette. (B) Assembly of the binary vectors for papaya ringspot virus FLA83 NIb expression in plants.

DETAILED DESCRIPTION OF THE INVENTION
Papaya ringspot virus (PRV) is a single-stranded (+) RNA plant virus that is translated into a single polyprotein. The viral RNA genome is approximately 10,000 bases in length. The expression strategy of potyviruses includes translation of a complete polyprotein from the positive sense viral genomic RNA. Translation of the genomic RNA produces a 330 kD protein which is subsequently cleaved into at least seven smaller viral proteins by a virally encoded protease. The virally encoded proteins include a 35 kD protein at the amino terminal end of the 330 kD protein which is thought to be involved in cell to cell transmission, H C protein is 56 kD in size and is believed to be involved in insect transmission and possess proteolytic activity, a 50 kD protein, a 90 kD cylindrical inclusion protein (CI) which is part of the replicase complex and possesses helicase activity, a 6 kD VPg protein which is covalently attached to the 5' end of the viral genomic RNA, a 49 kD NIa protein which functions as a protease, a 60 kD NIb protein which functions as a polymerase, and the coat protein (36 kD).
Two types of PRV have been established based on host range. One type is designated "P type"; it infects Caricacae (e.g., papaya), Cucurbitaceae (e.g., cucurbitis), and Chenopodiaceae (e.g., Chenopodium) (Wang et al., Phytopathology, 84, 1205 (1994)). A second type is designated "W type"; it infects only Cucurbitaceae and Chenopodiaceae (Wang et al., Phytopathology, 84, 1205 (1994)). Isolates of the P type include HA-severe, called HA-P herein (Wang et al., Arch Virol., 127, 345 (1992)), HA5-1, called USA P herein, YK (Wang et al., Phytopathology, 84, 1205 (1994)), and other isolates as described in Tennant et al. (Phytopatholovy, 84, 1359 (1994)). Isolates of the W type include FLA83, disclosed herein, PRV-W type (Yeh et al., Phytopathology, 74, 1081 (1984)) and PRV-W (Aust) (Bateson et al., Arch-Viol., 123, 101 (1992)).
To practice the present invention, the replicase (NIb) gene of a virus must be isolated from the viral genome and inserted into a vector. Thus, the present invention provides isolated and purified DNA molecules that encode the replicase of PRV FLA83. As used herein, a DNA molecule that encodes a coat protein gene includes nucleotides of the coding strand, also referred to as the "sense" strand, as well as nucleotides of the noncoding strand, complementary strand, also referred to as the "antisense" strand, either alone or in their base-paired configuration. Thus, a DNA molecule that encodes the replicase of PRV FLA83, for example, includes the DNA molecule having the nucleotide sequence of FIG. 1 [SEQ ID NO:1], a DNA molecule complementary to the nucleotide sequence of FIG. 1 [SEQ ID NO:1], as well as a DNA molecule which also encodes a PRV replicase and its complement which hybridizes with a PRV FLA83-specific DNA probe in hybridization buffer with 6.times.SSC, 5.times. Denhardt's reagent, 0.5% SDS and 100 .mu.g/ml denatured, fragmented salmon sperm DNA and remains bound when washed at 68.degree. C. in 0.1.times.SSC and 0.5% SDS (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989). Moreover, the DNA molecules of the present invention can include non-PRV replicase nucleotides that do not interfere with expression. Preferably, the isolated and purified DNA molecules of the present invention comprise a single coding region for the replicase. Thus, preferably the DNA molecules of the present invention are those "consisting essentially of" DNA that encodes the PRV replicase.
The PRV replicase gene does not contain the signals necessary for its expression once transferred and integrated into a plant genome. Accordingly, a vector must be constructed to provide the regulatory sequences such that they will be functional upon inserting a desired gene. When the expression vector/insert construct is assembled, it is used to transform plant cells which are then used to regenerate plants. These transgenic plants carry the viral gene in the expression vector/insert construct. The gene is expressed in the plant and increased resistance to viral infection is conferred thereby.
Several different methods exist to isolate the replicase gene. To do so, one having ordinary skill in the art can use information about the genomic organization of potyviruses to locate and isolate the replicase gene. The replicase gene is located in the 3' half of the genome, between the NIa gene and the coat protein gene. Additionally, the information related to proteolytic cleavage sites is used to determine the N-terminus of the replicase gene. The protease recognition sites are conserved in the potyviruses and have been determined to be either the dipeptide Gln-Ser, Gln-Gly, or Gln-Ala. The nucleotide sequences which encode these dipeptides can be determined.
Using methods well known in the art, a quantity of virus is grown and harvested. The viral RNA is then separated and the replicase gene isolated using a number of known procedures. A cDNA library is created using the viral RNA, by methods known to the art. The viral RNA is incubated with primers that hybridize to the viral RNA and reverse transcriptase, and a complementary DNA molecule is produced. A DNA complement of the complementary DNA molecule is produced and that sequence represents a DNA copy (cDNA) of the original viral RNA molecule. The DNA complement can be produced in a manner that results in a single double stranded cDNA or polymerase chain reactions can be used to amplify the DNA encoding the cDNA with the use of oligomer primers specific for the coat protein. These primers can include novel restriction sites used in subsequent cloning steps. Thus, a double stranded DNA molecule is generated which contains the sequence information of the viral RNA. These DNA molecules can be cloned in E. coli plasmid vectors after the additions of restriction enzyme linker molecules by DNA ligase. The various fragments are inserted into cloning vectors, such as well-characterized plasmids, which are then used to transform E. coli and create a cDNA library.
NIa and coat protein genes can be used as hybridization probes to screen the cDNA library to determine if any of the transformed bacteria contain DNA fragments with sequences coding for the replicase region. The cDNA inserts in any bacterial colonies which hybridize to both of these probes can be sequenced. The replicase gene is present in its entirety in colonies which have sequences that extend 5' to sequences which encode a N-terminal proteolytic cleavage site and 3' to sequences which encode a C-terminal proteolytic cleavage site for NIb.
Alternatively, cDNA fragments can be inserted in the sense oritentation into expression vectors. Antibodies against the protease can be used to screen the cDNA expression library and the gene can be isolated from colonies which express the protein.
Another molecular strategy to provide virus resistance in transgenic plants is based on antisense RNA. As is well known, a cell manufactures protein by transcribing the DNA of the gene encoding that protein to produce RNA, which is then processed to messenger RNA (mRNA) (e.g., by the removal of introns) and finally translated by ribosomes into protein. This process may be inhibited in the cell by the presense of antisense RNA. The term antisense RNA means an RNA sequence which is complementary to a sequence of bases in the mRNA in question in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, thus preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. This antisense RNA may be produced in the cell by transformation of the cell with an appropriate DNA construct arranged to transcribe the non-template strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).
The use of antisense RNA to downregulate the expression of specific plant genes is well known. Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference, e.g., lack of anthocyanin production in flower petals of petunia leading to colorless instead of colored petals (van der Krol et al., Nature, 333:866-869 (1988)); or at a more subtle biochemical level, e.g., change in the amount of polygalacturonase and reduction in depolymerization of pectin during tomato fruit ripening (Smith et al., Nature, 334:724-726 (1988)).
Another more recently described method of inhibiting gene expression in transgenic plants is the use of sense RNA transcribed from an exogenous template to downregulate the expression of specific plant genes (Jorgensen, Keystone Symposium "Improved Crop and Plant Products through Biotechnology", Abstract X1-022 (1994)). Thus, both antisense and sense RNA have been proven to be useful in achieving downregulation of gene expression in plants, and is encompassed by the present invention.
In the present invention, the DNA molecules encoding the replicase genes of PRV FLA83 W strain have been determined and the genes have been inserted into expression vectors. These expression cassettes can be individually placed into a vector that can be transmitted into plants, preferably a binary vector. Alternatively, two or more of PRV replicase genes can each be present in an expression cassette which can be placed into the same binary vector, or a PRV NIb expression cassette of the present invention can be placed into a binary vector with one or more viral gene expression cassettes. The expression vectors contain the necessary genetic regulatory sequences for expression of an inserted gene. The replicase gene is inserted such that those regulatory sequences are functional and the genes can be expressed when incorporated into a plant genome. For example, vectors of the present invention can contain combinations of expression cassettes that include DNA from a cucumber mosaic virus coat protein gene, a zuchini yellow mosiac virus coat protein gene, and a watermelon mosaic virus-2 coat protein gene.
Moreover, when combinations of viral gene expression cassettes are placed in the same binary plasmid, and that multigene cassette containing plasmid transformed into a plant, the multiple gene expression cassettes all preferably exhibit substantially the same degrees of efficacy when present in transgenic plants. For example, if one examines numerous transgenic lines containing two different intact viral gene cassettes, the transgenic line will be immune to infection by both viruses. Similarly, if a line exhibits a delay in symptom development to one virus, it will also exhibit a delay in symptom development to the second virus. Finally, if a line is susceptible to one of the viruses it will be susceptible to the other. This phenomenon is unexpected. If there were not a correlation between the efficacy of each gene in these multiple gene constructs this approach as a tool in plant breeding would probably be prohibitively difficult to use. Even with single gene constructs, one must test numerous transgenic plant lines to find one that displays the appropriate level of efficacy. The probability of finding a line with useful levels of expression can range from 10-50% (depending on the species involved). For further information refer to Applicants' assignees copending patent application Ser. No. 08/366,991 entitled "Transgenic Plants Expressing DNA Constructs Containing a Plurality of Genes to Impart Virus Resistance" filed on Dec. 30, 1994, now abandoned and incorporated by reference herein.
In order to express the viral gene, the necessary genetic regulatory sequences must be provided. Since the replicase of a potyvirus is produced by the post-translational processing of a polyprotein, the replicase gene isolated from viral RNA does not contain transcription and translation signals necessary for its expression once transferred and integrated into a plant genome. It must, therefore, be engineered to contain a plant expressible promoter, a translation initiation codon (ATG), and a plant functional poly(A) addition signal (AATAAA) 3' of its translation termination codon. In the present invention, the replicase genes are inserted into vectors which contain cloning sites for insertion 3' of the initiation codon and 5' of the poly(A) signal. The promoter is 5' of the initiation codon such that when structural genes are inserted at the cloning site, a functional unit is formed in which the inserted genes are expressed under the control of the various genetic regulatory sequences.
The segment of DNA referred to as the promoter is responsible for the regulation of the transcription of DNA into mRNA. A number of promoters which function in plant cells are known in the art and can be employed in the practice of the present invention. These promoters can be obtained from a variety of sources such as plants or plant viruses, and can include, but are not limited to, promoters isolated from the caulimovirus group such as the cauliflower mosaic virus 35S promoter (CaMV35S), the enhanced cauliflower mosaic virus 35S promoter (enh CaMV35S), the figwort mosaic virus full-length transcript promoter (FMV35S), and the promoter isolated from the chlorophyll a/b binding protein. Other useful promoters include promoters which are capable of expressing the potyvirus proteins in an inducible manner or in a tissue-specific manner in certain cell types in which the infection is known to occur. For example, the inducible promoters from phenylalanine ammonia lyase, chalcone synthase, hydroxyproline rich glycoprotein, extensin, pathogenesis-related proteins (e.g. PR-1a), and wound-inducible protease inhibitor from potato may be useful.
Preferred promoters for use in the present replicase-containing cassettes include the constitutive promoters from CaMV, the Ti genes nopaline synthase (Bevan et al., Nucleic Acids Res. II, 369 (1983)) and octopine synthase (Depicker et al., J. Mol. Appl. Genet., 1, 561 (1982)), and the bean storage protein gene phaseolin. The poly(A) addition signals from these genes are also suitable for use in the present cassettes. The particular promoter selected is preferably capable of causing sufficient expression of the DNA coding sequences to which it is operably linked, to result in the production of amounts of the proteins or RNAs effective to provide viral resistance, but not so much as to be detrimental to the cell in which they are expressed. The promoters selected should be capable of functioning in tissues including, but not limited to, epidermal, vascular, and mesophyll tissues. The actual choice of the promoter is not critical, as long as it has sufficient transcriptional activity to accomplish the expression of the preselected proteins and/or their respectives RNAs and subsequent conferral of viral resistance to the plants.
The nontranslated leader sequence can be derived from any suitable source and can be specifically modified to increase the translation of the mRNA. The 5' nontranslated region can be obtained from the promoter selected to express the gene, an unrelated promoter, the native leader sequence of the gene or coding region to be expressed, viral RNAs, suitable eucaryotic genes, or a synthetic gene sequence. The present invention is not limited to the constructs presented in the following examples. The nontranslated leader sequence can also be derived from an unrelated promoter or viral coding region as described.
The termination region or 3' nontranslated region which is employed is one which will cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3' end of the transcribed mRNA sequence. The termination region can be native with the promoter region, native with the structural gene, or can be derived from another source, and preferably include a terminator and a sequence coding for polyadenylation. Suitable 3' nontranslated regions of the chimeric plant gene include but are not limited to: (1) the 3' transcribed, nontranslated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene; and (2) plant genes like the soybean 7S storage protein genes.
Preferably, the expression cassettes of the present invention are engineered to contain a constitutive promoter 5' to its translation initiation codon (ATG) and a poly(A) addition signal (AATAAA) 3' to its translation termination codon. Several promoters which function in plants are available, however, the preferred promoter is the 35S constitutive promoters from cauliflower mosaic virus (CaMV). The poly (A) signal can be obtained from the CaMV 35S gene or from any number of well characterized plant genes, i.e., nopaline synthase, octopine synthase, and the bean storage protein gene phaseolin. The constructions are similar to that used for the expression of the CMV C coat protein in PCT Patent Application PCT/US88/04321, published on Jun. 29, 1989 as WO 89/05858, claiming the benefit of U.S. Ser. No. 135,591, filed Dec. 21, 1987, entitled "Cucumber Mosaic Virus Coat Protein Gene", and the CMV WL coat protein in PCT Patent Application PCT/US89/03288, published on Mar. 8, 1990 as WO 90/02185, claiming the benefit of U.S. Ser. No. 234,404, filed Aug. 19, 1988, entitled "Cucumber Mosaic Virus Coat Protein Gene."
Selectable marker genes can be incorporated into the present expression cassettes and used to select for those cells or plants which have become transformed. The marker gene employed may express resistance to an antibiotic, such as kanamycin, gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracyline, chloramphenicol, and the like. Other markers could be employed in addition to or in the alternative, such as, for example, a gene coding for herbicide tolerance such as tolerance to glyphosate, sulfonylurea, phosphinothricin, or bromoxynil. Additional means of selection could include resistance to methotrexate, heavy metals, complementation providing prototrophy to an auxotrophic host, and the like.
The particular marker employed will be one which will allow for the selection of transformed cells as opposed to those cells which are not transformed. Depending on the number of different host species one or more markers can be employed, where different conditions of selection would be useful to select the different host, and would be known to those of skill in the art. A screenable marker such as the .beta.-glucuronidase gene can be used in place of, or with, a selectable marker. Cells transformed with this gene can be identified by the production of a blue product on treatment with 5-bromo-4-chloro-3-indoyl-.beta.-D-glucuronide (X-Gluc).
In developing the present expression construct, i.e., expression cassette, the various components of the expression construct such as the DNA molecules, linkers, or fragments thereof will normally be inserted into a convenient cloning vector, such as a plasmid or phage, which is capable of replication in a bacterial host, such as E. coli. Numerous cloning vectors exist that have been described in the literature. After each cloning, the cloning vector can be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, resection, insertion, in vitro mutagenesis, addition of polylinker fragments, and the like, in order to provide a vector which will meet a particular need.
For Agrobacterium-mediated transformation, the expression cassette will be included in a vector, and flanked by fragments of the Agrobacterium Ti or Ri plasmid, representing the right and, optionally the left, borders of the Ti or Ri plasmid transferred DNA (T-DNA). This facilitates integration of the present chimeric DNA sequences into the genome of the host plant cell. This vector will also contain sequences that facilitate replication of the plasmid in Agrobacterium cells, as well as in E. coli cells.
All DNA manipulations are typically carried out in E. coli cells, and the final plasmid bearing the potyvirus protein expression cassette is moved into Agrobacterium cells by direct DNA transformation, conjugation, and the like. These Agrobacterium cells will contain a second plasmid, also derived from Ti or Ri plasmids. This second plasmid will carry all the vir genes required for transfer of the foreign DNA into plant cells. Suitable plant transformation cloning vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as generally disclosed in Glassman et al. (U.S. Pat. No. 5,258,300), or Agrobacterium rhizogenes.
A variety of techniques are available for the introduction of the genetic material into or transformation of the plant cell host. However, the particular manner of introduction of the plant vector into the host is not critical to the practice of the present invention, and any method which provides for efficient transformation can be employed. In addition to transformation using plant transformation vectors derived from the tumor-inducing (Ti) or root-inducing (Ri) plasmids of Agrobacterium, alternative methods could be used to insert the DNA constructs of the present invention into plant cells. Such methods may include, for example, the use of liposomes electroporation, chemicals that increase the free uptake of DNA (Paszkowski et al., EMBO J., 3, 2717 (1984)), microinjection (Crossway et al., Mol. Gen. Genet., 202, 179 (1985)), electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA, 82, 824 (1985)), or high-velocity microprojectiles (Klein et al., Nature, 327, 70 (1987) and transformation using viruses or pollen.
The choice of plant tissue source or cultured plant cells for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is regenerable, in that it will retain the ability to regenerate whole, fertile plants following transformation.
The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA carrying the present potyvirus multi-gene expression cassette for an effective period of time. This can range from a less-than-one-second pulse of electricity for electroporation, to a two-to-three day co-cultivation in the presence of plasmid-bearing Aqrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet Corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.
Following treatment with DNA, the plant cells or tissue may be cultivated for varying lengths of time prior to selection, or may be immediately exposed to a selective agent such as those described hereinabove. Protocols involving exposure to Agrobacterium will also include an agent inhibitory to the growth of the Agrobacterium cells. Commonly used compounds are antibiotics such as cefotaxime and carbenicillin. The media used in the selection may be formulated to maintain transformed callus or suspension culture cells in an undifferentiated state, or to allow production of shoots from callus, leaf or stem segments, tuber disks, and the like.
Cells or callus observed to be growing in the presence of normally inhibitory concentrations of the selective agents are presumed to be transformed and may be subcultured several additional times on the same medium to remove nonresistant sections. The cells or calli can then be assayed for the presence of the viral gene cassette, or can be subjected to known plant regeneration protocols. In protocols involving the direct production of shoots, those shoots appearing on the selective media are presumed to be transformed and can be excised and rooted, either on selective medium suitable for the production of roots, or by simply dipping the excised shoot in a root-inducing compound and directly planting it in vermiculite.
In order to produce transgenic plants exhibiting viral resistance, the viral genes must be taken up into the plant cell and stably integrated within the plant genome. Plant cells and tissues selected for their resistance to an inhibitory agent are presumed to have acquired the selectable marker gene encoding this resistance during the transformation treatment. Since the marker gene is commonly linked to the viral genes, it can be assumed that the viral genes have similarly been acquired. Southern blot hybridization analysis using a probe specific to the viral genes can then be used to confirm that the foreign genes have been taken up and integrated into the genome of the plant cell. This technique may also give some indication of the number of copies of the gene that have been incorporated. Successful transcription of the foreign gene into mRNA can likewise be assayed using Northern blot hybridization analysis of total cellular RNA and/or cellular RNA that has been enriched in a polyadenylated region. mRNA molecules encompassed within the scope of the invention are those which contain viral specific sequences derived from the viral genes present in the transformed vector which are of the same polarity as that of the viral genomic RNA such that they are capable of base pairing with viral specific RNA of the opposite polarity to that of viral genomic RNA under conditions described in Chapter 7 of Sambrook et al. (1989). Moreover, mRNA molecules encompassed within the scope of the invention are those which contain viral specific sequences derived from the viral genes present in the transformed vector which are of the opposite polarity as that of the viral genomic RNA such that they are capable of base pairing with viral genomic RNA under conditions described in Chapter 7 in Sambrook et al. (1989).
The presence of a viral replicase can be assayed via infectivity studies as generally disclosed by Namba et al., Phytopathology 82:940 (1992), wherein plants are scored as symptomatic when any inoculated leaf shows veinclearing, mosaic or necrotic symptoms.
Seed from plants regenerated from tissue culture is grown in the field and self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines which are evaluated for viral resistance in the field under a range of environmental conditions. The commercial value of viral-resistant plants is greatest if many different hybrid combinations with resistance are available for sale. Additionally, hybrids adapted to one part of a country are not adapted to another part because of differences in such traits as maturity, disease and insect tolerance. Because of this, it is necessary to breed viral resistance into a large number of parental lines so that many hybrid combinations can be produced.
Adding viral resistance to agronomically elite lines is most efficiently accomplished when the genetic control of viral resistance is understood. This requires crossing resistant and sensitive plants and studying the pattern of inheritance in segregating generations to ascertain whether the trait is expressed as dominant or recessive, the number of genes involved, and any possible interaction between genes if more than one are required for expression. With respect to transgenic plants of the type disclosed herein, the transgenes exhibit dominant, single gene Mendelian behavior. This genetic analysis can be part of the initial efforts to convert agronomically elite, yet sensitive lines to resistant lines. A conversion process (backcrossing) is carried out by crossing the original transgenic resistant line with a sensitive elite line and crossing the progeny back to the sensitive parent. The progeny from this cross will segregate such that some plants carry the resistance gene(s) whereas some do not. Plants carrying the resistance gene(s) will be crossed again to the sensitive parent resulting in progeny which segregate for resistance and sensitivity once more. This is repeated until the original sensitive parent has been converted to a resistant line, yet possesses all of the other important attributes originally found in the sensitive parent. A separate backcrossing program is implemented for every sensitive elite line that is to be converted to a virus resistant line.
Subsequent to the backcrossing, the new resistant lines and the appropriate combinations of lines which make good commercial hybrids are evaluated for viral resistance, as well as for a battery of important agronomic traits. Resistant lines and hybrids are produced which are true to type of the original sensitive lines and hybrids. This requires evaluation under a range of environmental conditions under which the lines or hybrids will be grown commercially. Parental lines of hybrids that perform satisfactorily are increased and utilized for hybrid production using standard hybrid production practices.
The invention will be further described by reference to the following detailed examples. Enzymes were obtained from commercial sources and were used according to the vendor's recommendations or other variations known in the art. Other reagents, buffers, etc., were obtained from commercial sources, such as GIBCO-BRL, Bethesda, Md., and Sigma Chemical Co., St. Louis, Mo., unless otherwise specified.
Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail in, for example, in European Patent Application Publication Number 223,452, published Nov. 29, 1986, which is incorporated herein by reference. General references containing such standard techniques include the following: R. Wu, ed., Methods in Enzymology, Vol. 68 (1979); J. H. Miller, Experiments in Molecular Genetics (1972); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989); and D. M. Glover, ed., DNA Cloning Vol. II (1982).
FIG. 6 illustrates the constructions of this invention. Papaya Ringspot virus FLA83 W-type was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. on Aug. 3, 1998 and assigned ATCC Deposit Number 203076. This deposit was made in compliance with the requirements of the Budapest Treaty that the duration of the deposits should be for thirty (30) years from the date of deposit or for five (5) years after the last request for the deposit at the depository or for the enforceable life of a U.S. Patent that matures from this application, whichever is longer. The plant virus strain of FLA 83 W-type will be replenished should it become non-viable.
EXAMPLE
A. Isolation of PRV Fla83-W Viral RNA
7-day-old yellow crookneck squash plants grown in the greenhouse were inoculated with PRV strain W (watermelon) Florida-83; 21 days post inoculation leaves were harvested and PRV virus isolated. The procedure used is based on a modified method used by Purcifull et al. (Phytopathology, 69, 112 (1979)) for PRV type W isolation. Approximately 50 grams of fresh leaf tissue was homogenized in 100 ml 0.5 M potassium phosphate buffer (pH 7.5 "PB") containing 0.1% sodium sulphate, 25 ml chloroform, and 25 ml carbon tetrachloride. After centrifugation of the extract at 1,000.times.g for 5 minutes the pellet was resuspended in 50 ml of PB buffer and centrifuged again at 1,000.times.g for 5 minutes. The supernatants from both centrifugations were combined and centrifuged at 13,000.times.g for 15 minutes. To the resulting supernatant, Triton X-100 was added to a final concentration of 1% (v/v), polyethyleneglycol (PEG) 8,000 (Reagent grade, Sigma Chemical Co.) to a final concentration of 4%, (w/v) and NaCl to a final concentration of 100 mM. The suspension was stirred for 1 hour at 0-4.degree. C. This suspension was centrifuged at 10,000.times.g for 10 minutes.
The pellet was resuspended in 40 ml of PB. After centrifugation at 12,000.times.g for 10 minutes the pellet was discarded and virus was precipitated from the supernatant by adding PEG to a final concentration of 8% (w/v) and NaCl to a final concentration of 100 mM, and stirring for 0.5 hour at 0-4.degree. C. After centrifugation at 12,000.times.g for 10 minutes the pellets were resuspended with the aid of a tissue grinder in 5 ml of 20 mM PB and layered over a 30% Cs.sub.2 SO.sub.4 cushion. This was centrifuged in a Beckman Ti75 at 140,000.times.g for 18 hours at 5.degree. C. After centrifugation the virus band was harvested and dialyzed against 20 mM PB overnight at 4.degree. C. The dialyzed virus prepreparation was lysed and viral RNA precipitated by the addition of with LiCl (2 M final concentration). The viral RNA was recovered by centrifugation. Viral RNA was dissolved and precipitated by ethanol and resuspended in water.
B. Cloning and Engineering the PRV Replicase Gene
To obtain engineered genes of the PRV FLA83 replicase gene, the following steps were carried out: 1) single-stranded cDNA of PRV FLA83 was constructed; 2) replicase sequences were amplified by PCR; 3) the PRV replicase PCR product was cloned; 4) expression cassettes were inserted into binary vectors; 5) plants transgenic for the PRV replicase construct were produced; and 6) progeny of R.sub.o transgenic plants were challenged to identify protected lines.
cDNA clones of PRV FLA83 W RNA were constructed with the use of the cDNA ClonStruct.TM. cDNA Library Construction Kit (US Biochemical, Cleveland Ohio). Briefly, the process begins with first strand cDNA synthesis; the reaction was primed with the vector primer pTRXN PLUS (US Biochemical, Cleveland Ohio). Next, a C-tailing reaction is carried out to add homopolymers of dC to the 3' ends of the heteroduplex molecule of RNA-cDNA. Third, the heteroduplex was subjected to BstX I restriction digestion; the heteroduplex was then circularized with the use of T4 DNA Ligase. Fourth, second strand cDNA synthesis and repair was carried out with the use of DNA polymerase I, RNaase H and T4 DNA ligase. Fifth, recombinant plasmids were transformed into E. coli (BRL competent DH5alpha).
Colonies were screened by the in situ colony lift procedure (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989)) using 2 DNA probes: coat protein gene and NIa gene. The NIb gene is located between the coat protein gene and the NIa gene in potyviral genomes; clones that hybridize with both probes should include the NIb gene. Clones were selected that hybridized with both probes and were then subjected to restriction analysis. Clone PRVFLA83#71 includes the coat protein gene, NIb gene, and nearly all of the NIa gene. This clone was used for subsequent engineering steps.
Novel restriction sites were incorporated by polymerase chain reaction (PCR) amplification into the PRV FLA83 W strain NIb gene (FIG. 2). Primers RMM335 and RMM336 [SEQ ID NO:3 and 4, respectively] were designed to include novel EcoRI, BamHI and NcoI restriction sites. The manufacturer's protocol (Perkin-Elmer Cetus) was followed to amplify the NIb coding sequence from cDNA clone PRVFLA83#71. Following PCR amplification of PRV FLA 83 W NIb sequence, the amplified product was directly cloned into cloning vector pCRII (TA CLONING KIT available from Invitrogen Corp., San Diego, Calif.) and four clones isolated: PRFLA83NIbTA13, PRFLA83NIbTA15, PRVFLA83NIbTA17 and PRVFLA83NIbTA19.
The PRV NIb insert of clone PRVFLA83NIbTA15 was sequenced by the dideoxy chain termination method using the US Biochemical (Cleveland, Ohio) SEQUENASE Version 2 DNA Sequencing Kit. Both top and bottom strands were sequenced. The sequence obtained for clone PRVFLA83NIbTA15 includes a complete reading frame for NIb [SEQ ID NO:1] (FIG. 1). Comparison of PRVFLA83NIbTA15 with the other published PRV NIb nucleotide sequences (FIGS. 2 and 3) reveals that each of the genes sequenced to date is unique. Comparison of PRV NIb amino acid sequences (FIG. 3) shows that PRV FLA83 W differs from each of the other three PRV NIb genes sequenced to date (see * in FIG. 3 for differences).
A fragment harboring the NIb coding sequence for clone PRVFLA83NIbTA15 was excised as a partial NcoI fragment and inserted into the plant expression cassette pUC18 cpexpress to yield cpexpPRVFLA83 NIb-1 antisense (as) and cpexpPRFLA83 NIb-6 sense (s) cassettes (FIG. 6). Both sense and antisense PRV NIb expression cassettes were isolated as Bam HI fragments and subsequently inserted into the binary vector pPRBN (For further information on pPRBN, refer to Applicants' Assignees copending patent application Ser. No. 08/366,991 entitled "Transgenic Plants Expressing DNA Constructs Containing a Plurality of Genes to Impart Virus Resistance" filed on Dec. 30, 1994, now abandoned and incorporated by reference herein) into which coat protein genes for CMV-V27 (For further information on CMV coat proteins, see Applicants' Assignees copending patent application Ser. No. 08/367,789 entitled "Plants Resistant to V27, V33, or V34 Strains of Cucumber Mosaic Virus" filed on Dec. 30, 1994, now abandoned and incorporated by reference herein), ZYMV, and WMVII (For further information on ZYWV and WMV2 coat protein genes, see Applicants' Assignees copending patent application Ser. No. 08/232,846 entitled "Potyvirus Coat Protein Genes and Plants Transformed Therewith" filed on Apr. 25, 1994, and incorporated by reference herein) had already been inserted (CV27/Z72/WMBN22 or pEPG243) (Table 1). Insertion of cpexpPRVFLA83NIb-1 into pEPG243 gave CV27/Z72/PRVFLA83INb(as)/WMBN22 (pEPG246). Insertion of cpexpPRVFLA83NIb-6 (s) into PEPG243 gave CV27/Z72/PRVFLA83NIb(s)/WMBN22 (pEPG245) (FIG. 6). Insertion of PRV NIb gene sense (cpexpPRVFLA83NIb-6) and antisense (cpexpPRVFLA83NIb-1) cassettes into the binary CV33/Z72/WMBN22 (pEPG244) yielded CV33/Z72/PRVFLA83NIb(s)/WMBN22 (pEPG247) and CV33/Z72/PRVFLA83NIb(as)/WMBN22 (pEPG248) (FIG. 6).
TABLE 1______________________________________Binary Parental PlasmidFLA83 NIb Used pEPG# Site PRV______________________________________pPRBN pEPG243 (V-27ZW) BglII cpexpPRVFLA NIb-6 (s) 245 pPRBN pEPG243 (V-27ZW) BglII cpexpPRVFLA NIb-1 (as) 246 pPRBN pEPG244 (V-33ZW) BglII cpexpPRVFLA NIb-6 (s) 247 pPRBN pEPG244 (V-33ZW) BglII cpexpPRVFLA NIb-1 (as) 248______________________________________
C. Transfer of PRV Replicase Genes to Plants
Agrobacterium-mediated transfer of the plant expressible PRV replicase genes described herein was done using the methods described in PCT published application WO 89/05859, entitled "Agrobacterium Mediated Transformation of Germinating Plant Seeds". Binary plasmids pEPG245, pEPG246, pEPG247, and pEPG248 were transformed into Agrobacterium strains Mog301 and C58Z707. Transgenic plants have been produced containing the nucleotide sequence of PRV HA attenuated strain NIa gene. The gene is described in Quemada, et al., J. Gen. Virol. (1990). 71:203-210, incorporated by reference. Binary plasmids comprising this sequence include pEPG229 and pEPG233.
All publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 12 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1641 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..1640 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - CC ATG GTA AAG ATG AGT GGT AGT CGT TGG CTC - # TTC GAC AAA TTA CAC 47 Met Val Lys Met Ser Gly Ser Arg Trp - #Leu Phe Asp Lys Leu His 1 - # 5 - # 10 - # 15 - - GGC AAT TTG AAG GGT GTA AGT TCC GCT TCT AG - #C AAT TTG GTG ACA AAG 95 Gly Asn Leu Lys Gly Val Ser Ser Ala Ser Se - #r Asn Leu Val Thr Lys 20 - # 25 - # 30 - - CAC GTT GTT AAA GGC ATT TGT CCT CTC TTC AG - #G AAC TAT CTC GAG TGT 143 His Val Val Lys Gly Ile Cys Pro Leu Phe Ar - #g Asn Tyr Leu Glu Cys 35 - # 40 - # 45 - - GAT GAA GAG GCT AAG GAC TTC TTT AGT CCA CT - #T ATG GGT CAC TAC ATG 191 Asp Glu Glu Ala Lys Asp Phe Phe Ser Pro Le - #u Met Gly His Tyr Met 50 - # 55 - # 60 - - AAG AGT GTT CTG AGT AAG GAA GCA TAC ATT AA - #G GAT TTA TTG AAA TAT 239 Lys Ser Val Leu Ser Lys Glu Ala Tyr Ile Ly - #s Asp Leu Leu Lys Tyr 65 - # 70 - # 75 - - TCA AGT GAC ATC GTC GTT GGA GAA GTT AAC CA - #C GAC GTT TTT GAG GAT 287 Ser Ser Asp Ile Val Val Gly Glu Val Asn Hi - #s Asp Val Phe Glu Asp 80 - # 85 - # 90 - # 95 - - AGT GTT GCG CAA GTC GTC GAG CTG TTA AAT GA - #T CAC GAG TGC CCC GAG 335 Ser Val Ala Gln Val Val Glu Leu Leu Asn As - #p His Glu Cys Pro Glu 100 - # 105 - # 110 - - CTT GAA TAC ATT ACA GAT AGT GAG GTG ATT AT - #A CAA GCA TTG AAC ATG 383 Leu Glu Tyr Ile Thr Asp Ser Glu Val Ile Il - #e Gln Ala Leu Asn Met 115 - # 120 - # 125 - - GAT GCA GCT GTC GGA GCT TTA TAC ACC GGA AA - #G AAA AGG AAA TAT TTT 431 Asp Ala Ala Val Gly Ala Leu Tyr Thr Gly Ly - #s Lys Arg Lys Tyr Phe 130 - # 135 - # 140 - - GAG GGG TCA ACA GTG GAG CAC AGG CAA GCT CT - #C GTA CGG AAA AGC TGT 479 Glu Gly Ser Thr Val Glu His Arg Gln Ala Le - #u Val Arg Lys Ser Cys 145 - # 150 - # 155 - - GAG CGC CTC TAC GAA GGG AGA ATG GGA GTT TG - #G AAC GGT TCA CTG AAG 527 Glu Arg Leu Tyr Glu Gly Arg Met Gly Val Tr - #p Asn Gly Ser Leu Lys 160 1 - #65 1 - #70 1 -#75 - - GCT GAG TTG AGA CCA GCT GAA AAA GTG CTT GC - #T AAA AAG ACA AGATCA 575 Ala Glu Leu Arg Pro Ala Glu Lys Val Leu Al - #a Lys Lys Thr Arg Ser 180 - # 185 - # 190 - - TTC ACA GCA GCT CCT CTT GAC ACG CTG TTA GG - #A GCC AAA GTC TGC GTT 623 Phe Thr Ala Ala Pro Leu Asp Thr Leu Leu Gl - #y Ala Lys Val Cys Val 195 - # 200 - # 205 - - GAT GAT TTC AAC AAC TGG TTC TAC AGT AAG AA - #C ATG GAA TGT CCA TGG 671 Asp Asp Phe Asn Asn Trp Phe Tyr Ser Lys As - #n Met Glu Cys Pro Trp 210 - # 215 - # 220 - - ACT GTT GGA ATG ACA AAA TTC TAC AAA GGC TG - #G GAC GAG TTC CTG AGG 719 Thr Val Gly Met Thr Lys Phe Tyr Lys Gly Tr - #p Asp Glu Phe Leu Arg 225 - # 230 - # 235 - - AAA TTT CCT GAC GGC TGG GTG TAT TGT GAT GC - #A GAT GGC TCC CAG AAG 767 Lys Phe Pro Asp Gly Trp Val Tyr Cys Asp Al - #a Asp Gly Ser Gln Lys 240 2 - #45 2 - #50 2 -#55 - - GAT AGC TCA TTA ACA CCA TAC TTG TTG AAC GC - #T GTG CTA TCA ATTCGG 815 Asp Ser Ser Leu Thr Pro Tyr Leu Leu Asn Al - #a Val Leu Ser Ile Arg 260 - # 265 - # 270 - - TTA TGG GCG ATG GAG GAT TGG GAT ATT GGA GA - #G CAA ATG CTT AAG AAT 863 Leu Trp Ala Met Glu Asp Trp Asp Ile Gly Gl - #u Gln Met Leu Lys Asn 275 - # 280 - # 285 - - TTG TAT GGG GAA ATC ACT TAC ACG CCA ATA TT - #G ACA CCA GAT GGA ACA 911 Leu Tyr Gly Glu Ile Thr Tyr Thr Pro Ile Le - #u Thr Pro Asp Gly Thr 290 - # 295 - # 300 - - ATT GTC AAG AAG TTC AAA GGA AAT AAT AGT GG - #C CAA CCT TCG ACA GTC 959 Ile Val Lys Lys Phe Lys Gly Asn Asn Ser Gl - #y Gln Pro Ser Thr Val 305 - # 310 - # 315 - - GTT GAT AAT ACA TTG ATG GTT TTA ATC ACA AT - #G TAT TAC GCG CTG CGA 1007 Val Asp Asn Thr Leu Met Val Leu Ile Thr Me - #t Tyr Tyr Ala Leu Arg 320 3 - #25 3 - #30 3 -#35 - - AAG GCC GGT TAC GAT GCG AAA GCT CAG GAA GA - #T ATG TGT GTA TTTTAT 1055 Lys Ala Gly Tyr Asp Ala Lys Ala Gln Glu As - #p Met Cys Val Phe Tyr 340 - # 345 - # 350 - - ATA AAT GGT GAT GAT CTC TGT ATT GCC ATT CA - #C CCA GAT CAT GAG CAT 1103 Ile Asn Gly Asp Asp Leu Cys Ile Ala Ile Hi - #s Pro Asp His Glu His 355 - # 360 - # 365 - - GTT CTT GAC TCA TTC TCT AGT TCA TTT GCT GA - #G CTT GGG CTT AAA TAT 1151 Val Leu Asp Ser Phe Ser Ser Ser Phe Ala Gl - #u Leu Gly Leu Lys Tyr 370 - # 375 - # 380 - - GAT TTC ACA CAA AGG CAC CGG AAT AAA CAG GA - #T TTG TGG TTT ATG TCA 1199 Asp Phe Thr Gln Arg His Arg Asn Lys Gln As - #p Leu Trp Phe Met Ser 385 - # 390 - # 395 - - CAT CGA GGT ATT CTG ATT GAT GAC ATT TAC AT - #T CCG AAA CTT GAA CCT 1247 His Arg Gly Ile Leu Ile Asp Asp Ile Tyr Il - #e Pro Lys Leu Glu Pro 400 4 - #05 4 - #10 4 -#15 - - GAG AGA ATT GTT GCA ATT CTT GAA TGG GAC AA - #A TCT AAG CTT CCGGAG 1295 Glu Arg Ile Val Ala Ile Leu Glu Trp Asp Ly - #s Ser Lys Leu Pro Glu 420 - # 425 - # 430 - - CAT CGA TTG GAG GCG ATC ACA GCA GCG ATG AT - #A GAG TCA TGG GGT TAT 1343 His Arg Leu Glu Ala Ile Thr Ala Ala Met Il - #e Glu Ser Trp Gly Tyr 435 - # 440 - # 445 - - GGT GAG TTA ACA CAC CAA ATT CGC AGA TTT TA - #T CAA TGG GTT CTT GAG 1391 Gly Glu Leu Thr His Gln Ile Arg Arg Phe Ty - #r Gln Trp Val Leu Glu 450 - # 455 - # 460 - - CAA GCT CCG TTC AAT GAG TTG GCG AAA CAA GG - #G AGG GCC CCA TAC GTC 1439 Gln Ala Pro Phe Asn Glu Leu Ala Lys Gln Gl - #y Arg Ala Pro Tyr Val 465 - # 470 - # 475 - - TCG GAA GTT GGA TTA AGA AGG TTG TAT ACG AG - #T GAA CGC GGA TCA GTG 1487 Ser Glu Val Gly Leu Arg Arg Leu Tyr Thr Se - #r Glu Arg Gly Ser Val 480 4 - #85 4 - #90 4 -#95 - - GAT GAA TTG GAA GCG TAT ATA GAT AAA TAT TT - #T GAG CGT GAG AGGGGA 1535 Asp Glu Leu Glu Ala Tyr Ile Asp Lys Tyr Ph - #e Glu Arg Glu Arg Gly 500 - # 505 - # 510 - - GAC TCA CCC GAA GTA CTG GTG TAC CAT GAA TC - #A AGG AGT ACT GAT GAT 1583 Asp Ser Pro Glu Val Leu Val Tyr His Glu Se - #r Arg Ser Thr Asp Asp 515 - # 520 - # 525 - - TAT GAA CTT GTT CGT GTC AAC AAT ACA CAT GT - #G TTT CAT CAG CTA AAG 1631 Tyr Glu Leu Val Arg Val Asn Asn Thr His Va - #l Phe His Gln Leu Lys 530 - # 535 - # 540 - - CTA GCC ATG G - # - #- # 1641 Leu Ala Met 545 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 546 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Val Lys Met Ser Gly Ser Arg Trp Leu Ph - #e Asp Lys Leu His Gly 1 5 - # 10 - # 15 - - Asn Leu Lys Gly Val Ser Ser Ala Ser Ser As - #n Leu Val Thr Lys His 20 - # 25 - # 30 - - Val Val Lys Gly Ile Cys Pro Leu Phe Arg As - #n Tyr Leu Glu Cys Asp 35 - # 40 - # 45 - - Glu Glu Ala Lys Asp Phe Phe Ser Pro Leu Me - #t Gly His Tyr Met Lys 50 - # 55 - # 60 - - Ser Val Leu Ser Lys Glu Ala Tyr Ile Lys As - #p Leu Leu Lys Tyr Ser 65 - # 70 - # 75 - # 80 - - Ser Asp Ile Val Val Gly Glu Val Asn His As - #p Val Phe Glu Asp Ser 85 - # 90 - # 95 - - Val Ala Gln Val Val Glu Leu Leu Asn Asp Hi - #s Glu Cys Pro Glu Leu 100 - # 105 - # 110 - - Glu Tyr Ile Thr Asp Ser Glu Val Ile Ile Gl - #n Ala Leu Asn Met Asp 115 - # 120 - # 125 - - Ala Ala Val Gly Ala Leu Tyr Thr Gly Lys Ly - #s Arg Lys Tyr Phe Glu 130 - # 135 - # 140 - - Gly Ser Thr Val Glu His Arg Gln Ala Leu Va - #l Arg Lys Ser Cys Glu 145 1 - #50 1 - #55 1 -#60 - - Arg Leu Tyr Glu Gly Arg Met Gly Val Trp As - #n Gly Ser Leu LysAla 165 - # 170 - # 175 - - Glu Leu Arg Pro Ala Glu Lys Val Leu Ala Ly - #s Lys Thr Arg Ser Phe 180 - # 185 - # 190 - - Thr Ala Ala Pro Leu Asp Thr Leu Leu Gly Al - #a Lys Val Cys Val Asp 195 - # 200 - # 205 - - Asp Phe Asn Asn Trp Phe Tyr Ser Lys Asn Me - #t Glu Cys Pro Trp Thr 210 - # 215 - # 220 - - Val Gly Met Thr Lys Phe Tyr Lys Gly Trp As - #p Glu Phe Leu Arg Lys 225 2 - #30 2 - #35 2 -#40 - - Phe Pro Asp Gly Trp Val Tyr Cys Asp Ala As - #p Gly Ser Gln LysAsp 245 - # 250 - # 255 - - Ser Ser Leu Thr Pro Tyr Leu Leu Asn Ala Va - #l Leu Ser Ile Arg Leu 260 - # 265 - # 270 - - Trp Ala Met Glu Asp Trp Asp Ile Gly Glu Gl - #n Met Leu Lys Asn Leu 275 - # 280 - # 285 - - Tyr Gly Glu Ile Thr Tyr Thr Pro Ile Leu Th - #r Pro Asp Gly Thr Ile 290 - # 295 - # 300 - - Val Lys Lys Phe Lys Gly Asn Asn Ser Gly Gl - #n Pro Ser Thr Val Val 305 3 - #10 3 - #15 3 -#20 - - Asp Asn Thr Leu Met Val Leu Ile Thr Met Ty - #r Tyr Ala Leu ArgLys 325 - # 330 - # 335 - - Ala Gly Tyr Asp Ala Lys Ala Gln Glu Asp Me - #t Cys Val Phe Tyr Ile 340 - # 345 - # 350 - - Asn Gly Asp Asp Leu Cys Ile Ala Ile His Pr - #o Asp His Glu His Val 355 - # 360 - # 365 - - Leu Asp Ser Phe Ser Ser Ser Phe Ala Glu Le - #u Gly Leu Lys Tyr Asp 370 - # 375 - # 380 - - Phe Thr Gln Arg His Arg Asn Lys Gln Asp Le - #u Trp Phe Met Ser His 385 3 - #90 3 - #95 4 -#00 - - Arg Gly Ile Leu Ile Asp Asp Ile Tyr Ile Pr - #o Lys Leu Glu ProGlu 405 - # 410 - # 415 - - Arg Ile Val Ala Ile Leu Glu Trp Asp Lys Se - #r Lys Leu Pro Glu His 420 - # 425 - # 430 - - Arg Leu Glu Ala Ile Thr Ala Ala Met Ile Gl - #u Ser Trp Gly Tyr Gly 435 - # 440 - # 445 - - Glu Leu Thr His Gln Ile Arg Arg Phe Tyr Gl - #n Trp Val Leu Glu Gln 450 - # 455 - # 460 - - Ala Pro Phe Asn Glu Leu Ala Lys Gln Gly Ar - #g Ala Pro Tyr Val Ser 465 4 - #70 4 - #75 4 -#80 - - Glu Val Gly Leu Arg Arg Leu Tyr Thr Ser Gl - #u Arg Gly Ser ValAsp 485 - # 490 - # 495 - - Glu Leu Glu Ala Tyr Ile Asp Lys Tyr Phe Gl - #u Arg Glu Arg Gly Asp 500 - # 505 - # 510 - - Ser Pro Glu Val Leu Val Tyr His Glu Ser Ar - #g Ser Thr Asp Asp Tyr 515 - # 520 - # 525 - - Glu Leu Val Arg Val Asn Asn Thr His Val Ph - #e His Gln Leu Lys Leu 530 - # 535 - # 540 - - Ala Met 545 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - TTAATGAATT CCCCATGGTA AAGATGAGTG GTAGTCGT - #- # 38 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - CACAAAGTAG TCGATTTCGA TCGGTACCCT AGGCGACCAA AC - # - # 42 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1700 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - TTTTAACGCG CAAAAGGAAG TTAATCAATT GAATGTTTTC GAGCAAAGTG GT -#AGTCGTTG 60 - - GCTCTTTGAC AAATTACACG GCAATTTGAA AGGAGTTAGC TCCGCTCCTA GC -#AATTTGGT 120 - - GACAAAGCAC GTTGTTAAAG GAATTTGTCC TCTTTTCAGG AACTATCTCG AG -#TGTGATGA 180 - - AGAGGCTAAA GCTTTCTTTA GTCCACTTAT GGGTCACTAC ATGAAGAGTG TT -#CTGAGCAA 240 - - GGAAGCGTAC ATTAAGGATT TATTGAAATA TTCAAGTGAT ATTGTCGTTG GA -#GAAGTCAA 300 - - CCATGATGTT TTTGAGGATA GTGTTGCGCA AGTTATCGAG CTGTTAAATG AT -#CATGAGTG 360 - - TCCCGAACTT GAATACATTA CAGACAGTGA AGTGATTATA CAAGCCTTGA AC -#ATGGATGC 420 - - AGCTGTCGGA GCCTTATATA CGGGTTTGTT TTGGAAATAT TTTGAGGGAT CA -#ACAGTGGA 480 - - GCATAGACAA GCTCTTGTAC GGAAAAGCTG TGAGCGTCTC TACGAAGGGA GA -#ATGGGCGT 540 - - CTGGAACGGT TCGCTGAAGG CAGAACTGAG ACCAGCTGAG AAAGTGCTCG CG -#AAAAAGAC 600 - - AAGGTCATTT ACAGCAGCCC CTCTTGACAC ACTATTAGGA GCCAAAGTCT GC -#GTTGATGA 660 - - TTTCAACAAC TGGTTTTACA GTAAGAATAT GGAGTGCCCA TGGACCGTCG GG -#ATGACAAA 720 - - ATTTTACAAA GGCTGGGATG AGTTCCTGAG GAAATTTCCT GACGGCTGGG TG -#TACTGTGA 780 - - TGCAGATGGT TCCCAGTTCG ATAGCTCATT AACACCATAC TTGTTGAATG CT -#GTGCTATC 840 - - AATTCGGTTA TGGGCGATGG AGGATTGGGA TATTGGAGAG CAAATGCTTA AG -#AACTTGTA 900 - - TGGGGAAATC ACTTACACGC CAATATTGAC ACCAGATGGA ACAATTGTCA AG -#AAATTCAA 960 - - GGGCAATAAT AGTGGCCAAC CTTCGACAGT TGTTGATAAT ACATTAATGG TT -#TTAATCAC 1020 - - AATGTATTAC GCACTACGGA AGGCTGGTTA CGATACGAAG ACTCAAGAAG AT -#ATGTGTGT 1080 - - ATTTTATATC AATGGTGATG ATCTCTGTAT TGCCATTCAC CCGGATCATG AG -#CATGTTCT 1140 - - TGACTCATTC TCTAGTTCAT TTGCTGAGCT TGGGCTTAAG TATGATTTCG CA -#CAAAGGCA 1200 - - TCGGAATAAA CAGAATTTGT GGTTTATGTC GCATCGAGGT ATTCTGATTG AT -#GACATTTA 1260 - - CATTCCAAAA CTTGAACCTG AGCGAATTGT CGCAATTCTT GAATGGGACA AA -#TCTAAGCT 1320 - - TCCGGAGCAT CGATTGGAGG CAATCACAGC GGCAATGATA GAGTCATGGG GT -#CATGGTGA 1380 - - TCTAACACAC CAGATTCGCA GATTTTACCA ATGGGTTCTT GAGCAAGCTC GA -#TTCAATGA 1440 - - GTTGGCGAAA CAAGGAAGGG CCCCATACGT CTCGGAAGTT GGATTAAGAA GA -#TTGTACAC 1500 - - AAGTGAACGT GGATCAATGG ACGAATTAGA AGCGTATATA GATAAATACT TT -#GAGCGTGA 1560 - - GAGAGGAGAC TCGCCCGAAT TACTAGTGTA CCATGAATCA AGGAGCACTG AT -#GATTATCA 1620 - - ACTTGTTTGT AGCAACAATA CGCATGTGTT TCATCAGTCC AAGAATGAAG CT -#GTGGATGC 1680 - - TGCTTTGAAT GAAAAACTCA - # - # 170 - #0 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1700 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - TTTTAACGCA CAAAAGGAAG TTAATCAATT GAATGTTTTC GAGCAAAGTG GT -#GGTCGTTG 60 - - GCTCTTTGAC AAATTACACG GCAATTTGAA AGGAGTTAGC TCCGCTCCTA GC -#AATTTGGT 120 - - GACAAAGCAC GTTGTTAAAG GAATTTGTCC TCTTTTCAGG AACTATCTCG AG -#TGTGATGA 180 - - AGAGGCTAAA GCTTTCTTTA GTCCACTTAT GGGTCACTAC ATGAAGAGTG TT -#CTGAGCAA 240 - - GGAAGCGTAC ATTAAGGATT TATTGAAATA TTCAAGTGAT ATTGTCGTTG GA -#GAAGTCAA 300 - - CCATGATGTT TTTGAGGATA GTGTTGCGCA AGTTATCGAG CTGTTAAATG AT -#CATGAGTG 360 - - TCCCGAACTT GAATACATAA CAGACAGTGA AGTGATTATA CAAGCCTTGA AC -#ATGGATGC 420 - - AGCTGTCGGA GCCTTATATA CGGGAAAGAA AAGGAAATAT TTTGAGGGAT CA -#ACAGTGGA 480 - - GCATAGACAA GCTCTTGTAC GGAAAAGCTG TGAGCGTCTC TACGAAGGGA GA -#ATGGGCGT 540 - - CTGGAACGGT TCGCTGAAGG CAGAACTGAG ACCAGCTGAG AAAGTGCTCG CG -#AAAAAGAC 600 - - AAGGTCATTT ACAGCAGCCC CTCTTGACAC ACTATTAGGA GCCAAAGTCT GC -#GTTGATGA 660 - - TTTCAACAAC TGGTTTTACA GTAAGAATAT GGAGTGCCCA TGGACCGTCG GG -#ATGACAAA 720 - - ATTTTACAAA GGCTGGGATG AGTTCCTGAA GAAATTTCCT GACGGCTGGG TG -#TACTGTGA 780 - - TGCAGATGGT TCCCAGTTCG ATAGCTCATT AACACCATAC TTGTTGAATG CT -#GTGCTATC 840 - - AATTCGGTTA TGGGCGATGG AGGATTGGGA TATTGGAGAG CAAATGCTTA AG -#AACTTGTA 900 - - CGGGGAAATC ACTTACACGC CAATACTGAC GCCAGATGGA ACAATTGTCA AG -#AAATTCAA 960 - - GGGCAATAAT AGTGGCCAAC CTTCGACAGT TGTTGATAAT ACATTGATGG TT -#TTAATCAC 1020 - - AATGTATTAC GCACTACGGA AGGCTGGTTA CGATACGAAG ACTCAAGAAG AT -#ATGTGTGT 1080 - - ATTTTATATC AATGGTGATG ATCTCTGTAT TGCCATTCAC CCGGATCATG AG -#CATGTTCT 1140 - - TGACTCATTC TCTAGTTCAT TTGCTGAGCT TGGGCTTAAG TATGATTTCG CA -#CAAAGGCA 1200 - - TCGGAATAAA CAGAATTTGT GGTTTATGTC GCATCGAGGT ATTCTGATTG AT -#GACATTTA 1260 - - CATTCCAAAA CTTGAACCTG AGCGAATTGT CGCAATTCTT GAATGGGACA AA -#TCTAAGCT 1320 - - TCCGGAGCAT CGATTGGAGG CAATCACAGC GGCAATGATA GAGTCATGGG GT -#TATGGTGA 1380 - - TCTAACACAC CAGATTCGTA GATTTTACCA ATGGGTTCTT GAGCAAGCTC CA -#TTCAATGA 1440 - - GTTGGCGAAA CAAGGAAGGG CCCCATACGT CTCGGAAGTT GGATTAAGAA GA -#TTGTACAC 1500 - - AAGTGAACGT GGATCAATGG ACGAATTAGA AGCGTATATA GATAAATACT TT -#GAGCGTGA 1560 - - GAGAGGAGAC TCGCCCGAAT TACTAGTGTA CCATGAATCA AGGGGCACTG AT -#GATTATCA 1620 - - ACTTGTTTGT AGCAACAATA CGCATGTGTT TCATCAGTCC AAGAATGAAG CT -#GTGGATGC 1680 - - TGGTTTGAAT GAAAAACTCA - # - # 170 - #0 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1208 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - TCTTGTACGG AAAAGCTGTG AGCGTCTCTA CGAAGGGAGA ATGGGCGTTT GG -#AACGGTTC 60 - - GTTGAAGGCA GAACTGAGAC CAGCTGAAAA AGTGCTCGCG AAAAAGACAA GG -#TCATTTAC 120 - - AGCAGCTCCT CTTGACACAC TATTAGGAGC CAAAGTCTGC GTTGATGATT TT -#AACAACTG 180 - - GTTTTACAGT AAGAATATGG AGTGCCCATG GACCGTCGGA ATGACAAAAT TT -#TACAAAGG 240 - - CTGGGACGAG TTCCTGAGGA AATTTCCTGA CGGCTGGGTG TACTGTGATG CA -#GATGGTTC 300 - - CCAGTTCGAT AGCTCATTAA CACCATACTT GTTGAATGCT GTGCTATCAA TT -#CGGTTATG 360 - - GGCGATGGAG GATTGGGATA TTGGAGAGCA AATGCTTAAG AACTTGTATG GG -#GAAATCAC 420 - - TTACACGCCA ATATTGACAC CAGATGGAAC AATTGTCAAG AAATTCAAGG GC -#AATAATAG 480 - - TGGCCAACCT TCGACAGTTG TTGATAATAC ATTGATGGTT TTAATCACAA TG -#TATTACGC 540 - - ACTACGGAAG GCTGGTTACG ATACGAAGAC TCAAGAAGAT ATGTGTGTAT TT -#TATATCAA 600 - - TGGTGATGAT CTCTGTATTG CCATTCACCC GGATCATGAG CATGTTCTTG AC -#TCATTCTC 660 - - TAGATCGTTT GCTGAGCTTG GGCTTAAGTA TGATTTCACA CAAAGGCATC GG -#AATAAACA 720 - - GAATTTGTGG TTTATGTCGC ATCGAGGTAT TCTGATTGAT GACATTTACA TT -#CCAAAACT 780 - - TGAACCTGAG CGAATTGTCG CAATTCTTGA ATGGGACAAA TCTAAGCTTC CG -#GAGCATCG 840 - - ATTGGAAGCA ATCACAGCGG CAATGATAGA GTCATGGGGT TATGGTGATC TA -#ACACACCA 900 - - GATTCGCAGA TTTTACCAAT GGGTTCTTGA GCAAGCTCCA TTCAATGAGT TG -#GCGAAACA 960 - - AGGAAGGGCC CCATACGTCT CGGAAGTTGG ATTAAGAAGA TTGTACACAA GT -#GAACGTGG 1020 - - ATCAATGGAT GAATTAGAAG CGTATATAGA TAAATACTTT GAGCGTGAGA GA -#GGAGACTC 1080 - - ACCCGAATTA CTAGTGTACC ATGAATCAAG GAGCACTGAT GATTATCAAC TT -#GTTTGCAG 1140 - - TAACAATACA CATGTGTTTC ATCAGTCCAA AAATGAAGCT GTGGATACTG GT -#TTGAATGA 1200 - - AAAATTCA - # - #- # 1208 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 399 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - Leu Val Arg Lys Ser Cys Glu Arg Leu Tyr Gl - #u Gly Arg Met GlyVal 1 5 - # 10 - # 15 - - Trp Asn Gly Ser Leu Lys Ala Glu Leu Arg Pr - #o Ala Glu Lys Val Leu 20 - # 25 - # 30 - - Ala Lys Lys Thr Arg Ser Phe Thr Ala Ala Pr - #o Leu Asp Thr Leu Leu 35 - # 40 - # 45 - - Gly Ala Lys Val Cys Val Asp Asp Phe Asn As - #n Trp Phe Tyr Ser Lys 50 - # 55 - # 60 - - Asn Met Glu Cys Pro Trp Thr Val Gly Met Th - #r Lys Phe Tyr Lys Gly 65 - #70 - #75 - #80 - - Trp Asp Glu Phe Leu Arg Lys Phe Pro Asp Gl - #y Trp Val Tyr Cys Asp 85 - # 90 - # 95 - - Ala Asp Gly Ser Gln Phe Asp Ser Ser Leu Th - #r Pro Tyr Leu Leu Asn 100 - # 105 - # 110 - - Ala Val Leu Ser Ile Arg Leu Trp Ala Met Gl - #u Asp Trp Asp Ile Gly 115 - # 120 - # 125 - - Glu Gln Met Leu Lys Asn Leu Tyr Gly Glu Il - #e Thr Tyr Thr Pro Ile 130 - # 135 - # 140 - - Leu Thr Pro Asp Gly Thr Ile Val Lys Lys Ph - #e Lys Gly Asn Asn Ser 145 1 - #50 1 - #55 1 -#60 - - Gly Gln Pro Ser Thr Val Val Asp Asn Thr Le - #u Met Val Leu IleThr 165 - # 170 - # 175 - - Met Tyr Tyr Ala Leu Arg Lys Ala Gly Tyr As - #p Thr Lys Thr Gln Glu 180 - # 185 - # 190 - - Asp Met Cys Val Phe Tyr Ile Asn Gly Asp As - #p Leu Cys Ile Ala Ile 195 - # 200 - # 205 - - His Pro Asp His Glu His Val Leu Asp Ser Ph - #e Ser Arg Ser Phe Ala 210 - # 215 - # 220 - - Glu Leu Gly Leu Lys Tyr Asp Phe Thr Gln Ar - #g His Arg Asn Lys Gln 225 2 - #30 2 - #35 2 -#40 - - Asn Leu Trp Phe Met Ser His Arg Gly Ile Le - #u Ile Asp Asp IleTyr 245 - # 250 - # 255 - - Ile Pro Lys Leu Glu Pro Glu Arg Ile Val Al - #a Ile Leu Glu Trp Asp 260 - # 265 - # 270 - - Lys Ser Lys Leu Pro Glu His Arg Leu Glu Al - #a Ile Thr Ala Ala Met 275 - # 280 - # 285 - - Ile Glu Ser Trp Gly Tyr Gly Asp Leu Thr Hi - #s Gln Ile Arg Arg Phe 290 - # 295 - # 300 - - Tyr Gln Trp Val Leu Glu Gln Ala Pro Phe As - #n Glu Leu Ala Lys Gln 305 3 - #10 3 - #15 3 -#20 - - Gly Arg Ala Pro Tyr Val Ser Glu Val Gly Le - #u Arg Arg Leu TyrThr 325 - # 330 - # 335 - - Ser Glu Arg Gly Ser Met Asp Glu Leu Glu Al - #a Tyr Ile Asp Lys Tyr 340 - # 345 - # 350 - - Phe Glu Arg Glu Arg Gly Asp Ser Pro Glu Le - #u Leu Val Tyr His Glu 355 - # 360 - # 365 - - Ser Arg Ser Thr Asp Asp Tyr Gln Leu Val Cy - #s Ser Asn Asn Thr His 370 - # 375 - # 380 - - Val Phe His Gln Ser Lys Asn Glu Ala Val As - #p Thr Gly Leu Asn 385 3 - #90 3 - #95 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 600 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - Trp Ser Tyr Asn Ile Asn Glu Leu Ser Trp Gl - #y Ala Leu Lys Val Trp 1 5 - # 10 - # 15 - - Glu Ser Arg Pro Glu Ala Ile Phe Asn Ala Gl - #n Lys Glu Val Asn Gln 20 - # 25 - # 30 - - Leu Asn Val Phe Glu Gln Ser Gly Ser Arg Tr - #p Leu Phe Asp Lys Leu 35 - # 40 - # 45 - - His Gly Asn Leu Lys Gly Val Ser Ser Ala Pr - #o Ser Asn Leu Val Thr 50 - # 55 - # 60 - - Lys His Val Val Lys Gly Ile Cys Pro Leu Ph - #e Arg Asn Tyr Leu Glu 65 - #70 - #75 - #80 - - Cys Asp Glu Glu Ala Lys Ala Phe Phe Ser Pr - #o Leu Met Gly His Tyr 85 - # 90 - # 95 - - Met Lys Ser Val Leu Ser Lys Glu Ala Tyr Il - #e Lys Asp Leu Leu Lys 100 - # 105 - # 110 - - Tyr Ser Ser Asp Ile Val Val Gly Glu Val As - #n His Asp Val Phe Glu 115 - # 120 - # 125 - - Asp Ser Val Ala Gln Val Ile Glu Leu Leu As - #n Asp His Glu Cys Pro 130 - # 135 - # 140 - - Glu Leu Glu Tyr Ile Thr Asp Ser Glu Val Il - #e Ile Gln Ala Leu Asn 145 1 - #50 1 - #55 1 -#60 - - Met Asp Ala Ala Val Gly Ala Leu Tyr Thr Gl - #y Leu Phe Trp LysTyr 165 - # 170 - # 175 - - Phe Glu Gly Ser Thr Val Glu His Arg Gln Al - #a Leu Val Arg Lys Ser 180 - # 185 - # 190 - - Cys Glu Arg Leu Tyr Glu Gly Arg Met Gly Va - #l Trp Asn Gly Ser Leu 195 - # 200 - # 205 - - Lys Ala Glu Leu Arg Pro Ala Glu Lys Val Le - #u Ala Lys Lys Thr Arg 210 - # 215 - # 220 - - Ser Phe Thr Ala Ala Pro Leu Asp Thr Leu Le - #u Gly Ala Lys Val Cys 225 2 - #30 2 - #35 2 -#40 - - Val Asp Asp Phe Asn Asn Trp Phe Tyr Ser Ly - #s Asn Met Glu CysPro 245 - # 250 - # 255 - - Trp Thr Val Gly Met Thr Lys Phe Tyr Lys Gl - #y Trp Asp Glu Phe Leu 260 - # 265 - # 270 - - Arg Lys Phe Pro Asp Gly Trp Val Tyr Cys As - #p Ala Asp Gly Ser Gln 275 - # 280 - # 285 - - Phe Asp Ser Ser Leu Thr Pro Tyr Leu Leu As - #n Ala Val Leu Ser Ile 290 - # 295 - # 300 - - Arg Leu Trp Ala Met Glu Asp Trp Asp Ile Gl - #y Glu Gln Met Leu Lys 305 3 - #10 3 - #15 3 -#20 - - Asn Leu Tyr Gly Glu Ile Thr Tyr Thr Pro Il - #e Leu Thr Pro AspGly 325 - # 330 - # 335 - - Thr Ile Val Lys Lys Phe Lys Gly Asn Asn Se - #r Gly Gln Pro Ser Thr 340 - # 345 - # 350 - - Val Val Asp Asn Thr Leu Met Val Leu Ile Th - #r Met Tyr Tyr Ala Leu 355 - # 360 - # 365 - - Arg Lys Ala Gly Tyr Asp Thr Lys Thr Gln Gl - #u Asp Met Cys Val Phe 370 - # 375 - # 380 - - Tyr Ile Asn Gly Asp Asp Leu Cys Ile Ala Il - #e His Pro Asp His Glu 385 3 - #90 3 - #95 4 -#00 - - His Val Leu Asp Ser Phe Ser Ser Ser Phe Al - #a Glu Leu Gly LeuLys 405 - # 410 - # 415 - - Tyr Asp Phe Ala Gln Arg His Arg Asn Lys Gl - #n Asn Leu Trp Phe Met 420 - # 425 - # 430 - - Ser His Arg Gly Ile Leu Ile Asp Asp Ile Ty - #r Ile Pro Lys Leu Glu 435 - # 440 - # 445 - - Pro Glu Arg Ile Val Ala Ile Leu Glu Trp As - #p Lys Ser Lys Leu Pro 450 - # 455 - # 460 - - Glu His Arg Leu Glu Ala Ile Thr Ala Ala Me - #t Ile Glu Ser Trp Gly 465 4 - #70 4 - #75 4 -#80 - - His Gly Asp Leu Thr His Gln Ile Arg Arg Ph - #e Tyr Gln Trp ValLeu 485 - # 490 - # 495 - - Glu Gln Ala Pro Phe Asn Glu Leu Ala Lys Gl - #n Gly Arg Ala Pro Tyr 500 - # 505 - # 510 - - Val Ser Glu Val Gly Leu Arg Arg Leu Tyr Th - #r Ser Glu Arg Gly Ser 515 - # 520 - # 525 - - Met Asp Glu Leu Glu Ala Tyr Ile Asp Lys Ty - #r Phe Glu Arg Glu Arg 530 - # 535 - # 540 - - Gly Asp Ser Pro Glu Leu Leu Val Tyr His Gl - #u Ser Arg Ser Thr Asp 545 5 - #50 5 - #55 5 -#60 - - Asp Tyr Gln Leu Val Cys Ser Asn Asn Thr Hi - #s Val Phe His GlnSer 565 - # 570 - # 575 - - Lys Asn Glu Ala Val Asp Ala Gly Leu Asn Gl - #u Lys Leu Lys Glu Lys 580 - # 585 - # 590 - - Glu Asn Gln Lys Glu Lys Glu Lys 595 - # 600 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 590 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - Gly Ala Leu Lys Val Trp Glu Ser Arg Pro Gl - #u Ala Ile Phe Asn Ala 1 5 - # 10 - # 15 - - Gln Lys Glu Val Asn Gln Leu Asn Val Phe Gl - #u Gln Ser Gly Gly Arg 20 - # 25 - # 30 - - Trp Leu Phe Asp Lys Leu His Gly Asn Leu Ly - #s Gly Val Ser Ser Ala 35 - # 40 - # 45 - - Pro Ser Asn Leu Val Thr Lys His Val Val Ly - #s Gly Ile Cys Pro Leu 50 - # 55 - # 60 - - Phe Arg Asn Tyr Leu Glu Cys Asp Glu Glu Al - #a Lys Ala Phe Phe Ser 65 - #70 - #75 - #80 - - Pro Leu Met Gly His Tyr Met Lys Ser Val Le - #u Ser Lys Glu Ala Tyr 85 - # 90 - # 95 - - Ile Lys Asp Leu Leu Lys Tyr Ser Ser Asp Il - #e Val Val Gly Glu Val 100 - # 105 - # 110 - - Asn His Asp Val Phe Glu Asp Ser Val Ala Gl - #n Val Ile Glu Leu Leu 115 - # 120 - # 125 - - Asn Asp His Glu Cys Pro Glu Leu Glu Tyr Il - #e Thr Asp Ser Glu Val 130 - # 135 - # 140 - - Ile Ile Gln Ala Leu Asn Met Asp Ala Ala Va - #l Gly Ala Leu Tyr Thr 145 1 - #50 1 - #55 1 -#60 - - Gly Lys Lys Arg Lys Tyr Phe Glu Gly Ser Th - #r Val Glu His ArgGln 165 - # 170 - # 175 - - Ala Leu Val Arg Lys Ser Cys Glu Arg Leu Ty - #r Glu Gly Arg Met Gly 180 - # 185 - # 190 - - Val Trp Asn Gly Ser Leu Lys Ala Glu Leu Ar - #g Pro Ala Glu Lys Val 195 - # 200 - # 205 - - Leu Ala Lys Lys Thr Arg Ser Phe Thr Ala Al - #a Pro Leu Asp Thr Leu 210 - # 215 - # 220 - - Leu Gly Ala Lys Val Cys Val Asp Asp Phe As - #n Asn Trp Phe Tyr Ser 225 2 - #30 2 - #35 2 -#40 - - Lys Asn Met Glu Cys Pro Trp Thr Val Gly Me - #t Thr Lys Phe TyrLys 245 - # 250 - # 255 - - Gly Trp Asp Glu Phe Leu Lys Lys Phe Pro As - #p Gly Trp Val Tyr Cys 260 - # 265 - # 270 - - Asp Ala Asp Gly Ser Gln Phe Asp Ser Ser Le - #u Thr Pro Tyr Leu Leu 275 - # 280 - # 285 - - Asn Ala Val Leu Ser Ile Arg Leu Trp Ala Me - #t Glu Asp Trp Asp Ile 290 - # 295 - # 300 - - Gly Glu Gln Met Leu Lys Asn Leu Tyr Gly Gl - #u Ile Thr Tyr Thr Pro 305 3 - #10 3 - #15 3 -#20 - - Ile Leu Thr Pro Asp Gly Thr Ile Val Lys Ly - #s Phe Lys Gly AsnAsn 325 - # 330 - # 335 - - Ser Gly Gln Pro Ser Thr Val Val Asp Asn Th - #r Leu Met Val Leu Ile 340 - # 345 - # 350 - - Thr Met Tyr Tyr Ala Leu Arg Lys Ala Gly Ty - #r Asp Thr Lys Thr Gln 355 - # 360 - # 365 - - Glu Asp Met Cys Val Phe Tyr Ile Asn Gly As - #p Asp Leu Cys Ile Ala 370 - # 375 - # 380 - - Ile His Pro Asp His Glu His Val Leu Asp Se - #r Phe Ser Ser Ser Phe 385 3 - #90 3 - #95 4 -#00 - - Ala Glu Leu Gly Leu Lys Tyr Asp Phe Ala Gl - #n Arg His Arg AsnLys 405 - # 410 - # 415 - - Gln Asn Leu Trp Phe Met Ser His Arg Gly Il - #e Leu Ile Asp Asp Ile 420 - # 425 - # 430 - - Tyr Ile Pro Lys Leu Glu Pro Glu Arg Ile Va - #l Ala Ile Leu Glu Trp 435 - # 440 - # 445 - - Asp Lys Ser Lys Leu Pro Glu His Arg Leu Gl - #u Ala Ile Thr Ala Ala 450 - # 455 - # 460 - - Met Ile Glu Ser Trp Gly Tyr Gly Asp Leu Th - #r His Gln Ile Arg Arg 465 4 - #70 4 - #75 4 -#80 - - Phe Tyr Gln Trp Val Leu Glu Gln Ala Pro Ph - #e Asn Glu Leu AlaLys 485 - # 490 - # 495 - - Gln Gly Arg Ala Pro Tyr Val Ser Glu Val Gl - #y Leu Arg Arg Leu Tyr 500 - # 505 - # 510 - - Thr Ser Glu Arg Gly Ser Met Asp Glu Leu Gl - #u Ala Tyr Ile Asp Lys 515 - # 520 - # 525 - - Tyr Phe Glu Arg Glu Arg Gly Asp Ser Pro Gl - #u Leu Leu Val Tyr His 530 - # 535 - # 540 - - Glu Ser Arg Gly Thr Asp Asp Tyr Gln Leu Va - #l Cys Ser Asn Asn Thr 545 5 - #50 5 - #55 5 -#60 - - His Val Phe His Gln Ser Lys Asn Glu Ala Va - #l Asp Ala Gly LeuAsn 565 - # 570 - # 575 - - Glu Lys Leu Lys Glu Lys Glu Lys Gln Lys Gl - #u Lys Glu Lys 580 - # 585 - # 590 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1669 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - GGTGGAGCAA GCTAAGCATT CTGCATGGAT GTTTGAAGCC TTGACAGGAA AT -#TTGCAAGC 60 - - TGTCGCAACA ATGAAGAGCC AATTAGTAAC CAAGCATGTA GTTAAAGGAG AG -#TGTCGACA 120 - - CTTCACAGAA TTTCTGACTG TGGATGCAGA GGCAGAGGCA GAGGCATTCT TC -#AGGCCTTT 180 - - GATGGATGCG TATGGGAAAA GCTTGCTAAA TAGAGATGCG TACATCAAGG AC -#ATAATGAA 240 - - GTATTCAAAA CCTATAGATG TTGGTGTCGT GGATCGGATG CATTTGAGGA AG -#CCATCAAT 300 - - AGGGTTATCA TCTACCTGCA ATGTGCACGG CTTCAAGAAG TGTGCATATG TC -#ACTGATGA 360 - - GCAAGAAATT TTCAAAGCGC TCAACATGAA AGCTGCAGTC GGAGCCAGTT AT -#GGGTGCAA 420 - - AAAGAAAGAC TATTTTGAGC ATTTCACTGA TGCAGATAAG GAAGAAATAG TC -#ATGCAAAG 480 - - CTGTCTGCGA TTGTATAAAG GTTTGCTTGG CATTTGGAAC GGATCATTGA AG -#GCAGAGCT 540 - - CCGGTGTAAG GAGAAGATAC TTGCAAATAA GACGAGGACG TTCACTGCTG CA -#CCTCTAGA 600 - - CACTTTGCTG GGTGGTAAAG TGTGTGTTGA TGACTTCAAT AATCAATTTT AT -#TCAAAGAA 660 - - TATTGAATGC TGTTGGACAG TTGGGATGAC TAAGTTTTAT GGTGGTTGGG AT -#AAACTGCT 720 - - TCGGCGTTTA CCTGAGAATT GGGTATACTG TGATGCTGAT GGCTCACAGT TT -#GATAGTTC 780 - - ACTAACTCCA TACCTAATCA ATGCTGTTCT CACCATCAGA AGCACATACA TG -#GAAGACTG 840 - - GGATGTGGGG TTGCAGATGC TGCGCAATTT ATACACTGAG ATTGTTTACA CA -#CCAATTTC 900 - - AACTCCAGAT GGAACAATTG TCAAGAAGTT TAGAGGTAAT AATAGTGGTC AA -#CCTTCTAC 960 - - CGTTGTGGAT AATTCTCTCA TGGTTGTCCT TGCTATGCAT TACGCTCTCA TT -#AAGGAGTG 1020 - - CGTTGAGTTT GAAGAAATCG ACAGCACGTG TGTATTCTTT GTTAATGGTG AT -#GACTTATT 1080 - - GATTGCTGTG AATCCGGAGA AAGAGAGCAT TCTCGATAGA ATGTCACAAC AT -#TTCTCAGA 1140 - - TCTTGGTTTG AACTATGATT TTTCGTCGAG AACAAGAAGG AAGGAGGAAT TG -#TGGTTCAT 1200 - - GTCCCATAGA GGCCTGCTAA TCGAGGGTAT GTACGTGCCA AAGCTTGAAG AA -#GAGAGAAT 1260 - - TGTATCCATT CTGCAATGGG ATAGAGCTGA TCTGCCAGAG CACAGATTAG AA -#GCGATTTG 1320 - - CGCAGCTATG ATAGAGTCCT GGGGTTATTC TGAACTAACA CACCAAATCA GG -#AGATTCTA 1380 - - CTCATGGTTA TTGCAACAGC AACCTTTTGC AACAATAGCG CAGGAAGGGA AG -#GCTCCTTA 1440 - - TATAGCAAGC ATGGCACTAA GGAAACTGTA TATGGATAGG GCTGTGGATG AG -#GAAGAGCT 1500 - - AAGAGCCTTC ACTGAAATGA TGGTCGCATT AGATGATGAG TTTGAGCTTG AC -#TCTTATGA 1560 - - AGTACACCAT CAAGCAAATG ACACAATTGA TGCAGGAGGA AGCAACAAGA AA -#GATGCAAA 1620 - - ACCAGAGCAG GGCAGCATCC AGCCAAACCC GAACAAAGGA AAGGATAAG - # 1669 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 600 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - Val Glu Gln Ala Lys His Ser Ala Trp Met Ph - #e Glu Ala Leu Thr Gly 1 5 - # 10 - # 15 - - Asn Leu Gln Ala Val Ala Thr Met Lys Ser Gl - #n Leu Val Thr Lys His 20 - # 25 - # 30 - - Val Val Lys Gly Glu Cys Arg His Phe Thr Gl - #u Phe Leu Thr Val Asp 35 - # 40 - # 45 - - Ala Glu Ala Glu Ala Glu Ala Phe Phe Arg Pr - #o Leu Met Asp Ala Tyr 50 - # 55 - # 60 - - Gly Lys Ser Leu Leu Asn Arg Asp Ala Tyr Il - #e Lys Asp Ile Met Lys 65 - #70 - #75 - #80 - - Tyr Ser Lys Pro Ile Asp Val Gly Val Val As - #p Arg Met His Leu Arg 85 - # 90 - # 95 - - Lys Pro Ser Ile Gly Leu Ser Ser Thr Cys As - #n Val His Gly Phe Lys 100 - # 105 - # 110 - - Lys Cys Ala Tyr Val Thr Asp Glu Gln Glu Il - #e Phe Lys Ala Leu Asn 115 - # 120 - # 125 - - Met Lys Ala Ala Val Gly Ala Ser Thr Gly Cy - #s Lys Lys Lys Asp Tyr 130 - # 135 - # 140 - - Phe Glu His Phe Thr Asp Ala Asp Lys Glu Gl - #u Ile Val Met Gln Ser 145 1 - #50 1 - #55 1 -#60 - - Cys Leu Arg Leu Tyr Lys Gly Leu Leu Gly Il - #e Trp Asn Gly SerLeu 165 - # 170 - # 175 - - Lys Ala Glu Leu Arg Cys Lys Glu Lys Ile Le - #u Ala Asn Lys Thr Arg 180 - # 185 - # 190 - - Thr Phe Thr Ala Ala Pro Leu Asp Thr Leu Le - #u Gly Gly Lys Val Cys 195 - # 200 - # 205 - - Val Asp Asp Phe Asn Asn Gln Phe Tyr Ser Ly - #s Asn Ile Glu Cys Cys 210 - # 215 - # 220 - - Trp Thr Val Gly Met Thr Lys Phe Tyr Gly Gl - #y Trp Asp Lys Leu Leu 225 2 - #30 2 - #35 2 -#40 - - Arg Arg Leu Pro Glu Asn Trp Val Tyr Cys As - #p Ala Asp Gly SerGln 245 - # 250 - # 255 - - Phe Asp Ser Ser Leu Thr Pro Tyr Leu Ile As - #n Ala Val Leu Thr Ile 260 - # 265 - # 270 - - Arg Ser Thr Tyr Met Glu Asp Trp Asp Val Gl - #y Leu Gln Met Leu Arg 275 - # 280 - # 285 - - Asn Leu Tyr Thr Glu Ile Val Tyr Thr Pro Il - #e Ser Thr Pro Asp Gly 290 - # 295 - # 300 - - Thr Ile Val Lys Lys Phe Arg Gly Asn Asn Se - #r Gly Gln Pro Ser Thr 305 3 - #10 3 - #15 3 -#20 - - Val Val Asp Asn Ser Leu Met Val Val Leu Al - #a Met His Tyr AlaLeu 325 - # 330 - # 335 - - Ile Lys Glu Cys Val Glu Phe Glu Glu Ile As - #p Ser Thr Cys Val Phe 340 - # 345 - # 350 - - Phe Val Asn Gly Asp Asp Leu Leu Ile Ala Va - #l Asn Pro Glu Lys Glu 355 - # 360 - # 365 - - Ser Ile Leu Asp Arg Met Ser Gln His Phe Se - #r Asp Leu Gly Leu Asn 370 - # 375 - # 380 - - Tyr Asp Phe Ser Ser Arg Thr Arg Arg Lys Gl - #u Glu Leu Trp Phe Met 385 3 - #90 3 - #95 4 -#00 - - Ser His Arg Gly Leu Leu Ile Glu Gly Met Ty - #r Val Pro Lys LeuGlu 405 - # 410 - # 415 - - Glu Glu Arg Ile Val Ser Ile Leu Gln Trp As - #p Arg Ala Asp Leu Pro 420 - # 425 - # 430 - - Glu His Arg Leu Glu Ala Ile Cys Ala Ala Me - #t Ile Glu Ser Trp Gly 435 - # 440 - # 445 - - Tyr Ser Glu Leu Thr His Gln Ile Arg Arg Ph - #e Tyr Ser Trp Leu Leu 450 - # 455 - # 460 - - Gln Gln Gln Pro Phe Ala Thr Ile Ala Gln Gl - #u Gly Lys Ala Pro Tyr 465 4 - #70 4 - #75 4 -#80 - - Ile Ala Ser Met Ala Leu Arg Lys Leu Tyr Me - #t Asp Arg Ala ValAsp 485 - # 490 - # 495 - - Glu Glu Glu Leu Arg Ala Phe Thr Glu Met Me - #t Val Ala Leu Asp Asp 500 - # 505 - # 510 - - Glu Phe Glu Leu Asp Ser Tyr Glu Val His Hi - #s Gln Ala Asn Asp Thr 515 - # 520 - # 525 - - Ile Asp Ala Gly Gly Ser Asn Lys Lys Asp Al - #a Lys Pro Glu Gln Gly 530 - # 535 - # 540 - - Ser Ile Gln Pro Asn Pro Asn Lys Gly Lys As - #p Lys Asp Val Asn Ala 545 5 - #50 5 - #55 5 -#60 - - Gly Thr Ser Gly Thr His Thr Val Pro Arg Il - #e Lys Ala Ile ThrSer 565 - # 570 - # 575 - - Lys Met Arg Met Pro Thr Ser Lys Gly Ala Th - #r Val Pro Asn Leu Glu 580 - # 585 - # 590 - - His Leu Leu Glu Tyr Ala Pro Gln 595 - # 600__________________________________________________________________________

Papaya ringspot virus replicase gene

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