MXPA99002187A

MXPA99002187A - A method for generating birnavirus from synthetic rna transcripts

Info

Publication number: MXPA99002187A
Application number: MXPA/A/1999/002187A
Authority: MX
Inventors: N Vakharia Vikram; Mundt Egbert
Original assignee: University Of Marylandbiotechnology Inst
Priority date: 1996-09-05
Filing date: 1999-03-05
Publication date: 2000-02-02

Abstract

A system for the generation of live Birnavirus such as infectious bursal disease virus (IBDV), a segmented double-stranded (ds)RNA virus of the Birnavirdae family, using synthetic transcripts derived from cloned DNA has been developed. Independent full-length cDNA clones were constructed which contained the entire coding and non-coding regions of RNA segments A and B of IBDV, respectively. Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hours post-transfection. The development of a reverse genetics system for dsRNA viruses will greatly facilitate studies of the regulation of viral gene expression pathogenesis, and design of a new generation of live and inactivated vaccines.

Description

A METHOD FOR GENERATING BIRNAVIRUS FROM SYNTHETIC RNA TRANSCRIPTS BACKGROUND OF THE INVENTION The infectious bursal disease virus (IBDV), a member of the Bimaviridae family, is the causative agent of a highly immunosuppressive disease in young chickens (Kibenge, FSB et al., J. Gen. Virol., 69, 1757 -1775 (19'88)). Infectious bursal disease (IBD) or Gumboro disease is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a group of chickens from 3 to ß weeks of age totally susceptible, the clinical disease causes severe immunosuppression and is responsible for losses due to growth damage, decreased feeding efficiency and death. Susceptible chickens less than 3 weeks old do not show external clinical signs of the disease but have a notable infection characterized by large lesions in the pouch. The virus that is associated with the symptoms of the disease is called infectious bursal disease virus (IBDV). IBDV is a pathogen of great economic importance for the national and world poultry industries. It causes severe immunodeficiency in young chickens by the destruction of B cell precursors of antibody production in the bursa of Fabricius. Immunosuppression causes an increase in susceptibility to other diseases and interferes with effective vaccination against Ne castle disease, Marek's disease and infectious bronchitis. There are two known serotypes of IBDV. Serotype I viruses are pathogenic for chickens while serotype II viruses infect chickens and turkeys. The infection of turkeys is currently of unknown clinical importance. IBDV belongs to a group of viruses called Bimaviridae that includes other bisected RNA viruses such as pancreatic infectious necrosis virus (fish), tellina virus and oyster virus (bivalve molluscs) and drosophila X virus (fruit fly). All these viruses contain genomes of double-stranded RNA of high molecular weight (MW). The IBDV virion capsid consists of various structural proteins. Nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship (Kibenge, F.S.B. et al., J. Gen. Virol., 69, 1757-1775 (1988)). The designation and molecular weights of viral proteins (VP) are those shown below.

Viral Protein Molecular Weight VP1 90 kDa VP2 41 kDa VP3 32 kDa VP4 28 kDa VP5 17 kDa Two segments of double-stranded RNA were identified in the IBDV genome. The IBDV genome consists of two segments of double-stranded RNA (ds) ranging from 2827 (segment B) to 3261 (segment A) nucleotide base pairs (Mundt, E. et al., Virology, 209, 10-18 (nineteen ninety five)). The larger segment A codes for a polyprotein that unfolds by autoproteolysis to form mature viral proteins VP2, VP3 and VP4 (Hudson, P.J. et al., Nucleic Acids Res., 14, 5001-5012 (1986)). VP2 and VP3 are the largest structural proteins of the virion. VP2 is the most important host protective immunogen of IBDV and contains the antigenic regions responsible for the induction of neutralization antibodies (Azad et al., Virology, 161, 145-152 (1987)). A second open reading frame (ORF), which precedes and partially overlaps the polyprotein gene, codes for a protein (VP5) of unknown function that is present in cells infected with IBDV (Mundt, E. et al., J. Gen. Virol., 76, 437-443 (1995)). The minor segment B codes for VPl, a multifunctional protein of 90-kDa with polymerase and enzyme-stopper activities (Spies, U., et al., Virus Res., 8, 127-140 (1987); Spies, U., et al., J. Gen. Virol., 71, 977-981 (1990)). It has been shown that the VP2 protein is the most important host protective immunogen of IBDV and that this contains the antigenic region responsible for the induction of neutralizing antibodies. The region containing the neutralization site has proven to be quite conformation dependent. It has been considered that the VP3 protein is a group specific antigen because it is recognized by the monoclonal antibodies directed against it from strains of both serotype I and serotype II viruses. The VP4 protein appears to be a virus-encoded protease that is involved in the processing of a polyprotein precursor to VP2 proteins, VP3 and VP4. Although the nucleotide sequences for segments A and B of the genome of various strains of IBDV have been published, it was only recently that the complete non-coding sequences 5'- and 3'- of both segments were determined. The non-coding region 5'- of the A and B segments of IBDV contain a consensus sequence of 32 nucleotides, while the 3'- non-coding terminal sequences of both segments are not related, but are conserved among the IBDV strains of the same serotype (Mundt, E., and collaborators, Virology, 209, 10-18 (1995)). These terminations may contain important sequences in packaging and in the regulation of IBDV gene expression, as demonstrated for other viruses containing dsRNAs such as plant and mammalian reoviruses and rotaviruses (Anzola, et al., Proc. Natl. Acad. Sci. USA, 84, 8301-8305 (1987); Zou, S., et al., Virology, 186, 377-388 (1992); Gorziglia, M.I., and collaborators, Proc. Natl. Acad. Sci. USA, 89, 5784-5788 (1992)). In recent years, several infectious animal RNA viruses have been generated from cloned cDNA using transcripts produced by DNA-dependent RNA polymerase (Boyer, J.C., et al., Virology, 198, 415-426 (1994)). For example, poliovirus, a positive strand RNA virus; the influenza virus, a RNA virus segmented negative strand; the rabies virus, a non-segmented negative-strand RNA virus; all were recovered from cDNAs from their respective genomes (van der Werf, S., et al Proc. Natl. Acad. Sci. USA, 83, 2330-2334 (1986); Enami, M. et al., Proc. Natl. Acad. Sci. USA, 87, 3802-3805 (1990), Schnell, MJ, et al., EMBO J., 13, 4195-4205 (1994)). For reovirus, transfection of the cells with a combination of SSARN, dsRNA and reovirus products translated in vi tro was shown to generate infectious viruses when supplemented with an auxiliary virus of different serotype (Roner, MR, et al., Virology, 179, 845-852 (1990)). However, to date, there have been no reports of an infectious virus recovered from segmented dsRNA genome from synthetic RNAs alone.

SUMMARY OF THE INVENTION This invention relates to infectious bursal disease virus (IBDV) that is associated with Gumboro disease of young chickens. More particularly, this invention relates to a system for the generation of infectious bursal disease virus (IBDV) using synthetic transcripts derived from cloned cDNA. The present invention will facilitate studies of the regulation of viral gene expression, pathogenesis and design of a new generation of live and inactivated vaccines.

DETAILED DESCRIPTION OF THE INVENTION In an effort to develop a reverse genetics system for IBDV, three independent full-length cDNA clones containing segment A of strain D78 serotype I or strain 23/82 serotype II and segment B of the strain were constructed. strain P2 serotype I, respectively. Synthetic RNAs of segments A and B were produced by transcription reaction in vi tro on plasmids linearized with T7 RNA polymerase. Transcripts from these segments, either treated or untreated with DNase or RNase, were evaluated for the generation of infectious virus by transfection of Vero cells. The present inventors have demonstrated that synthetic transcripts derived from cloned DNA corresponding to the complete genome of an animal virus segmented ARNds can give a virus of replication. The recovery of the infectious virus after the transfection of the cells with positive sense RNAs derived from cDNA cloned from a virus with a dsRNA genome (IBDV) completes the search to generate inverse infectious systems for RNA viruses. Several investigators have generated infectious animal RNA viruses from cloned cDNA (Boyer, J.C., et al., Virology, 198, 415-426 (1994)). Van der Werf et al. were the first to generate poliovirus, a positive-strand RNA virus, using synthetic RNA produced by T7 RNA polymerase in a cloned cDNA template (van der Werf, S., et al., Proc. Natl. Acad. Sci. USA, 83, 2330-2334 (1986)), later, Enami et al. rescued the influenza virus, a segmented negative strand RNA virus (Enami, M., et al., Proc. Natl. Acad. Sci. USA, 87, 3802-3805 (1990)); and Schnell et al. they generated rabies virus, a non-segmented negative strand RNA virus, from cDNAs cloned from their respective genomes (Schnell, M.J., et al., EMBO J., 13, 4195-4205 (1994)). Roner et al. developed an infectious system for a segmented reovirus ARNds by transfecting cells with a combination of ssRNA, synthetic dsRNA, reovirus products translated in vi tro and supplemented with an auxiliary virus of different serotype (Roner, MR et al., Virology, 179, 845-852 (1990)). The resulting virus was discriminated from the helper virus by plaque assay. However, in this system the use of an auxiliary virus was necessary. In contrast, the reverse genetics system of IBDV described herein does not require an auxiliary virus or other viral proteins. The transfection of cells with positive sense RNAs of the two segments was sufficient to generate the infectious virus (IBDV). The fate of one or four additional nucleotides, respectively, transcribed at the 3'-end of segment A was not determined. However, this did not prevent the replication of the viral dsRNA. Similar effects were observed by different investigators for positive-strand RNA viruses (Boyer, J. C, et al., Virology, 198, 415-426 (1994)). Transfection of positive sense RNAs from both segments in the same cell was necessary to successfully recover the IBDV. The transfected RNAs of both segments had to be translated by the cell translation machinery. The polyprotein of segment A was supposedly processed to the proteins VP2, VP3 and VP4 that form the viral capsid. The translated VP1 protein of segment B probably acted as RNA-dependent RNA polymerase and transcribed negative strands from synthetic positive strands of both segments and the reaction products formed dsRNA. Recently, Dobos reported that in vitro transcription by the virion RNA-dependent RNA polymerase of infectious pancreatic necrosis virus (IPNV), a prototype virus of the family Birnaviridae, is primed by VPl and then continues by way of a displacement mechanism. of asymmetric, semiconservative strand to synthesize only positive strands during the replication of the viral genome (Dobos, P., Virology, 208, 10-25 (1995)). The present system shows that the synthesis of negative strands continues in the positive strands. If the resulting transcribed negative strand RNA does or does not serve as a template for the transcription of positive strands, it remains a matter for further investigation. To prove that the infectious IBDV contained in the supernatant of transfected cells was derived in P774 effect of the synthetic transcripts, an artificial chimera was generated that contained segment A of a serotype II and segment B strain of a serotype I strain. The sequence analysis verified this genome combination. The results also indicate that the motifs of the terminal sequence described by Mundt and Müller are probably responsible for the replication, classification and packaging of the viral genome (Mundt, E. et al., Virology, 209, 10-18 (1995)). The presence of specific terminal sequences of a serotype obviously does not prevent the adequate replication of segment A serotype II by the action of the RNA-dependent RNA polymerase VPl of segment B serotype I. The ability to create recombinant viruses will help a lot to analyze the precise function of serotype-specific and serotype-common terminal sequences. The recovery of infectious IBDV demonstrates that only positive-strand RNAs from both segments are sufficient to initiate dsRNA replication. Thus, the results are in accordance with the general characteristics of the replication of reovirus and rotavirus in which positive-strand RNAs serve as a template for the synthesis of negative strand progeny to give dsRNA (Schonberg, M., et al. , Proc. Natl. Acad. Sci. Patton, JT, Virus Res., 6, 217-233 (1986); Chen, D., et al., J. Virol., 68, 7030-7039 (1994)). However, the strand displacement mechanisms, semiconservatives, proposed by Spies, et al. and Dobos can not be excluded (Spies, U., et al., Virus Res., 8, 127-140 (1987); Dobos, P., Virology, 208, 10-25 (1995)). The development of a reverse genetics system for IBDV will greatly facilitate future studies of gene expression, pathogenesis and will aid in the design of new generations of live and inactivated IBDV vaccines. In the sense in which it is used in the present application, the term "synthetic" when applied to nucleic acids indicates that it is a human-made nucleic acid in contrast to naturally occurring nucleic acids. The term does not imply limitation with respect to the manufacturing method, which can be chemical or biological as long as it involves human intervention. The term "cDNA" is intended to encompass any cDNA containing -segments A and B and the non-coding regions 5 '- and 3' - of segments A and B. The term "infectious" when applied to viruses indicates that the virus has the ability to reproduce. The virus can be pathogenic or non-pathogenic and still be infectious. The present invention provides a system for the generation of infectious bursal disease virus using synthetic RNA transcripts. This system can be used for the study of the regulation of viral gene expression, pathogenesis and for the design of a new generation of live and inactivated IBDV vaccines. The present invention provides a recombinant vector containing at least one cDNA copy according to the present invention. The recombinant vector may also comprise other necessary sequences such as expression control sequences, markers, amplification genes, signal sequences, promoters and the like, known in the art. Useful vectors for this purpose are plasmids and viruses such as báculoviruses, herpes viruses (HVT) and rash viruses, for example, poultry pustulation viruses, and the like. Also provided herein is a host cell transformed with the recombinant vector of the present invention or a host cell transfected with the synthetic RNA of the present invention. The host cell can be a eukaryotic or prokaryotic host cell. Suitable examples are E. coli, insect cell lines such as Sf-9, chicken embryo fibroblast cells (CEF), chicken embryo kidney cells (CEK), Vero cells of African green monkey and the like. An IBDV poultry vaccine comprising a protective amount for poultry of a virus or a portion of recombinantly produced virus is also part of this invention., where the virus is inactivated or modified in such a way that it is no longer virulent. The virus can be inactivated by physical or chemical means. Chemical inactivation can be achieved by treating the virus with, for example, enzymes, formaldehyde, β-propiolactone, ethylene imine or derivatives thereof, an organic solvent (eg, halogenated hydrocarbons) and / or a detergent. If necessary, the inactivating substance can be neutralized after the virus has been inactivated. Physical inactivation can be carried out by subjecting the viruses to radiation such as UV light, X-radiation, radiation- ?. The virus can be attenuated by known methods including serial subcultures, deletion of nucleic acid sequences and site-directed mutagenesis either before or after the production of the infectious virus to produce a virus that retains sufficient antigenicity but has decreased virulence. Physiologically acceptable vehicles for vaccination of poultry are known in the field of P774 technique and do not need to be described more in the present. Furthermore, to be physiologically acceptable in poultry the vehicle must not interfere with the immune response caused by the vaccine and / or with the expression of its polypeptide product. Other additives, such as adjuvants and stabilizers, among others, may also be contained in the vaccine in amounts known in the art. Preferably, adjuvants such as aluminum hydroxide, aluminum phosphate, vegetable and animal oils and the like are administered with the vaccine in amounts sufficient to enhance the immune response to IBDV. The amount of adjuvant added to the vaccine will vary depending on the nature of the adjuvant, in general it will vary between about 0.1 and 100 times the weight of the IBDV, preferably between about 1 and 10 times the weight of the IBDV. The vaccine of the present invention may also contain various stabilizers. Any suitable stabilizer may be used, including carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin or glucose; proteins such as albumin or casein; and buffers such as alkali metal phosphate and the like. A stabilizer is particularly advantageous when a preparation of the dry vaccine is prepared by P774 lyophilization. The vaccine can be administered by any known method to inoculate poultry, including by nasal route, ophthalmic route, by injection, in drinking water, in food, by exposure and the like. Preferably, the vaccine is administered by mass administration techniques such as placing the vaccine in drinking water or spraying the environment of the animals. When administered by injection, vaccines are preferably administered parenterally. Parenteral administration in the sense in which it is used herein means administration by intavenous, subcutaneous, intramuscular or itraperitoneal injection. The vaccine of the present invention is administered to poultry to prevent IBD at any time before or after incubation. Preferably, the vaccine is administered before birth and after the animal is approximately 6 weeks of age. Poultry is defined to include but not exclusively chickens, roosters, chickens, young chickens, broiler chickens, broiler chickens, laying hens, turkeys and ducks. The vaccine can be supplied in a sterile container in unitary form or in other quantities. It is preferably stored under refrigeration, below -20 ° C and more preferably below -70 ° C. This is thawed before use and can be refrozen immediately afterwards. For administration to poultry the recombinantly produced virus can be suspended in a vehicle in an amount between about 10 and 10 pfu / ml and more preferably between about 10 and 10 pfu / ml in a vehicle such as a saline solution . The inactivated vaccine may contain the antigenic equivalent of 10 to 10 pfu / ml suspended in a vehicle. Diluents and inert pharmaceutical carriers known in the art are examples of pharmaceutically acceptable carriers. Preferably, the vehicle or diluent is one that is compatible with the administration of the vaccine by the mass administration techniques. However, the vehicle or diluent may also be compatible with other methods of administration such as injection, eye drops and the like. The invention can also be used to produce vaccines combined with the IBDV material. The IBDV material can be combined with the antigenic material of Newcastle Disease Virus Infectious Bronchitis Virus, Reo virus Adeno virus and / or Marek virus. The aforementioned characteristics of P774 the present invention are further described in the following Examples. However, the present invention is not limited to the Examples and the variations will be apparent to those skilled in the art without departing from the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of cDNA constructs used for the synthesis of positive sense ssRNAs of IBDV with T7 RNA polymerase. The pUC19FLAD78 construct contains the cDNA of segment A of IBDV strain D78 and the recombinant plasmid pUC18FLA23 which contains the full-length cDNA of segment A of IBDV strain 23/82. Segment A of IBDV codes for the polyprotein (VP2-VP4-VP3) and the newly identified VP5 protein. Plasmid pUC18FLBP2 contains the B-segment cDNA of strain P2 which codes for RNA-dependent RNA polymerase (VPl). The specific sequences of the virus are underlined and the T7 promoter sequences are written in italics. The restriction sites are shown in bold and are identified. The cleavage sites of the linearized plasmids are shown by vertical arrows and the directions of transcription are marked by horizontal arrows. Figure 2 shows an agarose gel analysis P774 of the transcription reaction products that were used for the transfection of Vero cells. The synthetic RNAs were transcribed in vi tro using T7 RNA polymerase and linearized plasmids pUC19FLAD78 (lanes 2, 4 and 6) containing cDNA from segment A of IBDV strain D78 and pUC18FLBP2 (lanes 1, 3 and 5) containing cDNA from segment B of strain P2, respectively. After transcription, the reaction mixtures were treated with DNase (lanes 1 and 2), RNase (lanes 3 and 4) or left untreated (lanes 5 and 6). Two μl of the reaction products were analyzed on a 1% agarose gel. Lambda DNA, digested with Hind III / EcoR I, was used as a marker (M band). Figure 3 shows a comparison of nucleotide sequences of cloned RT-PCR fragments of segments A and B of the IBDV strain 23A / P2B chimeric (bold) with known sequences of segments A and B of strain 23/82 serotype II and strain P2 serotype I, respectively. The identities of the nucleotides are marked by two points. Figure 4 shows the DNA sequence of pUC18FLA23. Figure 5 shows the DNA sequence of pUC19FLAD78. Figure 6 shows the DNA sequence of pUC18FLBP2.

EXAMPLES Virus and Cells. Two strains of IBDV serotype I, the attenuated P2 strain from Germany and the vaccine strain D78 (Intervent International) and one strain serotype II, strain 23/82 non-pathogenic, were propagated in chicken embryo cells (CEC) and purified (Mundt, E. et al., Virology, 209, 10-18 (1995); Vakharia, VN, et al., Virus Res., 31, 265-273 (1994)). Vero cells were cultured in M199 medium supplemented with fetal calf serum (FCS) 5% and used for transfection experiments. The subsequent propagation of recovered virus and immunofluorescence studies were carried out on Vero cells (Mundt, E., et al., J. Gen. Virol., 76, 437-443 (1995)). For plaque assay, secondary CEC monolayers were prepared and used (Müller, H., et al., Virus Res., 4, 297-309 (1986)). Construction of Length cDNA Clones Complete IBDV genome. Clones of full-length cDNA from segments A and B of IBDV were prepared independently. CDNAs containing the complete coding region of RNA segment A of strain D78 were prepared using standard methods and cloning methods (Vakharia, V.N., et al., Virus Res., 31, 265-273 (1994)). Comparing the D78 terminal sequence with the recently published terminal sequences of other IBDV strains (Mundt, E. et al., Virology, 209, 10-18 (1995)), it was observed that the D78 cDNA clones lack P774 the first 17 and the last 10 nucleotides conserved at the 5'- and 3'- ends, respectively. Therefore, to construct a full-length cDNA clone of segment A, two first pairs (A5'-D78, A5-IPD78 and A3'-IPD78) were synthesized and used for PCR amplification (Table 1). The DNA segments were amplified according to the supplier protocol (New England Biolabs) using "Deep Vent Polymerase" (high fidelity thermophilic DNA polymerase). The amplified fragments were cloned into the EcoR I site of a pCRII vector (Invitrogen Corp.) to obtain plasmids pCRD78A5 'and pCRD78A3', respectively. Each plasmid was digested with EcoR I and Sal I and the resulting fragments were ligated in EcoR I digested with pUC19 to obtain the plasmid pUC19FLAD78 (SEQ ID NOS: 27 and 29) which now contains a full length cDNA copy of segment A coding for all structural proteins (VP2, VP4 and VP3, SEQ ID NO: 30) as well as the non-structural protein VP5 (SEQ ID NO: 28) (Figure 1). The first two pairs (A5'-23, A5IP23 and A3'-23, A3-IP23; see Table 1) were used for reverse transcription (RT) of viral genomic dsRNA of strain 23/82 using "Super Script RT II" (RNA-directed DNA polymerase with decreased RNAse H activity, GIBCO / BRL). The reaction products of RT were purified by phenol / chloroform extraction and ethanol precipitation. To obtain two cDNA fragments linked by primer pairs A5'-23, A5-IP23 and A3'-23, A3-IP23, respectively, the RT reaction products were amplified by PCR using "Deep Vent Polymerose". Both RT and PCR were carried out according to the supplier's protocol. The resulting PCR fragments were blunt end ligated into the vector pUC18 cleaved with Sma I to obtain pUC23A5 'and pUC23A3'. The 3'- end of segment A contained in the plasmid pUC23A3 'was ligated to the unfolded plasmid pUC23A5' with Hind III-BstB I to establish the full length cDNA of segment A of strain 23/82. The resulting plasmid was named pUC18FLA23 (SEQ ID NOS: 31 and 33) (Figure 1) and codes for structural proteins VP2, VP3 and VP4 (SEQ ID NO: 32) and non-structural proteins VP5 (SEQ ID NO: 34). To obtain cDNA clones of segment B of strain P2, two primer pairs (B5'-P2, B5-IPP2 and B3'-P2, B3-IPP2) were designed according to the published sequences and used for amplification RT-PCR (see Table 1) . Using genomic dsRNA as a template, cDNA fragments were synthesized and amplified according to the supplier's protocol (Perkin-Elmer Cetus). The amplified fragments were blunt endpoints ligated to the pBS vector cleaved with Sma I (Stratagene) to obtain clones pBSP2B5 'and pBSP2B3'. To build a full-length clone of segment B, P774 the 5 'end fragment of plasmid pBSP2B5' was first subcloned between the EcoR I and Pst I sites of the pUC18 vector to obtain pUCP2B5 '. Then the 3 'end fragment of plasmid pBSP2B3' was inserted between the unique Bgl II and Pst I sites of plasmid pUCP2B5 'to obtain a full length plasmid pUC18FLBP2 (SEQ ID NO: 25) coding for the VP1 protein (SEQ ID NO: 26) (Figure 1). The plasmids pUC18FLBP2, pUC18FLA23 and pUC19FLAD78 were completely sequenced using the DNA sequencing system "Sequenase" (US Biochem) and the sequence data were analyzed using the computer programs "DNASIS" (Pharmacia) or "PC / Gene" (Intelligenetics ). The integrity of the full-length constructs was analyzed by the coupled reticulocyte lysate system of transcription and translation in vi tro using T7 RNA polymerase (Promega). Transcription and Transfection of Synthetic RNAs. Plasmids pUC19FLAD78, pUC18FLA23 and pUC18FLBP2 were digested with enzymes BsrG I, Nsi I and Pst I (see Figure 1), respectively, and used as a template for transcription in vivo with T7 RNA polymerase (Promega). Briefly, cleavage assays with restriction enzymes were adjusted to 0.5% SDS and incubated with proteinase K (0.5 mg / ml) for 1 hour at 37 ° C. The linearized DNA templates (~ 3 μg) were recovered after precipitating with ethanol and were added separately to a transcription mixture (50 μl) containing 40 mM Tris-HCl (pH 7.-9), 10 mM NaCl, 6 mM MgCl2, 2 mM spermidine, 0.5 mM ATP, CTP and UTP, 0.1 mM GTP, 0.25 mM analog "cap" [m7G (5 ') PPP (5') G], 120 units of "RNasin" (ribonuclease inhibitor), 150 units of T7 RNA polymerase (Promega) and incubated for 1 hour at 37 ° C. The synthetic RNA transcripts were purified by phenol / chloroform extraction and ethanol precipitation. , the precipitation products were treated with either DNase or RNase (Promega) before the purification step Vero cells were grown to 80% confluence in 60 mm plates and washed once with phosphate buffered saline (PBS) Three ml of "OPTI-MEM I" (reduced serum medium containing HEPES ammobuffer, sodium bicarbonate, hypoxanthine, thymidine, pyruvate) Sodium, L-glutamine, microelements, growth factors and phenol red, from GIBCO / BRL) were added to the monolayers and the cells were incubated at 37 ° C for 1 hour in a C02 incubator. Simultaneously, 0.15 ml of "OPTI-MEM I" were incubated with 1.25 μg of "Lipofectin" reagent (N- [1- (2, 3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride and dioleylphosphatidylethanolamine, GIBCO / BRL) for 45 minutes in a polystyrene tube at room temperature. The synthetic RNA transcripts of the two segments resuspended in 0.15 ml of water treated with diethyl pyrocarbonate were added to the OPTI-MEM-Lipofectin mixture, mixed gently and incubated on ice for 5 min. After removing the "OPTI-MEM" from the monolayers in the 60 mm plates and replacing it with 1.5 ml of fresh "OPTI-MEM", the mixture containing the nucleic acids was added dropwise to the Vero cells and stirred gently. After 2 hours of incubation at 37 ° C, the mixture was replaced with M199 medium [CaCl2 (anhydrous), Fe (N03) 3 9H20, KCl, MgSO4 (anhydrous), NaCl, NaH2P04H20, NaHCO3, L-Alanine, L- Arginine HCl, L-Aspartic Acid, L-Cysteine HCl H20, L-Cysteine 2HC1, L-Glutamic Acid, L-Glutamine, Glycine, L-Histidine HCl H20, L-Hydroxyproline, L-Isoleucine, L-Leucine, L- Lysine HCl, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na2H20, L-Valine, Alpha Tocopherol P04 Na2, Ascorbic Acid, Biotin, Calciferol, D- Calcium Pantothenate, Choline Chloride, Folic Acid, I-Inositol, Menadione NaHS03 3H20, Niacin, Nicotinamide, para-a-benzoic acid, Pyridoxine HCl, Riboflavin, Thiamine HCl, Vitamin A Acetate, Adenine S04, Adenic Acid, ATP, Na2, Cholesterol, 2-Deoxy-Ribose, D-Glucose, Glutathione, Guanine HCl, Hypoxanthine Na, Red Phenol Na, Ribose, Sodium Acetate (anhydrous), Thymine, Tween 80, Uracil and Xanthine Na; from Mediatech, Inc.] containing 5% FCS (without rinsing cells) and the cells were re-incubated at 37 ° C for convenient time intervals. Identification of Generated IBDV. CEC was infected with the filtered supernatant (0.2 μm) of the Vero cells transfected with transcripts of pUC18FLA23 and pUC18FLP2B. At 16 hours post-infection, total cellular nucleic acids were isolated (Mundt, E. et al., Virology, 209, 10-18 (1995)). The primers were designed according to the published sequences and the RT-PCR fragments were amplified, cloned and sequenced (Mundt, E. et al., Virology, 209, 10-18 (1995)). The sequence data were analyzed using the computer program "DNASIS". Immunofluorescence Vero cells, grown on coverslips at 80% confluence, were infected with the supernatants derived from transfected Vero cells (after freeze-thaw) and incubated at 37 ° C for two days. The cells were then washed, fixed with acetone and treated with anti-IBVD rabbit serum. After washing, the cells were treated with fluorescein-labeled goat anti-rabbit antibody (Kirkegaard &Perry Lab.) And examined by fluorescence microscopy. Plate Test. Monocells of secondary CECs, cultured in 60 mm plates, were inoculated with the supernatants derived from the transfected Vero cells. After 1 hour of infection, the cells were washed once with PBS and covered with 0.8% noble agar (Difco) containing 10% tryptose phosphate broth, 2% FCS, 0.112% NaHCO 3, 10 units penicillin, 10 μg streptomycin. / ml, fungizone 0.25 μg / ml, neutral red 0.005%, phenol red 0.0015%. The cells were incubated at 37 ° C for 2 to 3 days until the plates could be observed and counted (Müller, H. et al., Virus Res., 4, 297-309 (1986)). Construction of Length cDNA clones Complete of the IBDV Genome. To develop the reverse genetics system for the IBDV dsRNA virus, two independent cDNA clones were constructed which contained segment A of strain D78 and segment B of strain P2 (Figure 1). Each plasmid encoded either the precursor of the structural proteins (VP2, VP4, VP3) and VP5 or only for the VPl protein (RNA-dependent RNA polymerase). The plasmid pUC18FLBP2 in digestion with Pst I and in vitro transcription by T7 RNA polymerase, could give RNA containing the correct 5'- and 3'-ends. Whereas, in digestion with BsrG I and transcription, the plasmid pUC19FLAD78 could give RNA containing the 5 '- correct end but with four additional nucleotides at the 3'- end. The coupled transcription and translation of the above plasmids in a rabbit reticulocyte system gave protein products that were correctly processed and migrated simultaneously with marker IBDV proteins after fractionation in SDS polyacrylamide gel and autoradiography (data not shown). Transcription, Transfection and Generation of Viruses Infectious Positive-sense transcripts of IBDV segment A and B were synthesized separately in vi tro with T7 RNA polymerase using linearized full length cDNA plasmid templates (see Figure 2). Although two species of RNA transcripts were observed for segment B on a neutral gel (lanes 1 and 5), fractionation of these samples on a denatured gel gave only one specific band of transcript (data not shown). To demonstrate that the sense-positive RNA transcripts of both segments are needed for the generation of infectious viruses, the transcript mixtures were incubated with different nucleases, as shown in Figure 2. The synthetic RNAs recovered after treating the products of transcription with DNase (bands 1 + 2), RNase (bands 3 + 4) or without treatment (bands 5 + 6) were used for the transfection of Vero cells. As a false control, lipofectin alone was used. At five days post-transfection, the cytopathic effect (CPE) was visible only in cells transfected with combined transcripts of P774 transcript treated or not with DNase, but not with transcript mixtures treated with RNase or control transfected in false. In addition, no CPE was detected when Vero cells were transfected with RNA from only one segment A or B (data not shown). These results demonstrated that the replication of IBDV that originates after the transfection of the Vero cells with SSARNs of positive sense of both segments of IBDV. To verify that the agent that causes CPE in Vero cells was in fact IBDV, Vero cells were frozen-thawed and supernatants were centrifuged and used to infect CEC or Vero cells.CEC infected with supernatants Derivatives of transfected Vero cells from transcription mixtures treated or not with DNase produced CPE in one day post-inoculation (Table 2). However, CPE could not be detected in the ECCs even after five days, with the Verna cell supernatants transfected mixtures of transcripts treated with RNase, transcription mixtures of untreated segments A or B and Vero cells transfected in fake.Similarly, when Vero cells in the coverslips were infected with the same supernatants in the same way as described above and examined by immunofluorescence staining after 2 days, only supernatants derived from Vero cells P774 transfected transcription mixtures treated or not with DNase gave positive immunofluorescence signal (Table 2). Recovery of Transfectant Viruses. To determine the time point for the recovery of infectious virus, Vero cells were transfected with RNA transcripts of segments A and B combined. At 4, 8, 16, 24, 36 and 48 hours post-transfection, the supernatants were examined for the presence of transfectant virus by infectivity and plaque assays, as shown in Table 3. Our results indicate that the virus It can be recovered as early as 36 hours after transfection. The virus titer was 2.3 x 10 pfu / ml which seems to decrease for samples obtained more than 48 hours after transfection. Generation of Chimeric Virus. To prove that the positive sense ssRNA of the two segments of IBDV are sufficient for the recovery of infectious virus, a chimeric IBDV was generated. Plasmid pUC18FLA23 which contained a full length sequence of segment A of strain serotype II was linearized by digestion of Nsi I and ssRNA was synthesized in vi tro using T7 RNA polymerase. The transcript ^ ssARN specifies the 5'-correct end but contains an additional residue at the 3'- end (Figure 1). Vero cells were transfected with segment A ssRNA of strain 23/82 serotype II and ssRNA of segment B of strain P2 serotype I. Five days after transfection when CPE was evident, the supernatant was clarified (after freezing -freeze) and was used to infect CEC. After a second subculture in CEC, virus genomic RNA was analyzed by RT-PCR and sequencing of the PCR products. Primers for segment A were designed to specifically amplify only A-segment sequences derived from the serotype II strain. The primer for segment B linked the sequences of the two serotypes. The amplified fragments were cloned and sequenced. The segments of segment A that were obtained showed a perfect coincidence with the sequences of the segment A of the 23/82 serotype II strain known, while the sequence of the segment B showed complete homology with respect to the sequences of segment B of the strain P2 serotype I published (Figure 3).

P774 Table 1. Oligonucleotides Used for the Construction of Full Length cDNA Clones of Genomic IBDV Segments A and B ? Composition and location of the oligonucleotide primers used for cloning. The sequences of the T7 promoter are marked in italics, the specific sequences of viruses are underlined and the restriction sites marked with bold. The orientation of the specific sequence of the primer virus is shown to sense (+) and antisense (-). The positions where the primers join (number of nucleotide) are according to the published sequences of strain P2 (2).

Table 2. Generation of IBDV Infections from Segment A and B Synthetic RNAs Transfected Material CPE Immunofluorescence Vero cells were transfected with synthetic RNAs of segments A and B derived from transcription reactions that were or were not treated with DNase or RNase. After five days, the supernatants were harvested, clarified by centrifugation and analyzed for the presence of virus. The infectivity of the recovered viruses was determined in CEC by the appearance of cytopathic effect (CPE) at 1-2 days post-inoculation. The specificity of the recovered viruses was determined by immunofluorescent staining of Vero cells infected with anti-IBDV rabbit serum.

Table 3. Post-Transfection Virus Recovery at Various Times Time in hours CPE Immunofluorescence pfu / ml post-transfection The Vero cells were transfected with synthetic RNAs of segment A and B in the same manner as described. The infectivity and specificity of the recovered viruses was detected by CPE in CEC and staining in immunofluorescence in Vero cells, respectively. Secondary CEC monolayers were used for the plaque assay after inoculating the cells with the supernatants derived from the transfected Vero cells. The approximate virus titre was calculated as plaque forming units per ml (pfu / ml).

P774 LISTINGS OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: VAKHARIA, Vikram N. MUNDT, Egbert (ii) TITLE OF THE INVENTION: A METHOD FOR GENERATING BIRNAVIRUS FROM SYNTHETIC RNA TRANSCRIPTS (iii) NUMBER OF SEQUENCES: 34 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: NIKAIDO, MARMELSTEIN, MURRAY & ORAM LLP (B) STREET: 655 Fifteenth Street, N. , Suite 330 - G Street Lobby (C) CITY: Washington (D) STATE: DC (E) COUNTRY: E.U.A. (F) POSTAL CODE: 20005-5701 (v) FORM FOR COMPUTER READING: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS ( D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (vi) DATA OF THIS APPLICATION (A) NUMBER OF APPLICATION: EU (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) INFORMATION OF POWDER / AGENT (A) NAME: KITTS, Monica C. (B) REGISTRATION NUMBER: 36,105 (C) REFERENCE / RECORD: P8172-6002 (ix) ) INFORMATION FOR TELECOMMUNICATION P774 (A) TELEPHONE: 202 / 638-5000 (B) TELEFAX: 202 / 638-4810 (2) INFORMATION FOR THE S? Q ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY : circular (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 GAATTCGGCT TTAATACGAC TCACTATAGG ATACGATCGG TCTGAC 46 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 AATTGGATCC GTTCGCGGGT CCCCTGTACA AAGCCGAATT C 41 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA P774 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 CGGCGAATTC ATGCATAGGG GACCCGCGAA CGGATC 36 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GTCAGACCGA TCGTATCCTA TAGTGAGTCG TATTAGAATT CTCT 44 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5 TTGCATGCCT GCAGGGGGCC CCCGCAGGCG AAG 33 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: circular P774 (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TCGTATCCTA TAGTGAGTCG TATTAGAATT C 31 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: GGAAGCCTGA GTGAGTTGAC TGACTACAGC TACAACGGGC TGATGTCAGC CACTGCGAAC 60 ATCAACGACA AGATCGGGAA CGTTCTAGTT GGAGAAGGGG TGACTGTTCT CAGTCTACCG 120 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: GGAAGCCTGA GTGAGTTGAC TGACTACAGC TACAACGGGC TGATGTCAGC CACTGCGAAC 60 ATCAACGACA AGATCGGGAA CGTTCTAGTT GGAGAAGGGG TGACTGTTCT CAGTCTACC 119 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs P774 (B) TYPE: nucleic acid (C) STRING TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: GGAAGCCTGA GTGAACTGAC AGATGTTAGC TACAATGGGT TGATGTCTGC AACAGCCAAC 60 ATCAACGACA AAATTGGGAA CGTCCTAGTA GGGGAAGGGG TCACCGTCCT CAGCTTACCC 120 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: TTTTCAATAG TCCACAGGCG CGAACGAAGA TCTCAGCAGC GTTCGGCATA AAGCCTACTG 60 CTGGACAAGA CGTGGAAGAA CTCTTGATCC CCAAAGTCTG GGTGCCACCT GAGGATCCGC 120 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 P774 TTTTCAACAG TCCACAGGCG CGAAGCACGA TCTCAGCAGC GTTCGGCATA AAGCCTACTG 50 CTGGACAAGA CGTGGAAGAA CTCTTGATCC CTAAAGTTTG GGTGCCACCT GAGGATCCGC 120 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: TTTTCAACAG TCCACAGGCO CGAAGCACGA TCTCAGCAGC GTTCGGCATA AAGCCTACTG 60 CTGGACAAGA CGTGGAAGAA CTCTTGATCC CTAAAGTTTG GGTGCCACCT GAGGATCCGC 120 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: TAATACGACT CACTATAGGA TACGATCGGT CTGACCCCGG GGGAGTCA 48 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple P774 (D) TOPOLOGY: linear (i i) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14 AGAGAATTCT AATACGACTC ACTATAGGAT ACGATCGGTC TGAC 44 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: TGTACAGGGG ACCCGCGAAC GGATCCAATT 30 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16 CGGCGAATTC ATGCATAGGG .GACCCGCGAA CGGATC 36 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid P774 (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17: CGTCGACTAC GGGATTCTGG 20 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: CAGAGGCAGT ACTCCGTCTG 20 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: AGTCGACGGG ATTCTTGCTT 20 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs P774 (B) TYPE: nucleic acid (C) STRING TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: GAAGGTGTGC GAGAGGAC 18 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: AGAGAATTCT AATACGACTC ACTATAGGAT ACGATGGGTC TGAC 44 (2) INFORMATION FOR SEQ ID NO: 22 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: CGATCTGCTG CAGGGGGCCC CCGC GGCGA AGG 33 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: CTTGAGACTC TTGTTCTCTA * -CTCC 24 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: ATACAGCAAA GATCTCGGG 19 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2827 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 112..2745 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: P774 GGATACGATG GGTCTGACCC TCTGGGAGTC ACGAATTAAC GTGGCTACTA GGGGCGATAC SO CCGCCGCTGG CCGCCACGTT AGTGGCTCCT CTTCTTGATG ATTCTGCCAC C ATG AGT 117 Met Ser 1 GAC ATT TTC AAC AGT CCA CAG GCG CGA AGC ACG ATC TCA GCA GCG TTC 165 Asp lie Phe Asn Ser Pro Gln Ala Arg Ser Thr lie Ser Ala Ala Phe 5 10 15 GGC ATA AAG CCT ACT GCT GGA CAA GAC GTG GAA GAA CTC TTG ATC CCT 213 Gly lie Lys Pro Thr Ala Gly Gln Asp Val Glu Glu. Leu Leu lie Pro 20 25 30 AAA GTT TGG GTG CCA CCT GAG GAT CCG CTT GCC AGC CCT AGT CGA CTG 261 Lys Val Trp Val Pro Pro Glu Asp Pro Leu Ala Ser Pro Ser Arg Leu 35 40 45 50 GCA AAG TTC CTC AGA GAG AAC GGC TAC AAA GTT TTG CAG CCA CGG TCT 309 Wing Lys Phe Leu Arg Glu Asn Gly Tyr Lys Val Leu Gln Pro Arg Ser 55 60 65 CTG CCC GAG AAT GAG GAT TAT GAG ACC GAC CAA ATA CTC CCA GAC TTA 357 Leu Pro Glu Asn Glu Glu Tlu Glu Thr Asp Gln lie Leu Pro Asp Leu 70 75 80 GCA TGG ATG CGA CAG ATA GAA GGG GCT GTT TTA AAA CCC ACT CTA TCT 405 Wing Trp Met Arg Gln lie Glu Gly Wing Val Leu Lys Pro Thr Leu Ser 85 90 95 CTC CCT ATT GGA GAT CAG GAG TAC TTC CCA AAG TAC TAC CAC ACA CAT 453 Leu Pro lie Gly Asp Gln Glu Tyr Phe Pro Lys Tyr Tyr Pro Thr His 100 105 110 CGC CCT AGC AAG GAG AAG CCC AAT GCG TAC CCG CCA GAC ATC GCA CTA 501 Arg Pro Ser Lys Glu Lys Pro Asn Ala Tyr Pro Pro Asp lie Ala Leu 115 120 125 130 P774 CTC AAG CAG ATG ATT TAC CTG TTT CTC CAG GTT CCA GAG GCC AAC GAG 549 Leu Lys Gln Met lie Tyr Leu Phe Leu Gln Val Pro Glu Wing Asn Glu 135 140 145 GGC CTA AAG GAT GAA GTA ACC CTC TTG ACC CAA AAC ATA AGG GAC AAG 597 Gly Leu Lys Asp Glu Val Thr Leu Leu Thr Gln Asn lie Arg Asp Lys 150 155 160 GCC TAT GGA AGT GGG ACC TAC ATG GGA CAA GCA AAT CGA CTT GTG GCC 645 Wing Tyr Gly Ser Gly Thr Tyr Met Gly Gln Wing Asn Arg Leu Val Wing 165 170 175 ATG AAG GAG GCC GCC ACT GGA AGA AAC CCA AAC AAG GAT CCT CTA AAG 693 Met Lys Glu Val Wing Thr Gly Arg Asn Pro Asn Lys Asp Pro Leu Lys 180 185 190 CTT GGG TAC ACT TTT GAO AGC ATC GCG CAG CTA CTT GAC ATC ACA CTA 741 Leu Gly Tyr Thr Phe Glu Ser lie Wing Gln Leu Leu Asp lie Thr Leu 195 200 205 210 CCG GTA GGC CCA CCC GGT GAG GAT GAC AAG CCC TGG GTG CCA CTC ACA 789 Pro Val Gly Pro Pro Gly Glu Asp Asp Lys Pro Trp Val Pro Leu Thr 215 220 225 AGA GTG CCG TCA CGG ATG TTG GTG CTG ACG GGA GAC GTA GAT GGC GAC 837 Arg Val Pro Ser Arg Met Leu Val Leu Thr Gly Ace p Val Asp Gly Asp 230 235 240 TTT GAG GTT GAA GAT TAC CTT CCC AAA ATC AAC CTC AAG TCA TCA AGT 885 Phe Glu Val Glu Asp Tyr Leu Pro Lys As Asn Leu Lys Ser Ser 245. 250 255 GGA CTA CCA TAT GTA GGT CGC ACC AAA GGA GAG ACA ATT GGC GAG ATG 933 Gly Leu Pro Tyr Val Gly Arg Thr Lys Gly Glu Thr He Gly Glu Met 260 265 270 ATA GCT ATC TCA AAC CAG TTT CTC AGA GAG CTA TCA ACA CTG TTG AAG 981 He Ala lie Ser Asn Gln Phe Leu Arg Glu Leu Ser Thr Leu Leu Lys 275 280 285 290 CAA GGT GCA GGG ACA AAG GGG TCA AAC AAG AAG AAG CTA CTC AGC ATG 1029 Gln Gly Wing Gly Thr Lys Gly Ser Asn Lys Lys Lys Leu Leu Ser Met 295 300 305 TTA AGT GAC TAT TGG TAC TTA TCA TGC GGG CTT TTG TTT CCA AAG GCT 1077 Leu Ser Asp Tyr Trp Tyr Leu Ser Cys Gly Leu Leu Phe Pro Lys Wing 310 315 320 GAA AGG TAC GAC AAA AGT ACA TGG CTC ACC AAG ACC CGG AAC ATA TGG 1125 Glu Arg Tyr Asp Lys Ser Thr Trp Leu Thr Lys Thr Arg Asn He Trp 325 330 335 P774 TCA GCT CCA TCC CCA ACA CAC CTC ATG ATC TCT ATG ATC ACC TGG CCC 1173 Ser Wing Pro Pro Thr His Leu Met He Met Met He Thr Trp Pro 340 345 350 GTG ATG TCC AAC AGC CCA AAT AAC GTG TTG AAC ATT GAA GGG TGT CCA 1221 Val Met Ser Asn Ser Pro Asn Asn Val Leu Asn He Glu Gly Cys Pro 355 360 365 370 TCA CTC TAC AAA TTC AAC CCG TTC AGA GGA GGG TTG AAC AGG ATC GTC 1269 Ser Leu Tyr Lys Phe Asn Pro Phe Arg Gly Gly Leu Asn Arg lie Val 375 380 385 GAG TGG ATA TTG GCC CCG GAA GAA CCC AAG GCT CTT GTA TAT GCG GAC 1317 Glu Trp He Leu Wing Pro Glu Glu Pro Lys Wing Leu Val Tyr Wing Asp 390 395 400 AAC ATA TAC ATT GTC CAC TCA AAC ACG TGG TAC TCA ATT GAC CTA GAG 1365 Asn He Tyr He Val His Ser Asn Thr Trp Tyr Ser He Asp Leu Glu 405 410 415 AAG GGT GAG GAC AAC TGC ACT CGC CAA CAC ATG CA CA GCC GCA ATG TAC 1413 Lys Gly Glu Wing Asn Cys Thr Arg Gln His Met Gln Wing Wing Met Tyr 420 425 430 TAC ATA CTC ACC AGA GGG TGG TCA GAC AAC GGC GAC CCA ATG TTC AAT 1461 Tyr He Leu Thr Arg Gly Trp Ser Asp Asn Gly As p Pro Met Phe Asn 435 440 445 450 CAA ACA TGG GCC ACC TTT GCC ATG AAC ATT GCC CCT GCT CTA GTG GTG 1509 Gln Thr Trp Wing Thr Phe Wing Met Asn He Wing Pro Wing Pro Val Leu Val 455. 460 465 GAC TCA TCG TGC CTG ATA ATG AAC CTG CAA ATT AAG ACC TAT GGT CAA 1557 Asp Ser Ser Cys Leu He Met Asn Leu Gln He Lys Thr Tyr Gly Gln 470 475 480 GGC AGC GGG AAT GCA GCC ACG TTC ATC AAC AAC CAC CTC TTG AGC ACA 1605 Gly Ser Gly Asn Wing Wing Thr Phe He Asn Asn His Leu Leu Ser Thr 485 490 495 CTA GTG CTT GAC CAG TGG AAC CTG ATG AGA CAG CCC AGA CAC GAC AGC 1653 Leu Val Leu Asp Gln Trp Asn Leu Met Arg Gln Pro Arg Pro Asp Ser 500 505 510 GAG GAG TTC AAA TCA ATT GAG GAC AAG CTA GGT ATC AAC TTT AAG ATT 1701 Glu Glu Phe Lys Ser He Glu Asp Lys Leu Gly He Asn Phe Lys He 515 520 525 530 GAG AGG TCC ATT GAT GAT ATC AGG GGC AAG CTG AGA CAG CTT GTC CTC 1749 Glu Arg Ser He Asp Asp He Axg Gly Lys Leu Arg Gln Leu Val Leu 535 540 545 P774 CTT GCA CAA CC GGG TAC CTG AGT GGG GGG GTT GAA CCA GAA CAA TCC 1797 Leu Wing Gln Pro Gly Tyr Leu Ser Gly Gly Val Glu Pro Glu Gln Ser 550 555 560 AGC CCA ACT GTT GAG CTT GAC CTA CTA GGG TGG TCA GCT ACA TAC AGC 1845 Ser Pro Thr Val Glu Leu Asp Leu Leu Gly Trp Ser Wing Thx Tyr Ser 565 570 575 AAA GAT CTC GGG ATC TAT GTG CCG GTG CTT GAC AAG GAA CGC CTA TTT 1893 Lys Asp Leu Gly He Tyr Val Pro Val Leu Asp Lys Glu Arg Leu Phe 580 585 590 TGT TCT GCT GCG TAT CCC AAG GGA GTA GAG AAC AAG AGT CTC AAG TCC 1941 Cys Ser Ala Ala Tyr Pro Lys Gly Val Glu Asn Lys Ser Leu Lys Ser 595 600 605 610 AAA GTC GGG ATC GAG CAG GCA TAC AAG GTA GTC AGG TAT GAG GCG TTG 1989 Lys Val Gly lie Glu Gln Ala Tyr Lys Val Val Arg Tyr Glu Ala Leu 615 620 625 AGG TTG GTA GGT GGT TGG AAC TAC CCA CTC CTG AAC AAA GCC TGC AAG 2037 Arg Leu Val Gly Gly Trp Asn Tyr Pro Leu Leu Asn Lys Wing Cys Lys 630 635 640 AAT AAC GCA GGC GCC GCT CGG CGG CAT CTG GAG GCC AAG GGG TTC CCA 2085 Asn Asn Wing Gly Ala Wing Arg Arg His Leu Glu Wing Lys Gly Phe Pro 645 650 655 CTC GAC GAG TTC CTA GCC GAG TGG TCT GAG CTG TCA GAG TTC GGT GAG 2133 Leu Asp Glu Phe Leu Wing Glu Trp Ser Glu Leu Ser Glu Phe Gly Glu 6 60 665 670 GCC TTC GAA GGC TTC AAT ATC AAG CTG ACC GTA ACA TCT GAG AGC CTA 2181 Wing Phe Glu Gly Phe Asn He Lys Leu Thr Val Thr Ser Glu Ser Leu 675 680 685 690 GCC GAA CTG AAC AAG CCA GTA CCC CCC AAG CCC CCA AAT GTC AAC AGA 2229 Wing Glu Leu Asn Lys Pro Val Pro Pro Lys Pro Pro Asn Val Asn Arg 695 700 705 CCA GTC AAC ACT GGG GGA CTC AAG GCA GTC AGC AAC GCC CTC AAG ACC 2277 Pro Val Asn Thr Gly Gly Leu Lys Wing Val Ser Asn Wing Leu Lys Thr 710 715 720 GGT CGG TAC AGG AAC GAA GCC GGA CTG AGT GGT CTC GTC CTT CTA GCC 2325 Gly Arg Tyr Arg Asn Glu Wing Gly Leu Ser Gly Leu Val Leu Leu Wing 725 730 735 ACA GCA AGA AGC CGT CTG CAA GAT GCA GTT AAG GCC AAG GCA GAA GCC 2373 Thr Ala Arg Ser Arg Leu Gln Asp Ala Val Lys Ala Lys Ala Glu Ala 740 745 750 P774 GAG AAA CTC CAC AAG TCC AAG CCA GAC GAC CCC GAT GAC GAC TGG TTC 2421 Glu Lys Leu His Lys Ser Lys Pro Asp Asp Pro Asp Wing Asp Trp Phe 755 760 765 770 GAA AGA TCA GAA ACT CTG TCA GAC CTT CTG GAG AAA GCC GAC ATC GCC 2 69 Glu Arg Ser Glu Thr Leu Ser Asp Leu Leu Glu Lys Wing Asp He Wing 775 780 785 AGC AAG GTC GCC CAC TCA GCA CTC GTG GAA ACA AGC GAC GCC CTT GAA 2517 Ser Lys Val Ala His Ser Ala Leu Val Glu Thr Ser Asp Wing Leu Glu 790 795 800 GCA GTT CAG TCG ACT TCC GTG TAC ACC CCC AAG TAC CCA GAA GTC AAG 2565 Wing Val Gln Ser Thr Ser Val Tyr Thr Pro Lys Tyr Pro Glu Val Lys 805 810 815 AAC CCA CAG ACC GCC TCC AAC CCC GTT GTT GGG CTC CAC CTG CCC GCC 2613 Asn Pro Gln Thr Wing AS Asn Pro Val Val Gly Leu His Leu Pro Wing 820 825 830 AAG AGA GCC ACC GGT GTC CAG GCC GCT CTT CTC GGA GCA GGA ACG AGC 2661 Lys Arg Wing Thr Gly Val Gln Wing Wing Leu Leu Gly Wing Gly Thr Ser T35 840 845 850 AGA CCA ATG GGG ATG GAG GCC CCA ACA CGG TCC AAG AAC GCC GTG AAA 2709 Arg Pro Met Gly Met Glu Wing Pro Thr Arg Ser Lys Asn Wing Val Lys 855 860 865 ATG GCC AAA CGG CGG CAA CGC CAA AAG GAG AGC CGC TAACAGCCAT 2755 • Met Ala Lys Arg Arg Gln Arg Gln Lys Glu Ser Arg 870 875 GATGGGAACC ACTCAAGAAG AGGACACTAA TCCCAGACCC CGTATCCCCG GCCTTCGCCT 2815 GCGGGGGCCC CC 2827 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 878 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: Met Ser Asp He Phe Asn Ser Pro Gln Ala Arg Ser Thr He Ser Wing 1 5 10 15 Wing Phe Gly He Lys Pro Thr Wing Gly Gln Asp Val Glu Glu Leu Leu P774 20 25 30 He Pro Lys Val Trp Val Pro Pro Glu Asp Pro Leu Wing Ser Pro Ser 35 40 45 Arg Leu Wing Lys Phe Leu Arg Glu Asn Gly Tyr Lys Val Leu Gln Pro 50 55 60 A-rg Ser Leu Pro Glu Asn Glu Glu Tyr Glu Thr Asp Gln He Leu Pro 65 70 75 80 Asp Leu Wing Trp Met Arg Gln He Glu Gly Wing Val Leu Lys Pro Thr 85 90 95 Leu Ser Leu Pro He Gly Asp Gln Glu Tyr Phe Pro Lys Tyr Tyr Pro 100 105 not Thr His Arg Pro Ser Lys Glu Lys Pro Asn Wing Tyr Pro Pro Asp He 115 120 125 Wing Leu Leu Lys Gln Met He Tyr Leu Phe Leu Gln Val Pro Glu Wing 130 135 140 Asn Glu Gly Leu Lys Asp Glu Val Thr Leu Leu Thr Gln Asn He Arg 145 150 155 160 Asp Lys Wing Tyr Gly Ser Gly Thr Tyr Met Gly Gln Wing Asn Arg Leu 165 170 175 Val Wing Met Lys Glu Val Wing Thr Gly Arg Asn Pro Asn Lys Asp Pro 180 _ 185 190 Leu Lys Leu Gly Tyr Thr Phe Glu Ser He Wing Gln Leu Leu Asp He 195 200 205 Thr Leu Pro Val Gly Pro Pro Gly Glu Asp Asp Lys Pro Trp Val Pro 210 215 220 Leu Thr Arg Val Pro Ser Arg Met Leu Val Leu Thr Gly Asp Val Asp 225 230 235 240 Gly Asp Phe Glu Val Glu Asp Tyr Leu Pro Lys He Asn Leu Lys Ser 245 250 255 Being Ser Gly Leu Pro Tyr Val Gly Arg Thr Lys Gly Glu Thr He Gly 260 265 270 Glu Met He Wing He Being Asn Gln Phe Leu Arg Glu Leu Ser Thr Leu 275 280 285 Leu Lys Gln Gly Wing Gly Thr Lys Gly Ser Asn Lys Lys Lys Leu Leu P774 290 295 300 Ser Met Leu Ser Asp Tyr Trp Tyr Leu Ser Cys Gly Leu Leu Phe Pro 305 310 315 320 Lys Ala Glu Arg Tyr Asp Lys Ser Thr Trp Leu Thr Lys Thr Arg Asn 325 330 335 He Trp Be Wing Pro Pro Thr His Leu Met He Met Met He Thr 340 345 350 Trp Pro Val Met Ser Asn Ser Pro Asn Asn Val Leu Asn He Glu Gly 355 360 365 Cys Pro Ser Leu Tyr Lys Phe Asn Pro Phe Arg Gly Gly Leu Asn Arg 370 375 380 He Val Glu Trp He Leu Ala Pro Glu Glu Pro Lys Ala Leu Val Tyr 385 390 395 400 Wing Asp Asn He Tyr He Val His Ser Asn Thr Trp Tyr Ser He Asp 405 410 415 Leu Glu Lys Gly Glu Wing Asn Cys Thr Arg Gln His Met Gln Wing Wing 420 425 430 Met Tyr Tyr He Leu Thr Arg Gly Trp Ser Asp Asn Gly Asp Pro Met 435 440 445 Phe Asn Gln Thr Trp Wing Thr Phe Wing Met Asn He Ala Pro Ala Leu 450 455 460 Val Val Asp Ser Ser Cys Leu He Met Asn Leu Gln He Lys Thr Tyr 465 470 475 480 Gly Gln Gly Ser Gly Asn Wing Wing Thr Phe He Asn Asn His Leu Leu 485 490 495 Be Thr Leu Val Leu Asp Gln Trp Asn Leu Met Arg Gln Pro Arg Pro 500 505 510 Asp Ser Glu Glu Phe Lys Ser He Glu Asp Lys Leu Gly He Asn Phe 515 520 525Arg Ser He Asp Asp He Arg Gly Lys Leu Arg Gln Leu 530 535 540 Val Leu Leu Ala Gln Pro Gly Tyr Leu Ser Gly Gly Val Glu Pro Glu 545 550 555 560 Gln Ser Ser Pro Thr Val Glu Leu Asp Leu Leu Gly Trp Ser Ala Thr 74 565 570 575 Tyr Ser Lys Asp Leu Gly He Tyr Val Pro Val Leu Asp Lys Glu Arg 580 585 590 Leu Phe Cys Ser Ala Ala Tyr Pro Lys Gly Val Glu Asn Lys Ser Leu 595 600 605 Lye Ser Lys Val Gly He Glu Gln Ala Tyr Val Lys Val Arg Tyr slu 610 615 620 Wing Leu Arg Leu Val Gly Gly Trp Asn Tyr Pro Leu Leu Asn Lys Wing 625 630 635 640 Cys Lys Asn Asn Wing Gly Wing Wing Arg Arg His Leu Glu Wing Lys Gly 645 650 655 Phe Pro Leu Asp Glu Phe Leu Wing Glu Trp Ser Glu Leu Ser Glu Phe 660 665 670 Gly Glu Wing Phe Glu Gly Phe Asn He Lys Leu Thr Val Thr Ser Glu 575 680 685 Ser Leu Wing Glu Leu Asn Lys Pro Val Pro Pro Lys Pro Pro Asn Val 690 695 700 Asn Arg Pro Val Asn Thr Gly Gly Leu Lys Wing Val Ser Asn Ala Leu 705 710 715 720 Lys Thr Gly Arg Tyr Arg Asn Glu Wing Gly Leu Ser Gly Leu Val Leu 725 730 735 Leu Ala Thr Ala Arg Ser Arg Leu Gln Asp Wing Val Lys Wing Lys Wing 740 745 750 Glu Wing Glu Lys Leu His Lys Ser Lys Pro Asp Asp Pro Asp Wing Asp 755 760 765 Trp Phe Glu Arg Ser Glu Thr Leu Ser Asp Leu Leu Glu Lys Wing Asp 770 775 780 He Wing Ser Lys Val Wing His Ser Wing Leu Val Glu Thr Ser Asp Wing 785 790 795 800 Leu Glu Ala Val Gln Ser Thr Ser Val Tyr Thr Pro Lys Tyr Pro Glu 805 810 815 Val Lys Asn Pro Gln Thr Ala Ser Asn Pro Val Val Gly Leu His Leu 820 825 830 Pro Ala Lys Arg Ala Thr Gly Val Gln Ala Ala Leu Leu Gly Ala Gly 4 835 840 845 Thr Ser Arg Pro Met Gly Met Glu Wing Pro Thr Arg Ser Lys Asn Wing 850 855 860 Val Lys Met Wing Lys Arg Arg Gln Arg Gln Lys Glu Ser Arg 865 870 875 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3261 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 97.531 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27 P774 GGATACGATC GGTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCGTCAA GGCCTTGTTC 60 CAGGATGGGA CTCCTCCTTC TACAACGCTA TCATTG ATG GTT AGT AGA GAT CAG 114 Met Val Ser Arg Asp Gln 880 ACA AAC GAT CGC AGC GAT GAC AAA CCT GCA AGA TCA AAC CCA ACA GAT 162 Thr Asn Asp Arg Ser Asp Asp Lys Pro Wing Arg Ser Asn Pro Thr Asp 885 890 895 900 TGT TCC GTT CAT ACG GAG CCT TCT GAT GCC AAC AAC CGG ACC GGC GTC 21C Cys Ser Val His Thr Glu Pro Ser Asp Ala Asn. Asn Arg Thr Gly Val 905 910 915 CAT TCC GGA CGA CAC CCT GGA GAA GCA CAC TCT CAG GTC AGA GAC CTC 25. His Ser Gly Arg His Pro Gly Glu Ala His Ser Gln Val Arg Asp Leu 920 925 930 GAC CTA CAA TTT GAC TGT GGG GGA CAC AGG GTC AGG GCT AAT TGT CTT 30 »Asp Leu Gln Phe Asp Cys Gly Gly His Arg Val Arg Wing Asn Cys Leu 935 940 945 TTT CCC TGG ATT CCC TGG CTC AAT TGT GGG TGC TCA CTA CAC ACT GCA 35! Phe Pro Trp lie Pro Trp Leu Asn Cys Gly Cys Ser Leu His Thr Ala 950 955 960 GGG CAA TGG GAA CTA CAA GTT CGA TCA GAT GCT CCT GAC TGC CCA GAA 402 Gly Gln Trp Glu Leu Gln Val Arg Ser Aep Wing Pro Asp Cys Pro Glu 965 970 975 980 CCT ACC GGC CAG TTA CAA CTA CTG CAG GCT AGT GAG TCG GAG TCT CAC 450 Pro Thr Gly Gln Leu Gln Leu Leu Gln Wing Ser Glu Ser Glu Ser His 985 990 995 AGT GAG GTC AAG CAC ACT TCC TGG TGG CGT TTA TGC ACT AAA CGG CAC 498 Ser Glu Val Lys His Thr Ser Trp Trp Arg Leu Cys Thr Lys Arg His 1000 1005 1010 CAT AAA CGC GCT CTT CCA AGG AAG CCT GAG TGAACTGACA GATGTTAGCT 551 His Lys Arg Arg Asp Leu Pro Arg Lys Pro Glu 1015 1020 ACAATGGGTT GATGTCTGCA ACAGCCAACA TCAACGACAA AATTGGGAAC GTCCTAGTAG_611_GGGAAGGGGT CACCGTCCTC AGCTTACCCA CATCATATGA TCTTGGGTAT GTGAGGCTTG 671 GTGACCCCAT TCCCGCAATA GGGCTTGACC CAAAAATGGT AGCCACATGT GACAGCAGTG 731 ACAGGCCCAG AGTCTACACC ATAACTGCAG CCGATGATTA CCAATTCTCA TCACAGTACC 791 AACCAGGTGG GGTAACAATC ACACTGTTCT CAGCCAACAT TGATGCCATC ACAAGCCTCA 851 GCGTTGGGGG AGAGCTCGTG TTTCAAACAA GCGTCCACGG CCTTGTACTG GGCGCCACCA 911 TCTACCTCAT AGGCTTTGAT GGGACAACGG TAATCACCAG GGCTGTGGCC GCAAACAATG 971 GGCTGACGAC CGGCACCGAC AACCTTATGC CATTCAATCT TGTGATTCCA ACAAACGAGA 1031 TAACCCAGCC AATCACATCC ATCAAACTGG AGATAGTGAC CTCCAAAAGT GGTGGTCAGG 1091 CAGGGGATCA GATGTCATGG TCGGCAAGAG GGAGCCTAGC AGTGACGATC CATGGTGGCA 1151 ACTATCCAGG GGCCCTCCGT CCCGTCACGC TAGTGGCCTA CGAAAGAGTG GCAACAGGAT 1211 CCGTCGTTAC GGTCGCTGGG GTGAGCAACT TCGAGCTGAT CCCAAATCCT GAACTAGCAA 1271 AGAACCTGGT TACAGAATAC GGCCGATTTG ACCCAGGAGC CATGAACTAC ACAAAATTGA 1331 TACTGAGTGA GAGGGACCGT CTTGGCATCA AGACCGTCTG GCCAACAAGG GAGTACACTG 1391 ACTTTCGTGA ATACTTCATG GAGGTGGCCG ACCTCAACTC TCCCCTGAAG ATTGCAGGAG 1451 CATTCGGCTT CAAAGACATA ATCCGGGCCA TAAGGAGGAT AGCTGTGCCG GTGGTCTCCA 1511 CATTGTTCCC ACCTGCCGCT CCCCTAGCCC ATGCAATTGG GGAAGGTGTA GACTACCTGC 1571 TGGGCGATGA GGCACAGGCT GCTTCAGGAA CTGCTCGAGC CGCGTCAGGA AAAGCAAGAG 1631 P774 CTGCCTCAGG CCGCATAAGG CAGCTGACTC TCGCCGCCGA CAAGGGGTAC GAGGTAGTCG 1691 CGAATCTATT CCAGGTGCCC CAGAATCCCG TAGTCGACGG GATTCTTGCT TCACCTGGGG 1751 TACTCCGCGG TGCACACAAC CTCGACTGCG TGTTAAGAGA GGGTGCCACG CTATTCCCTG 1811 TGGTTATTAC GACAGTGGAA GACGCCATGA CACCCAAAGC ATTGAACAGC AAAATGTTTG 1871 CTGTCATTGA AGGCGTGCGA GAAGACCTCC AACCTCCATC TCAAAGAGGA TCCTTCATAC 1931 GAACTCTCTC TGGACACAGA GTCTATGGAT ATGCTCCAGA TGGGGTACTT CCACTGGAGA 1991 CTGGGAGAGA CTACACCGTT GTCCCAATAG ATGATGTCTG GGACGACAGC ATTATGCTGT 2051 CCAAAGATCC CATACCTCCT ATTGTGGGAA ACAGTGGAAA TCTAGCCATA GCTTAC TGG 2111 ATGTGTTTCG ACCCAAAGTC CCAATCCATG TGGCTATGAC GGGAGCCCTC AATGCTTGTG 2171 GCGAGATTGA GAAAGTAAGC TTTAGAAGCA CCAAGCTCGC CACTGCACAC CGACTTGGCC 2231 TTAGGTTGGC TGGTCCCGGA GCATTCGATG TAAACACCGG GCCCAACTGG GCAACGTTCA 2291 TCAAACGTTT CCCTCACAAT CCACGCGACT GGGACAGGCT CCCCTACCTC AACCTACCAT 2351 ACCTTCCACC CAATGCAGGA CGCCAGTACC ACCTTGCCAT GGCTGCATCA GAGTTCAAAG 2411 AGACCCCCGA ACTCGAGAGT GCCGTCAGAG CAATGGAAGC AGCAGCCAAC GTGGACCCAC 2471 TATTCCAATC TGCACTCAGT GTGTTCATGT GGCTGGAAGA GAATGGGATT GTGACTGACA 2531 TGGCCAACTT CGCACTCAGC GACCCGAACG CCCATCGGAT GCGAAATTTT CTTGCAAACG 2591 CACCACAAGC AGGCAGCAAG TCGCAAAGGG CCAAGTACGG GACAGCAGGC TACGGAGTGG 2651 AGGCTCGGGG CCCCACACCA GAGGAAGCAC AGAGGGAAAA AGACACACGG ATCTCAAAGA 2711 AGATGGAGAC CATGGGCATC TACTTTGCAA CACCAGAATG GGTAGCACTC AATGGGCACC 2771 GAGGGCCAAG CCCCGGCCAG CTAAAGTACT GGCAGAACAC ACGAGAAATA CCGGACCCAA 2831 ACGAGGACTA TCTAGACTAC GTGCATGCAG AGAAGAGCCG GTTGGCATCA GAAGAACAAA 2891 TCCTAAGGGC AGCTACGTCG ATCTACGGGG CTCCAGGACA GGCAGAGCCA CCCCAAGCTT 2951 TCATAGACGA AGTTGCCAAA GTCTATGAAA TCAACCATGG ACGTGGCCCA AACCAAGAAC 3011 AGATGAAAGA TCTGCTCTTG ACTGCGATGG AGATGAAGCA TCGCAATCCC AGGCGGGCTC 3071 TACCAAAGCC CAAGCCAAAA CCCAATGCTC CAACACAGAG ACCCCCTGGT CGGCTGGGCC 3131 GCTGGATCAG GACCGTCTCT GATGAGGACC TTGAGTGAGG CTCCTGGGAG TCTCCCGACA 3191 P774 CCACCCGCGC AGGTGTGGAC ACCAATTCGG CCTTACAACA TCCCAAATTG GATCCGTTCG 3251 CGGGTCCCCT 3261 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 145 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: pro tein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28 Met Val Ser Arg Asp Gln Thr Asn Asp Arg Ser Asp Aep Lys Pro Wing 1 5 10 15 Arg Ser Asn Pro Thr Asp Cys Ser Val His Thr Glu Pro Ser Asp Wing 20 25 30 Asn Asn Arg Thr Gly Val His Ser Gly Arg His Pro Gly Glu Wing His 35 40 45 Ser Gln Val Arg Asp Leu Aep Leu Gln Phe Asp Cys Gly Gly His Arg 50 55 60 Val Arg Wing Asn Cys Leu Phe Pro Trp He Pro Trp Leu Asn Cys Gly 65 70 75 80 Cys Ser Leu His Thr Ala Gly Gln Trp Glu Leu Gln Val Arg Ser Asp 85 90 95 P774 Wing Pro Asp Cys Pro Glu Pro Thr Gly Gln Leu Gln Leu Leu Gln Wing 100 105 110 Ser Glu Ser Glu Ser His Ser Glu Val Lys His Thr Ser Trp Trp Arg 115 120 125 Leu Cys Thr Lys Arg His His Lys Arg Arg Asp Leu Pro Arg Lys Pro 130 135 140 Glu 145 (2) INFORMATION FOR SEQ ID NO: 29 CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3261 base pairs (B) TYPE: amino acids (C) CHAIN TYPE: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (ix) FEATURES: (A) NAME / KEY: CDS (B) LOCATION: 131..3166 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29 P774 GGATACGATC GGTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCGTCAA GGCCTTGTTC 60 CAGGATGGGA CTCCTCCTTC TACAACGCTA TCATTGATGG TTAGTAGAGA TCAGACAAAC 120 GATCGCAGCG ATG ACA AAC CTG CAA GAT CAA ACC CAA CAG ATT GTT CCG 169 Met Thr Asn Leu Gln Asp Gln Thr Gln Gln lie Val Pro 150 155 TTC ATA CGG AGC CTT CTG ATG CCA ACA ACC GGA CCG GCG TCC ATT CCG 217 Phe lie Arg Ser Leu Leu Met Pro Thr Thr Gly Pro Wing Pro lie 160 165 170 GAC GAC ACC CTG GAG AAG CAC ACT CTC AGG TCA GAG ACC TCG ACC TAC 265 Asp Asp Thr Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr 175 180 185 190 AAT TTG ACT GTG GGG ACA GGG TCA GGG CTA ATT GTC TTT TTC CCT 313 Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu lie Val Phe Phe Pro 195 200 205 GGA TTC CCT GGC TCA ATT GTG GGT GCT CAC TAC ACA CTG CAG GGC AAT 361 Gly Phe Pro Gly Ser lie Val Gly Wing His Tyr Thr Leu Gln Gly Asn 210 215 220 GGG AAC TAC AAG TTC GAT CAG ATG CTC CTG ACT GCC CAG AAC CTA CCG 409 Gly Asn Tyr Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro 225 230 235 GCC AGT TAC AAC TAC TGC AGG CTA GTG AGT CGG AGT CTC ACA GTG AGG 457 Wing Ser Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg 240 245 250 TCA AGC ACA CTT CCT GGT GGC GTT TAT GCA CTA AAC GGC ACC ATA AAC 505 Ser Ser Thr Leu Pro Gly Val Tyr Ala Leu Asn Gly Thr lie Asn 255 260 265 270 GCC GTG ACC TTC CAA GGA AGC CTG AGT GA A CTG ACA GAT GTT AGC TAC 553 P774 Wing Val Thr Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr 275 280 285 AAT GGG TTG ATG TCT GCA ACA GCC AAC ATC AAC GAC AAA ATT GGG AAC 601 Asn Gly Leu Met Ser Ala Thr Ala Asn lie Asn Asp Lys lie Gly Asn 290 295 300 GTC CTA GTA GGG GAA GGG GTC ACC GTC CTC AGC TTA CCC ACA TCA TAT 649 Val Leu Val Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr 305 310 315 GAT CTT GGG TAT GTG AGG CTT GGT GAC CCC ATT CCC GCA ATA GGG CTT 697 Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro lie Pro Wing He Gly Leu 320 325 330 GAC CCA AAA ATG GTA GCC ACA TGT GAC AGC AGT GAC AGG CCC AGA GTC 745 Asp Pro Lys Met Val Wing Thr Cys Asp Ser As Asp Arg Pro Arg Val 335 340 345 350 TAC ACC ATA ACT GCA GCC GAT GAT TAC CAA TTC TCA TCA CAG TAC CAA 793 Tyr Thr He Thr Wing Wing Asp Asp Tyr Gln Phe Be Ser Gln Tyr Gln 355 360 365 CCA GGT GGG GTA ACA ATC ACA CTG TTC TCA GCC AAC ATT GAT GCC ATC 841 Pro Gly Gly Val Thr He Thr Leu Phe Ser Wing Asn He Asp Wing He 370 375 380 ACA AGC CTC AGC GTT GGG GGA GTC CTC GTG TTT CAA ACA AGC GTC CAC 889 Thr Ser Leu Ser Val Gly Glu Glu Leu Val Phe Gln Thr Ser Val His 385: - 390 395 GGC CTT GTA CTG GGC GCC ACC ATC TAC CTC ATA GGC TTT GAT GGG ACA 937 Gly Leu Val Leu Gly Wing Thr He Tyr Leu He Gly Phe Asp Gly Thr 400 405 410 ACG GTA ATC ACC AGG GCT GTG GCC GAC AAC AAT GGG CTG ACG ACC GGC 985 Thr Val He Thr Arg Wing Val Wing Wing Asn Asn Gly Leu Thr T hr Gly 415 420 425 430 ACC GAC AAC CTT ATG CCA TTC AAT CTT GTG ATT CCA ACA AAC GAG ATA 1033 Thr Asp Asn Leu Met Pro Phe Asn Leu Val He Pro Thr Asn Glu He 435 440 445 ACC CAG CCA ATC ACA TCC ATC AAA CTG GAG ATA GTG ACC TCC AAA AGT 1081 Thr Gln Pro He Thr Ser He Lys Leu Glu He Val Thr Ser Lys Ser 450 455 460 GGT GGT CAG GCA GGG GAT CAG ATG TCA TGG TCG GCA AGA GGG AGC CTA 1129 Gly Gly Gln Wing Gly Asp Gln Met Ser Trp Ser Ala Arg Gly Ser Leu 465 470 475 P774 GCA GTG ACG ATC CAT GGT GGC AAC TAT CCA GGG GCC CTC CGT CCC GTC 1177 Wing Val Thr He His Gly Gly Asn Tyr Pro Gly Wing Leu Arg Pro Val 480 485 490 ACG CTA GTG GCC TAC GAA AGA GTG GCA ACA GGA TCC GTC GTT ACG GTC 1225 Thr Leu Val Wing Tyr Glu Arg Val Wing Thr Gly Ser Val Val Thr Val 495 500 505 510 GCT GGG GTG AGC AAC TTC GAG CTG ATC CCA AAT CCT GAA CTA GCA AAG 1273 Wing Gly Val Ser Asn Phe Glu Leu He Pro Asn Pro Glu Leu Wing Lys 515 520 525 AAC CTG GTT ACA GAA TAC GGC CGA TTT GAC CCA GGA GCC ATG AAC TAC 1321 Asn Leu Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Wing Met Asn Tyr 530 535 540 ACA AAA TTG ATA CTG AGT GAG AGG GAC CGT CTT GGC ATC AAG ACC GTC 1369 Thr Lys Leu He Leu Ser Glu Arg Asp Arg Leu Gly He Lys Thr Val 545 550 555 TGG CCA ACA AGG GAG TAC ACT GAC TTT CGT GAA TAC TTC ATG GAG GTG 1417 Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val 560 565 570 GCC GAC CTC AAC TCT CCC CTG AAG ATT GCA GGA GCA TTC GGC TTC AAA 1465 Wing Asp Leu Asn Ser Pro Leu Lys He Wing Gly Wing Phe Gly Phe Lys 575 580 585 590 GAC ATA ATC CGG GCC ATA AGG AGG ATA GCT GTG CCG GTG GTC TCC ACA 1513 Asp He He Arg Wing He Arg Arg He Wing Val Val Pro Ser Thr 595 500 605 TTG TTC CTC CCT GCC GCT CCC CTA GCC CAT GCA ATT GGG GAA GGT GTA 1561 Leu Phe Pro Pro Ala Wing Pro Leu Wing His Wing He Gly Glu Gly Val 610 615 620 GAC TAC CTG GGC GAT GAG GG CAG GCT GCT TCA GGA ACT GCT CGA 1609 Asp Tyr Leu Glu Asp Glu Wing Ala Gln Wing Wing Gly Thr Wing Arg 625 630 635 GCC GCG TCA GGA AAA GCA AGA GCT GCC TCA GGC CGC ATA AGG CAG CTG 1657 Ala Ala Ser Gly Lys Ala Arg Ala Ala Ser Gly Arg He Arg Gln Leu 640 645 650 ACT CTC GCC GCC GAC AAG GGG TAC GAG GTA GTC GCG AAT CTA TTC CAG 1705 Thr Leu Ala Wing Asp Lys Gly Tyr Glu Val Val Wing Asn Leu Phe Gln 655 660 665 670 GTG CCC CAG AAT CCC GTA GTC GGG ATT CTT GCT TCA CCT GGG GTA 1753 Val Pro Gln Asn Pro Val Val Asp Gly He Leu Wing Ser Pro Gly Val 675 680 685 P774 CTC CGC GGT GCA CAC AAC CTC GAC TGC GTG TTA AGA GAG GGT GCC ACG 1801 Leu Arg Gly Wing His Asn Leu Asp Cys Val Leu Arg Glu Gly Wing Thr 690 695 700 CTA TTC CCT GTG GTT ATT ACG GTG GAA GAC GCC ATG ACA CCC AAA 1849 Leu Phe Pro Val Val Thr Thr Val Glu Asp Wing Met Thr Pro Lys 705 710 715 GCA TTG AAC AGC AAA ATG TTT GCT GTC ATT GAA GGC GTG CGA GAA GAC 1897 Ala Leu Asn Ser Lys Met Phe Ala Val He Glu Gly Val Arg Glu Asp 720 725 730 CTC CAA CCT CCA TCT CAA AGA GGA TTC TTC ATA CGA ACT CTC TCT GGA 1945 Leu Gln Pro Pro Ser Gln Arg Gly Ser Phe He Arg Thr Leu Ser Gly 735 740 745 750 CAC AGA GTC TAT GGA TAT GCT CCA GAT GGG GTA CTT CCA CTG GAG ACT 1993 His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr 755 760 765 GGG AGA GAC TAC ACC GTT GTC CCA ATA GAT GAT GTC GG GAC GAC AGC 2041 Gly Arg Asp Tyr Thr Val Val Pro As Asp Asp Val Trp Asp Asp Ser 770 775 780 ATT ATG CTG TCC AAA GAT CCC ATA CCT ATT GTG GGA AAC AGT GGA 2089 He Met Leu Ser Lys Asp Pro He Pro Pro He Va l Gly Asn Ser Gly 785 790 795 AAT CTA GCC ATA GCT TAC ATG GAT GTG TTT CGA CCC AAA GTC CCA ATC 2137 Asn Leu Wing Wing Tyr Met Asp Val Phe Arg Pro Val Val Le Pro 800 805 810 CAT GTG GCT ATG ACG GGA GCC CTC AAT GCT TGT GGC GAG ATT GAG AAA 2185 His Val Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Glu He Glu Lys 815 820 825 830 GTA AGC TTT AGA AGC ACC AAG CTC GCC ACT GCA CAC CGA CTT GGC CTT 2233 Val Ser Phe Arg Be Thr Lys Leu Wing Thr Wing His Arg Leu Gly Leu 835 840 845 AGG TTG GCT GGT CCC GGA GCA TTC GAT AAC ACC GGG CCC AAC TGG 2281 Arg Leu Wing Gly Pro Gly Wing Phe Asp Val Asn Thr Gly Pro Asn Trp 850 855 860 GCA ACG TTC ATC AAA CGT TTC CCT CAC AAT CCA CGC GAC TGG GAC AGG 2329 Wing Thr Phe He Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg 865 870 875 CTC CCC TAC CTC AAC CTA CCA TAC CTT CCA CCC AAT GCA GGA CGC CAG 2377 Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Asn Wing Gly Arg Gln 880 885 890 P774 CT CAC CTT GCC ATG GCT GCA TCA GAG TTC AAA GAG ACC CCC GAA CTC 2425 Tyr His Leu Wing Ala Wing Wing Ser Glu Phe Lys Glu Thr Pro Glu Leu 895 900 905 910 GAG AGT GCC GTC AGA GCA ATG GAA GCA GCC GCC AAC GTG GAC CCA CTA 2473 Glu Ser Wing Val Arg Wing Met Glu Wing Wing Wing Asn Val Asp Pro Leu 915 920 925 TTC CAA TCT GCA CTC AGT GTG TTC ATG TGG CTG GAA GAG AAT GGG ATT 2521 Phe Gln Ser Ala Leu Ser Val Phe Met Trp Leu Glu Glu Asn Gly He 930 935 940 GTG ACT GAC ATG GCC AAC TTC GCA CTC AGC GAC CCG AAC GCC CAT CGG 2569 Val Thr Asp Met Wing Asn Phe Ala Leu Ser Asp Pro Asn Wing His Arg 945 950 955 ATG CGA AAT TTT CTT GCA AAC GCA CCA CAA GCA GGC AGC AAG TCG CAA 2617 Met Arg Asn Phe Leu Wing Asn Wing Pro Gln Wing Gly Ser Lys Ser Gln 960 965 970 AGG GCC AAG TAC GGG ACA GCA GGC TAC GGA GTG GAG GCT CGG GGC CCC 2665 Arg Ala Lys Tyr Gly Thr Ala Gly Tyr Gly Val Glu Ala Arg Gly Pro 975 980 985 990 ACA CCA GAG GAA GCA CAG AGG GAA AAA GAC ACA CGG ATC TCA AAG AAG 2713 Thr Pro Glu Glu Glue Ala Glu Arg Glu Lys Asp Thr Arg He Ser Lys Lys 995 1000 1005 ATG GAG ACC ATG GGC ATC TAC TTT GCA ACA CCA GAA TGG GTA GCA CTC 2761 Met Glu Thr Met Gly He Tyr Phe Wing Thr Pro Glu Trp Val Wing Leu 1010 1015 1020 AAT GGG CAC CGA GGG CCA AGC CCC GGC CAG CTA AAG TAC TGG CAG AAC 2809 Asn Gly His Arg Gly Pro Ser Pro Gly Gln Leu Lys Tyr Trp Gln Asn 1025 1030 1035 ACA CGA GAA ATA CCG GAC CCA AAC GAG GAC TAT CTA GAC TAC GTG CAT 2857 Thr Arg Glu He Pro Asp Pro Asn Glu Asp Tyr Leu Asp Tyr Val His 1040 1045 1050 GCA GAG AAG AGC CGG TTG GCA TCA GAA GAA CAA ATC CTA AGG GCA GCT 2905 Wing Glu Lys Ser Arg Leu Wing Ser Glu Glu Gln He Leu Arg Wing Wing 1055 1060 1065 1070 ACG TCG ATC TAC GGG GCT CCA GGA CAG GCA CAC CCC CAA GCT TTC 2953 Thr Ser He Tyr Gly Wing Pro Gly Gln Wing Glu Pro Pro Gln Wing Phe 1075 1080 1085 ATA GAC GAA GTT GCC AAA GTC TAT GAA ATC AAC CAT GGA CGT GGC CCA 3001 He Asp Glu Val Wing Lys Val Tyr Glu He Asn His Gly Arg Gly Pro 1090 1095 1100 P774 AAC CAA GAA CAG ATG AAA GAT CTG CTC TTG ACT GCG ATG GAG ATG AAG 3049 Asn Gln Glu Gln Met Lys Asp Leu Leu Leu Thr Wing Met Glu Met Lys 1105 1110 1115 CAT CGC AAT CCC AGG CGG GCT CTA CCA AAG CCC AAG CCA AAA CCC AAT 3097 His Arg Asn Pro Arg Arg Wing Leu Pro Lys Pro Pro Lys Pro Asn 1120 1125 1130 GCT CCA ACA CAG AGA CCC CCT GGT CGG CTG GGC CGC TGG ATC AGG ACC 3145 Pro Wing Pro Gln Arg Pro Pro Gly Arg Leu Gly Arg Trp He Arg Thr 1135 H40 1145 1150 GTC TCT GAT GAG GAC CTT GAG TGAGGCTCCT GGGAGTCTCC CGACACCACC 3196 Val Ser Asp Glu Asp Leu Glu 1155 CGCGCAGGTG TGGACACCAA TTCGGCCTTA CAACATCCCA AATTGGATCC GTTCGCGGGT 3256 CCCCT 3 61 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1012 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30 P774 Met Thr Asn Leu Gln Asp Gln Thr Gln Gln He Val Pro Phe He Arg 1 5 10 15 Be Leu Leu Met Pro Thr Thr Gly Pro Wing Be Pro Asp Asp Thr 20 25 30 Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr 35 40 45 Val Gly Asp Thr Gly Ser Gly Leu He Val Phe Phe Pro Gly Phe Pro 50 55 60 Gly Ser He Val Gly Ala His Tyr Thr Leu Gln Gly Asn Gly Asn Tyr 65 70 75 80 Lys Phe Asp Gln Met Leu Leu Thr Wing Gln Asn Leu Pro Wing Ser Tyr 85 90 95 Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr 100 105 110 4 Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr He Asn Ala Val Thr 115 120 125 Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu 130 135 140 Met Ser Ala Thr Ala Asn He Asn Asp Lys He Gly Asn Val Leu Val 145 150 155 160 Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly 165 170 175 Tyr Val Arg Leu Gly Asp Pro He Pro Wing He Gly Leu Asp Pro Lys 180 185 190 Met Val Wing Thr Cys Asp Ser As Asp Arg Pro Arg Val Tyr Thr He 195 200 205 Thr Wing Wing Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly 210 215 220 Val Thr He Thr Leu Phe Ser Wing Asn He Asp Wing He Thr Ser Leu 225 230 235 240 Ser Val Gly Gly Glu Leu Val Phe Gln Thr Ser Val His Gly Leu Val 245 250 255 Leu Gly Wing Thr He Tyr Leu He Gly Phe Asp Gly Thr Thr Val He 250 265 270 Thr Arg Wing Val Wing Wing Asn Asn Gly Leu Thr Thr Gly Thr Asp Asn 275 '• 280 285 Leu Met Pro Phe Asn Leu Val He Pro Thr Asn Glu He Thr Gln Pro 290 295 300 He Thr Ser He Lys Leu Glu He Val Thr Ser Lys Ser Gly Gly Gln 305 310 315 320 Wing Gly Asp Gln Met Ser Trp Be Wing Arg Gly Ser Leu Wing Val Thr 325 330 335 He His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val 340 345 350 Wing Tyr Glu Arg Val Wing Thr Gly Val Val Thr Val Wing Val Gly Val 355 360 365 Ser Asn Phe Glu Leu He Pro Asn Pro Glu Leu Ala Lys Asn Leu Val 370 375 380 Thr Glu Tyr Gly Arg Phe Asp Pro Gly Wing Met Asn Tyr Thr Lys Leu P774 385 390 395 400 He Leu Ser Glu Arg Asp Arg Leu Gly He Lys Thr Val Trp Pro Thr 405 410 415 Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Wing Asp Leu 420 425 430 Asn Ser Pro Leu Lys He Wing Gly Wing Phe Gly Phe Lys Asp He He 435 440 445 Arg Wing He Arg Arg He Wing Val Pro Val Val Ser Thr Leu Phe Pro 450 455 460 Pro Wing Wing Pro Leu Wing His Wing He Gly Glu Gly Val Asp Tyr Leu 465 470 475 480 Leu Gly Asp Glu Ala Gln Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser 485 490 495 Gly Lys Wing Arg Wing Wing Be Gly Arg He Arg Gln Leu Thr Leu Wing 500 505 510 Wing Asp Lys Gly Tyr Glu Val Val Wing Asn Leu Phe Gln Val Pro Gln 515 520 525 Asn Pro Val Val Asp Gly He Leu Wing Ser Pro Gly Val Leu Arg Gly 530 535 540 Wing His Asn Leu Asp Cys Val Leu Arg Glu Gly Wing Thr Leu Phe Pro 545 550 555 560 Val Val He Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu Asn 565 570 575 Be Lys Met Phe Wing Val He Glu Gly Val Arg Glu Asp Leu Gln Pro 580 585 590 Pro Ser Gln Arg Gly Ser Phe He Arg Thr Leu Ser Gly His Arg Val 595 600 605 Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg Asp 610 615 620 Tyr Thr Val Val Pro He Asp Asp Val Trp Asp Asp Ser He Met Leu 625 630 635 640 Ser Lys Asp Pro He Pro Pro He Val Gly Asn Ser Gly Asn Leu Wing 645 650 655 He has Ala Tyr Met Asp Val Phe Arg Pro Lys Val Pro He His Vai Ala 660 665 670 P774 Met Thr Gly Ala Leu Asn Ala Cys Gly Glu He Glu Lys Val Ser Phe 675 680 685 Arg Ser Thr Lys Leu Wing Thr Wing His Arg Leu Gly Leu Arg Leu Wing 690 695 700 Gly Pro Gly Wing Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe 705 710 715 720 He Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr 725 730 735 Leu Asn Leu Pro Tyr Leu Pro Pro Asn Wing Gly Arg Gln Tyr His Leu 740 745 750 Wing Wing Ala Wing Glu Phe Lys Glu Thr Pro Glu Leu Glu Wing 755 760 765 Val Arg Wing Met Glu Ala Wing Wing Asn Val Asp Pro Leu Phe Gln Ser 770 775 780 Wing Leu Ser Val Phe Met Trp Leu Glu Glu Asn Gly He Val Thr Asp 785 790 795 800 Met Ala Asn Phe Ala Leu Ser Asp Pro Asn Ala His Arg Met Arg Asn 805 810 815 Phe Leu Wing Asn Wing Pro Gln Wing Gly Ser Lys Ser Gln Arg Wing Lys 820 825 830 Tyr Gly Thr Wing Gly Tyr Gly Val Glu Wing Arg Gly Pro Thr Pro Glu 835: - 840 845 Glu Wing Gln Arg Glu Lys Asp Thr Arg He Be Lys Lys Met Glu Thr T50 855 860 Met Gly He Tyr Phe Wing Thr Pro Glu Trp Val Wing Leu Asn Gly His 865 870 875 880 Arg Gly Pro Ser Pro Gly Gln Leu Lys Tyr Trp Gln Asn Thr Arg Glu 885 890 895 He Pro Asp Pro Asn Glu Asp Tyr Leu Asp Tyr Val His Wing Glu Lys 900 905 910 Ser Arg Leu Wing Ser Glu Glu Gln He Leu Arg Wing Wing Thr Ser He 915 920 925 Tyr Gly Wing Pro Gly Gln Wing Glu Pro Pro Gln Wing Phe He Asp Glu 930 935 940 Val Ala Lys Val Tyr Glu He Asn His Gly Arg Gly Pro Asn Gln Glu 4 945 950 955 960 Gln Met Lys Asp Leu Leu Leu Thr Wing Met Glu Met Lys His Arg Asn 965 970 975 Pro Arg Arg Wing Leu Pro Lys Pro Pro Lys Pro Asn Wing Pro Thr 980 985 990 Gln Arg Pro Pro Gly Arg Leu Gly Arg Trp He Arg Thr Val Ser Asp 995 1000 1005 Glu Asp Leu Glu 1010 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3264 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: cDNA (ix) FEATURES: (A) NAME / KEY: CDS P774 (B) LOCATION: 97. . 531 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31 GGATACGATC GGTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCATCAC TGCCTTGTTC 60 CTGGTTGGAA CTCCTCTTTC TGCTGTACTA TCGTTG ATG GTG AGT AGA GAT CAG 114 Met Val Ser Arg Asp Gln 1015 ACA AAC GAT CGC AGC GAT GAC AAA CCT GAT GGA TCA CAC CCA ACA GAT 162 Thr Asn Asp Arg Ser Asp Asp Lys Pro Asp Gly Ser His Pro Thr Asp 1020 1025 1030 TGT TCC GTT CAT ACG GAG CCT TCT GAT GCC AAC GAC CGG ACC GGC GTC 210 Cys Ser Val His Thr Glu Pro Ser Asp Wing Asn Asp Arg Thr Gly Val 1035 1040 1045 1050 CAT TCC GGA CGA CAC CCT GGA GAA GCA CAC ACT CAG GTC CGA AAC CTC 258 His Ser Gly Arg His Pro Gly Glu Ala His Thr Gln Val Arg Asn Leu 1055 1060 1065 GAC TTA CAA CTT GAC TGT AGG GGA TAC AGG GTC AGG ACT AAT TGT CTT 306 P774 Asp Leu Gln Leu Asp Cys Arg Gly Tyr Arg Val Arg Thr Asn Cys Leu 1070 1075 1080 TTT CCC TGG ATT CCC TGG TTC AGT TGT AGG TGC TCA CTA CAC ACT GCA 354 Phe Pro Trp He Pro Trp Phe Ser Cys Arg Cys Ser Leu His Thr Wing 1085 1090 1095 GAG CAG TGG GAA CTA CCA ATT CGA CCA GAT GCT CCT GAC AGC GCA GAA 402 Glu Gln Trp Glu Leu Pro He Arg Pro Asp Wing Pro Asp Ser Wing Glu 1100 1105 1110 CCT GCC TGC CAG CTA CAA CTA CTG CAG GCT AGT GAG CAG GAG TCT AAC 450 Pro Wing Cys Gln Leu Gln Leu Leu Gln Wing Ser Glu Gln Glu Ser Asn 1115 1120 1125 1130 CGT ACG GTC AAG CAC ACT CCC TGG TGG CGT TTA TGC ACT AAA CGG AAC 498 Arg Thr Val Lys His Thr Pro Trp Trp Arg Leu Cys Thr Lys Arg Asn 1135 1140 1145 CAT AAA CGC AGT GAC CTT CCA CGG AAG CCT GAG TGAGTTGACT GACTACAGCT 551 His Lys Arg Ser Asp Leu Pro Arg Lys Pro Glu 1150 1155 ACAACGGGCT GATGTCAGCC ACTGCGAACA TCAACGACAA GATCGGGAAC GTTCTAGTTG 611 GAGAAGGGGT GACTGTTCTC AGTCTACCGA CTTCATATGA CCTTAGTTAT GTGAGACTCG 671 GTGACCCCAT CCCCGCAGCA GGACTCGACC CGAAGTTGAT GGCCACGTGC GACAGTAGTG 731 ACAGACCCAG AGTCTACACC ATAACAGCTG CAGATGAATA CCAATTCTCG TCACAACTCA 791 TCCCGAGTGG CGTGAAGACC 'ACACTGTTCT CCGCCAACAT CGATGCTCTC ACCAGCTTCA 851 GCGTTGGTGG TGAGCTTGTC TTCAGCCAAG TAACGATCCA AAGCATTGAA GTGGACGTCA 911 CCATTCACTT CATTGGGTTT GACGGGACAG ACGTAGCAGT CAAGGCAGTT GCAACAGACT 971 TTGGGCTGAC AACTGGGACA AACAACCTTG TGCCATTCAA CCTGGTGGTC CCAACAAATG 1031 AGATCACCCA GCCCATCACT TCCATGAAAC TAGAGGTTGT GACCTACAAG ATTGGCGGCA 1091 CCGCTGGTGA CCCAATATCA TGGACAGTGA GTGGTACACT AGCTGTGACG GTGCACGGAG 1151 GCAACTACCC TGGGGCTCTC CGTCCTGTCA CCCTGGTGGC CTATGAACGA GTGGCTGCAG 1211 GATCTGTTGT CACAGTTGCA GGGGTGAGCA ACTTCGAGCT AATCCCCAAC CCTGAGCTTG 1271 CAAAGAACCT AGTTACAGAG TATGGCCGCT TTGACCCCGG AGCAATGAAC TACACCAAAC 1331 TAATACTGAG TGAGAGAGAT CGTCTAGGCA TCAAGACAGT CTGGCCCACC AGGGAGTACA 1391 CCGATTTCAG GGAGTACTTC ATGGAGGTTG CAGATCTCAA CTCACCCCTA AAGATTGCAG 1451TTGG CTTTAAGGAC ATAATCCGAG CCATTCGGAA GATTGCGGTG CCAGTGGTAT 1511 CCACACTCTT CCCTCCAGCT GCACCCCTAG CACATGCAAT CGGAGAAGGT GTAGACTACC 1571 TCCTGGGCGA CGAGGCCCAA GCAGCCTCAG GGACAGCTCG AGCCGCGTCA GGAAAAGCTA 1631 GAGCTGCCTC AGGACGAATA AGGCAGCTAA CTCTCGCAGC TGACAAGGGG TGCGAGGTAG_1691_TCGCCAACAT GTTCCAGGTG CCCCAGAATC CCATTGTTGA TGGCATTCTG GCATCCCCAG 1751 GAATCCTGCG TGGCGCACAC AACCTCGACT GCGTGCTATG GGAGGGAGCC ACTCTTTTCC 1811 CTGTTGTCAT TACGACACTC GAGGATGAGC TGACCCCCAA GGCACTGAAC AGCAAAATGT 1871 TTGCTGTCAT TGAAGGTGTG CGAGAGGACC TCCAGCCTCC ATCCCAACGG GGATCCTTCA 1931 TTCGAACTCT CTCTGGCCAT AGAGTCTATG GCTATGCCCC AGACGGAGTA CTGCCTCTGG 1991 AGACCGGGAG AGACTACACC GTTGTCCCAA TTGATGATGT GTGGGACGAT AGCATAATGC 2051 TGTCGCAGGA CCCCATACCT CCAATCATAG GGAACAGCGG CAACCTAGCC ATAGCATACA 2111 TGGATGTCTT CAGGCCCAAG GTCCCCATCC ACGTGGCTAT GACAGGGGCC CTCAATGCCC 2171 GCGGTGAGAT CGAGAGTGTT ACGTTCCGCA GCACCAAACT CGCCACAGCC CACCGACTTG 2231 GCATGAAGTT AGCTGGTCCT GGAGCCTATG ACATTAATAC AGGACCTAAC TGGGCAACGT 2291 TCGTCAAACG TTTCCCTCAC AATCCCCGAG ACTGGGACAG GTTGCCCTAC CTCAACCTTC 2351 CTTATCTCCC ACCAACAGCA GGACGTCAGT TCCATCTAGC CCTGGCTGCC TCCGAGTTCA 2411 AAGAGACCCC AGAACTCGAA GACGCTGTGC GCGCAATGGA TGCCGCTGCA AATGCCGACC 2471 CATTGTTCCG CTCAGCTCTC CAGGTCTTCA TGTGGTTGGA AGAAAACGGG ATTGTGACCG 2531 ACATGGCTAA CTTCGCCCTC AGCGACCCAA ACGCGCATAG GATGAAAAAC TTCCTAGCAA 2591 ACGCACCCCA GGCTGGAAGC AAGTCGCAGA GGGCCAAGTA TGGCACGGCA GGCTACGGAG 2651 TGGAGGCTCG AGGCCCCACA CCAGAAGAGG CACAGAGGGA AAAAGACACA CGGATCTCCA 2711 AGAAGATGGA AACAATGGGC ATCTACTTCG CGACACCGGA ATGGGTGGCT CTCAACGGGC 2771 ACCGAGGCCC AAGCCCCGGC CAACTCAAGT ACTGGCAAAA CACAAGAGAA ATACCAGAGC 2831 CCAATGAGGA CTACCCAGAC TATGTGCACG CGGAGAAGAG CCGGTTGGCG TCAGAAGAAC 2891 AGATCCTACG GGCAGCCACG TCGATCTACG GGGCTCCAGG ACAGGCTGAA CCACCCCAGG 2951 CCTTCATAGA CGAGGTCGCC AGGGTCTATG AAATCAACCA TGGGCGTGGT CCAAACCAGG 3011 P774 AGCAGATGAA GGACCTGCTC CTGACTGCGA TGGAGATGAA GCATCGCAAT CCCAGGCGGG 3071 CTCCACCAAA GCCAAAGCCA AAACCCAATG CTCCATCACA GAGACCCCCT GGACGGCTGG 3131 GCCGCTGGAT CAGGACGGTC TCCGACGAGG ACTTGGAGTG AGGCTCCTGG GAGTCTCCCG 3191 ACACTACCCG CGCAGGTGTG GACACCAATT CGGCCTTCTA CCATCCCAAA TTGGATCCGT 3251 TCGCGGGTCC CCT 3264 (2) INFORMATION FOR SEQ ID NO: 32 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 145 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32 Met Val Ser Arg Asp Gln Thr Asn Asp Arg Ser Asp Asp Lys Pro Asp 1 5 10 15 Gly Ser His Pro Thr Asp Cys Ser Val His Thr Glu Pro Ser Asp Wing 20 25 30 Asn Asp Arg Thr Gly Val His Ser Gly Arg His Pro Gly Glu Ala His 35 40 45 Thr Gln Val Arg Asn Leu Asp Leu Gln Leu Asp Cys Arg Gly Tyr Arg 50 55 60 P774 Val Arg Thr Asn Cys Leu Phe Pro Trp He Pro Trp Phe Ser Cys Arg 65 70 75 80 Cys Ser Leu Hie Thr Ala Glu Gln Trp Glu Leu Pro He Arg Pro Asp 85 90 95 Wing Pro Asp Wing Wing Glu Pro Wing Cys Gln Leu Gln Leu Leu Gln Wing 100 105 110 Ser Glu Gln Glu Ser Asn Arg Thr Val Lys His Thr Pro Trp Trp Arg 115 120 125 Leu Cys Thr Lys Arg Asn His Lys Arg Ser Asp Leu Pro Arg Lys Pro 130 35 140 Glu 145 (2) INFORMATION FOR SEQ ID NO: 33 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3264 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: circular ( ii) TYPE OF MOLECULE: CADN (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 131..3169 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 33 GGATACGATC GGTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCATCAC TGCCTTGTTC 60 CTGGTTGGAA CTCCTCTTTC TGCTGTACTA TCGTTGATGG TGAGTAGAGA TCAGACAAAC 120 GATCGCAGCG ATG ACA AAC CTG ATG GAT CAC ACC CAA CAG ATT GTT CCG 169 Met Thr Asn Leu Met Asp His Thr Gln Gln He Val Pro 150 155 TTC ATA CGG AGC CTT CTG ATG CCA ACG ACC GGA CCG GCG TCC ATT CCG 217 Phe He Arg Be Leu Leu Met Pro Pro Thr Thr Gly Pro Wing Pro Pro 160 165 170 GAC GAC ACC CTG GAG AAG CAC ACA CTC AGG TCC GAA ACC TCG ACT TAC 265 Asp Asp Thr Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr 175 180 185 190 AAC TTG ACT GTA GGG GAT ACA GGG TCA GGA CTA ATT GTC TTT TTC CCT 313 Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu He Val Phe Phe Pro 195 200 205 GGA TTC CCT GGT TCA GTT GTA GGT GCT CAC TAC ACA CTG CAG AGC AGT 361 Gly Phe Pro Gly Ser Val Val Gly Wing His Tyr Thr Leu Gln Ser Ser 210 215 220 GGG AAC TAC CAA TTC GAC CAG ATG CTC CTG ACA GCG CAG AAC CTG CCT 409 Gly Asn Tyr Gln Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro 225 230 235 GCC AGC TAC AAC TAC TGC AGG CTA GTG AGC AGG AGT CTA ACC GTA CGG 457 Wing Ser Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg 240 245 250 P774 TCA AGC ACA CTC CCT GGT GGC GTT TAT GCA CTA AAC GGA ACC ATA AAC 505 Ser Ser Thr Leu Pro Gly Val Tyr Ala Leu Asn Gly Thr He Asn 255 260 265 270 GCA GTG ACC TTC CAC GGA AGC CTG AGT GAG TTG ACT GAC TAC AGC TAC 553 Wing Val Thr Phe His Gly Ser Leu Ser Glu Leu Thr Asp Tyr Ser Tyr 275 280 285 AAC GGG CTG ATG TCA GCC ACT GCG AAC ATC AAC GAC AAG ATC GGG AAC 601 Aen Gly Leu Met Ser Ala Thr Ala Asn He Asn Asp Lys He Gly Asn 290 295 300 GTT CTA GTT GGA GAA GGG GTG ACT GTT CTC AGT CTA CCG ACT TCA TAT 649 Val Leu Val Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr 305 310 315 GAC CTT AGT TAT GTG AGA CTC GGT GAC CCC ATC CCC GCA GGA GTC CTC 697 Asp Leu Ser Tyr Val Arg Leu Gly Asp Pro He Pro Wing Wing Gly Leu 320 325 330 GAC CCG AAG TTG ATG GCC ACG TGC GAC AGT AGT GAC AGA CCC AGA GTC 745 Asp Pro Lys Leu Met Wing Thr Cys Asp Ser As Asp Arg Pro Arg Val 335 340 345 350 TAC ACC ATA GCC ACA GCT GAT GAA TAC CAA TTC TCG TCA CAA CTC ATC 793 Tyr Thr He Thr Wing Wing Asp Glu Tyr Gln Phe Ser Se r Gln Leu He 355 360 365 CCG AGT GGC GTG AAG ACC ACA CTG TTC TCC GCC AAC ATC GAT GCT CTC 841 Pro Ser Gly Val Lys Thr Thu Leu Phe Ser Wing Asn lie Asp Wing Leu 370 375 380 ACC AGC TTC AGC GTT GGT GGT GAG CTT GTC TTC AGC CAA GTA ACG ATC 889 Thr Ser Phe Ser Val Gly Gly Glu Leu Val Phe Ser Gln Val Thr He 385 390 395 CAA AGC ATT GAA GTG GAC GTC ACC ATT CAC TTC ATT GGG TTT GAC GGG 937 Gln Ser He Glu Val Asp Val Thr He His Phe He Gly Phe Asp Gly 400 405 410 ACA GAC GTA GTC GTC AAG GCA GTT GCA ACA GAC TTT GGG CTG ACA ACT 985 Thr Asp Val Wing Val Lys Wing Val Wing Thr Asp Phe Gly Leu Thr Thr 415 420 425 430 GGG ACA AAC AAC CTT GTG CCA TTC AAC CTG GTG GTC CCA ACA AAT GAG 1033 Gly Thr Asn Asn Leu Val Pro Phe Asn Leu Val Val Pro Thr Asn Glu 435 440 445 ATC ACC CAG CCC ATC ACT TCC ATG AAA CTA GAG GTT GTG ACC TAC AAG 1081 He Thr Gln Pro He Thr Ser Met Lys Leu Glu Val Val Thr Tyr Lys 450 455 460 ATT GGC GGC ACC GCT GGT GAC CCA ATA TCA TGG ACA GTG AGT GGT ACA 1129 He Gly Gly Thr Wing Gly Asp Pro He Ser Trp Thr Val Ser Gly Thr 465 470 475 CTA GCT GTG ACG GTG CAC GGA GGC AAC TAC CCT GGG GCT CTC CGT CCT 1177 Leu Wing Val Thr Val His Gly Gly Asn Tyr Pro Gly Wing Leu Arg Pro 480 485 490 GTC ACC CTG GTG GCC TAT GAA CGA GTG GCT GCA GTC TCT GTT GTC ACA 1225 Val Thr Leu Val Wing Tyr Glu Arg Val Wing Wing Gly Ser Val Val Thr 495 500 505 510 GTT GCA GGG GTG AGC AAC TTC GAG CTA ATC CCC AAC CCT GAG CTT GC 1273 Val Ala Gly Val Ser Asn Phe Glu Leu He Pro Asn Pro Glu Leu Ala 515 520 525 AAG CTA GTT ACA GAG TAT GGC CGC TTT GAC CCC GGA GCA ATG AAC 1321 Lys Asn Leu Val Thr Glu Tyr Gly Arg Phe Aep Pro Gly Wing Met Asn 530 535 540 TAC ACC AAA CTA ATA CTG AGT GAG AGA GAT CGT CTA GGC ATC AAG ACA 1369 Tyr Thr Lys Leu He Leu Ser Glu Arg Asp Arg Leu Gly He Lys Thr 545 550 555 GTC TGG CCC ACC AGG GAG TAC ACC GAT TTC AGG GAG TAC TTC ATG GAG 1417 Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu 560 565 570 GTT GCA GAT CTC AAC TCA CCC CTA AAG ATT GCA GGA GCA TTT GGC TTT 1465 Val Wing Asp Leu Asn Ser Pro Leu Lys He Wing Gly Wing Phe Gly Phe 575 580 585 590 AAG GAC ATA ATC CGA GCC ATT CGG AAG ATT GCG GTG CCA GTG GTA TCC 1513 Lys Asp He He Arg Wing He Arg Lys He Wing Val Val Pro Val Ser 595 600 605 ACA CTC TTC CCT CCA GCT CCC GCA CTA GCA CAT GCA ATC GGA GAA GGT 1561 Thr Leu Phe Pro Pro Wing Wing Pro Leu Wing His Wing He Gly Glu Gly 610 615 620 GTA GAC TAC CTC CTG GGC GAC GAG GCC CAA GCA GCC TCA GGG ACA GCT 1509 Val Asp Tyr Leu Leu Gly Asp Glu Wing Gln Wing Wing Ser Gly Thr Wing 625 630 635 CGA GCC GCG TCA GGA AAA GCT AGA GCT GCC TCA GGA CGA ATA AGG CAG 1657 Arg Wing Wing Ser Gly Lya To. Arg Ala Ala Ser Gly Arg He Arg Gln 640 645 650 CTA ACT CTC GCA GAC GAC AAG GGG TGC GAG GTA GTC GCC AAC ATG TTC 1705 Leu Thr Leu Wing Wing Asp Lys Gly Cys Glu Val Val Wing Asn Met Phe 655 660 665 670 CAG GTG CCC CAG AAT CCC ATT GTT GAT GGC ATT CTG GCA TCC CCA GGA 1753 Gln Val Pro Gln Asn Pro He Val Asp Gly He Leu Wing Ser Pro Gly 675 680 685 ATC CTG CGT GGC GCA CAC AAC CTC GAC TGC GTG CTA TGG GAG GGA GCC 1801 He Leu Arg Gly Wing His Asn Leu Asp Cys Val Leu Trp Glu Gly Wing 690 695 700 ACT CTT TTC CCT GTT GTC ATT ACG ACA CTC GAG GAT GAG CTG ACC CCC 1849 Thr Leu Phe Pro Val Val He Thr Thr Leu Glu Asp Glu Leu Thr Pro 705 710 715 AAG GCA CTG AAC AGC AAA ATG TTT GCT GTC ATT GAA GGT GTG CGA GAG 1897 Lys Ala Leu Asn Ser Lys Met Phe Ala Val He Glu Gly Val Arg Glu 720 725 730 GAC CTC CAG CCT CCA TCC CAG CGG GGA TCC TTC ATT CGA ACT CTC TCT 1945 Asp Leu Gln Pro Pro Ser Gln Arg Gly Ser Phe He Arg Thr Leu Ser 735 740 745 750 GGC CAT AGA GTC TAT GGC TAT GCC CCA GAC GTA GTA CTG CCT CTG GAG 1993 Gly His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu 755 760 765 ACC GGG AGA GAC TAC ACC GTT GTC CCA ATT GAT GTG TGG GAC GAT 2041 Thr Gly Arg Asp Tyr Thr Val Val Pro He Asp Asp Valp Asp Asp 770 775 780 AGC ATA ATG CTG TCG CAG GAC CCC ATA CCT CCA ATC ATA GGG AAC AGC 2089 Ser He Met Leu Ser Gln Asp Pro He Pro Pro He He Gly Asn Ser 785 790 795 GGC AAC CTA GCC ATA GCA T AC ATG GAT GTC TTC AGG CCC AAG GTC CCC 2137 Gly Asn Leu Wing Wing Tyr Met Asp Val Phe Arg Pro Lys Val Pro 800 805 810 ATC CAC GTG GCT ATG ACA GGG GCC CTC AAT GCC CGC GGT GAG ATC GAG 2185 He His Val Ala Met Thr Gly Ala Leu Asn Ala Arg Gly Glu He Glu 815 820 825 830 AGT GTT ACG TTC CGC AGC ACC AAA CTC GCC ACA GCC CAC CGA CTG GGC 2233 Ser Val Thr Phe Arg Ser Thr Lye Leu Ala Thr Ala His Arg Leu Gly 835 840 845 ATG AAG TTA GCT GGT CCT GGA GCC TAT GAC ATT AAT ACA GGA CCT AAC 2281 Met Lys Leu Wing Gly Pro Gly Wing Tyr Asp He Asn Thr Gly Pro Asn 850 855 860 TGG GCA ACG TTC GTC AAA CGT TTC CCT CAC AAT CCC CGA GAC TGG GAC 2329 Trp Wing Thr Phe Val Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp 865 870 875 74 AGG TTG CCC TAC CTC AAC CTT CCT TAT CTC CCA CCA ACA GCA GGA CGT 2377 Arg Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Thr Wing Gly Arg 880 885 890 CAG TTC CAT CTA GCC CTG GCT GCC TCC GAG TTC AAA GAG ACC CCA GAA 2425 Gln Phe His Leu Ala Leu Wing Wing Ser Glu Phe Lys Glu Thr Pro Glu 895 900 905 910 CTC GAA GAC GCT GTG CGC GCA ATG GAT GCC GCT GCA AAT GCC GAC CCA 2473 Leu Glu Asp Ala Val Arg Ala Met Asp Wing Wing Wing Asn Wing Asp Pro 915 920 925 TTG TTC CGC TCA GCT CTC CAG GTC TTC ATG TGG TTG GAA GAA AAC GGG 2521 Leu Phe Arg Be Ala Leu Gln Val Phe Met Trp Leu Glu Glu Asn Gly 930 935 940 ATT GTG ACC GAC ATG GCT AAC TTC GCC CTC AGC GAC CCA AAC GCG CAT 2569 He Val Thr Asp Met Wing Asn Phe Wing Leu Ser Asp Pro Asn Wing His 945 950 955 AGG ATG AAA AAC TTC CTA GCA AAC GCA CCC CAG GCT GGA AGC AAG TCG 2617 Arg Met Lys Asn Phe Leu Wing Asn Wing Pro Gln Wing Gly Ser Lys Ser 960 965 970 CAG AGG GCC AAG TAT GGC ACG GCA GGC TAC GGA GTG GAG GCT CGA GGC 2665 Gln Arg Wing Lys Tyr Gly Thr Wing Gly Tyr Gly Val Glu Wing Arg Gly 975 980 985 990 CCC ACA CCA GAA GAG GCA CAG AGG GAA AAA GAC ACA CGG ATC TCC AAG 2713 Pro Thr Pro Glu Glu Wing Gln Arg Glu Lys Asp Thr Arg He Ser Lys 995 1000 1005 AAG ATG GAA ACA ATG GGC ATC TAC TTC GCG ACA CCG GAA TGG GTG GCT 2761 Lys Met Glu Thr Met Gly He Tyr Phe Wing Thr Pro Glu Trp Val Wing 1010 1015 1020 CTC AAC GGG CAC CGA GGC CCA AGC CCC GGC CAA CTC AAG TAC TGG CAA 2809 Leu Asn Gly His Arg Gly Pro Ser Gly Gln Leu Lys Tyr Trp Gln 1025 1030 1035 AAC ACA AGA GAA ATA CCA GAG CCC AAT GAG GAC TAC CAC GAC TAT GTG 2857 Asn Thr Arg Glu He Pro Glu Pro Asn Glu Asp Tyr Pro Asp Tyr Val 1040 1045 1050 CAC GCG GAG AAG AGC CGG TTG GCG TCA GAA GAA CAG ATC CTA CGG GCA 2905 His Wing Glu Lys Ser Arg Leu Wing Ser Glu Glu Gln He Leu Arg Wing 1055 1060 1065 1070 GCC ACG TCG ATC TAC GGG GCT CCA GGA CAG GCT GAA CCA CCC CAG GCC 2953 Wing Thr Ser He Tyr Gly Wing Pro Gly Gln Wing Glu Pro Pro Gln Wing 1075 1080 1085 TTC ATA GAC GAG GTC GCC AGG GTC TAT GAA ATC AAC CAT GGG CGT GGT 3001 Phe He Asp Glu Val Wing Arg Val Tyr Glu He Asn His Gly Arg Gly 1090 1095 1100 CCA AAC CAG GAG CAG ATG AAG GAC CTG CTC CTG ACT GCG ATG GAG ATG 3049 Pro Asn Gln Glu Gln Met Lys Asp Leu Leu Leu Thr Wing Met Glu Met 1105 1110 1115 AAG CAT CGC AAT CCC AGG CGG GCT CCA CCA AAG CCA AAG CCA AAA CCC 3097 Lys His Arg Asn Pro Arg Arg Wing Pro Pro Lys Pro Lys Pro Lys Pro 1120 1125 1130 AAT GCT CCA TCA CAG AGA CCC CCT GGA CGG CTG GGC CGC TGG ATC AGG 3145 Asn Ala Pro Ser Gln Arg Pro Pro Gly Arg Leu Gly Arg Trp lie Arg 1135 1140 1145 1150 ACG GTC TCC GAC GAG GAC TTG GAG TGAGGCTCCT GGGAGTCTCC CGACACTACC 3199 Thr Val Ser Asp Glu Asp Leu Glu 1155 CGCGCAGGTG TGGACACCAA TTCGGCCTTC TACCATCCCA AATTGGATCC GTTCGCGGGT 3259 CCCCT 3264 (2) INFORMATION FOR SEQ ID NO: 34 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1013 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear P774 (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34 Met Thr Asn Leu Met Asp His Thr Gln Gln He Val Pro Phe He Arg 1 5 10 15 Be Leu Leu Met Pro Thr Thr Gly Pro Wing Be Pro Asp Asp Thr 20 25 30 Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr 35 40 45 Val Gly Asp Thr Gly Ser Gly Leu He Val Phe Phe Pro Gly Phe Pro 50 55 60 Gly Ser Val Val Gly Ala His Tyr Thr Leu Gln Ser Ser Gly Asn Tyr 65 70 75 80 Gln Phe Asp Gln Met Leu Leu Thr Wing Gln Asn Leu Pro Wing Being Tyr 74 85 90 95 Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr 100 105 lio Leu Pro Gly Val Tyr Ala Leu Asn Gly Thr He Asn Wing Val Thr 115 120 125 Phe His Gly Ser Leu Ser Glu Leu Thr Asp Tyr Ser Tyr Asn Gly Leu 130 135 140 Met Ser Ala Thr Ala Asn He Asn Asp Lys He Gly Asn Val Leu Val 145 150 155 160 Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Ser 165 170 175 Tyr Val Arg Leu Gly Asp Pro He Pro Wing Wing Gly Leu Asp Pro Lys 180 185 190 Leu Met Wing Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr He 195 200 205 Thr Wing Wing Asp Glu Tyr Gln Phe Ser Ser Gln Leu He Pro Ser Gly 210 215 220 Val Lys Thr Thr Leu Phe Ser Wing Asn lie Asp Ala Leu Thr Ser Phe 225 230 235 240 Ser Val Gly Gly Glu Leu Val Phe Ser Gln Val Thr He Gln Ser He 245 250 255 Glu Val Asp Val Thr He His Phe He Gly Phe Asp Gly Thr Asp Val 260 265 270 Wing Val Lys Wing Val Wing Thr Asp Phe Gly Leu Thr Thr Gly Thr Asn 275 280 285 Asn Leu Val Pro Phe Asn Leu Val Val Pro Thr Asn Glu He Thr Gln 290 295 300 Pro He Thr Ser Met Lys Leu Glu Val Val Thr Tyr Lys He Gly Gly 305 310 315 320 Thr Ala Gly Asp Pro Be Ser Trp Thr Val Ser Gly Thr Leu Ala Val 325 330 335 Thr Val His Gly Gly Asn Tyr Pro Gly Wing Leu Arg Pro Val Thr Leu 340 345 350 Val Wing Tyr Glu Arg Val Wing Wing Gly Ser Val Val Thr Val Wing Gly 355 360 365 74 Val Ser Asn Phe Glu Leu He Pro Asn Pro Glu Leu Ala Lys Asn Leu 370 375 380 Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Wing Met Asn Tyr Thr Lys 385 390 395 400 Leu He Leu Ser Glu Arg Asp Arg Leu Gly He Lys Thr Val Trp Pro 405 410 415 Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Wing Asp 420 425 430 Leu Asn Ser Pro Leu Lys He Wing Gly Wing Phe Gly Phe Lys Asp He 435 440 445 He Arg Wing He Arg Lys He Wing Val Pro Val Val Be Thr Leu Phe 450 455 460 Pro Pro Wing Pro Pro Leu Wing His Wing He Gly Glu Gly Val Asp Tyr 465 470 475 480 Leu Leu Gly Asp Glu Wing Gln Wing Wing Ser Gly Thr Wing Arg Wing Wing 485 490 495 Be Gly Lys Wing Arg Wing Wing Be Gly Arg He Arg Gln Leu Thr Leu 500 505 510 Wing Wing Asp Lys Gly Cys Glu Val Val Wing Asn Met Phe Gln Val Pro 515 520 525 Gln Asn Pro He Val Asp Gly He Leu Wing Ser Pro Gly He Leu Arg 530 535 540 Gly Ala His Asn Leu Asp Cys Val Leu Trp Glu Gly Ala Thr Leu Phe 545 550 555 560 Pro Val Val He Thr Thr Leu Glu Asp Glu Leu Thr Pro Lys Ala Leu 565 570 575 Asn Ser Lys Met Phe Wing Val He Glu Gly Val Arg Glu Asp Leu Gln 580 585 590 Pro Pro Ser Gln Arg Gly Ser Phe He Arg Thr Leu Ser Gly His Arg 595 600 605 Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg 610 615 620 Asp Tyr Thr Val Val Pro He Asp Asp Val Trp Asp Asp Ser lie Met 625 630 635 640 74 Leu Ser Gln Asp Pro He Pro Pro He He Gly Asn Ser Gly Asn Leu 645 650 655 Wing Wing Wing Tyr Met Asp Val Phe Arg Pro Lys Val Pro He His Val 660 665 670 Wing Met Thr Gly Wing Leu Asn Wing Arg Gly Glu He Glu Ser Val Thr 675 680 6B5 Phe Arg Ser Thr Lys Leu Wing Thr Wing His Arg Leu Gly Met Lys Leu 690 695 700 Wing Gly Pro Gly Wing Tyr Asp He Asn Thr Gly Pro Asn Trp Wing Thr 705 710 715 720 Phe Val Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro 725 730 735 Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Thr Wing Gly Arg Gln Phe His 740 745 750 Leu Wing Leu Wing Wing Ser Glu Phe Lys Glu Thr Pro Glu Leu Glu Asp 755 760 765 Wing Val Arg Wing Ala Asp Wing Wing Wing Asn Wing Asp Pro Leu Phe Arg 770 775 780 Be Ala Leu Gln Val Phe Met Trp Leu Glu Glu Asn Gly He Val Thr 785 790 795 800 Asp Met Wing Asn Phe A? A Leu Ser Asp Pro Asn Wing His Arg Met Lys 805 '810 815 Asn Phe Leu Ala Asn Ala Pro Gln Ala Gly Ser Lys Ser Gln Arg Wing 820 825 830 Lys Tyr Gly Thr Wing Gly Tyr Gly Val Glu Wing Arg Gly Pro Thr Pro 835 840 845 Glu Glu Wing Gln Arg Glu Lys Asp Thr Arg He Ser Lys Met Glu 850 855 860 Thr Met Gly He Tyr Phe Wing Thr Pro Glu Trp Val Wing Leu Asn Gly 865 870 875 880 His Arg Gly Pro Ser Pro Gly Gln Leu Lys Tyr Trp Gln Asn Thr Arg 885 890 895 Glu He Pro Glu Pro Asn Glu Asp Tyr Pro Asp Tyr Val His Wing Glu 900 905 910 74 l-ys Ser Arg l-aa Ala Se *. Gl-u Glu (Sis He Leu Apy Ala Wing Thr Ber 915 920 925 river and x: ßly Wing P3"or Dl-y Without Wing GITA Frß Pro Glt? 3-the Phe lie Asp <3? 1? 935 940 Glia Gl - i Mi-t I-ys Aep Le l-eu Leu Thr S-ln &t Glii "Wctt? Ya Hia Js-egr 965 S7Q 975 Aen Pro Arg A g Ale. Pro pro Lye Pro Lys Pro i-y-í pra A-sn Aia sra ffSQ 9T-5 S90 Sr-tr Glu A ^ - > ra -Pro Gly Arg L & Gly Arg Trp lie Arg Thr Val be BSS 1000 10 OS A = p Glu Aap your Glu 1010 4

Claims

CLAIMS - 1. A method for preparing live Birnaviruses, comprising the following steps: preparing one or more Birnavirus genome cDNA segments A and B transcribing one or more cDNAs to produce synthetic RNA transcripts, wherein the RNA transcripts are transcribed from Positive sense RNA of segments A and B, transfect host cells with the synthetic RNA transcripts, incubate those host cells in a culture medium, and isolate live infectious Birnavirus from the culture medium.
2. The method according to claim 1, wherein the Birnavirus is the Infectious Bursal Disease Virus (IBDV).
3. The method according to claim 1, wherein the host cells are green monkey Vero cells African The method according to claim 1, wherein the Birnavirus genome segments A and B are prepared independently from different strains of Birnavirus. P774 5. The method according to claim 4, wherein segment A is present in the plasmid PUC19FLAD78 or pUC18F A23. The method according to claim 4, wherein segment B is present in the plasmid PUC18F BP2. 7. A live chimeric Birnavirus, wherein said Birnavirus is made by a process comprising the steps of preparing one or more bDNA genomes of Birnavirus segments A and B, wherein those cDNAs are derived from more than one strain of Birnavirus, transcribed one or more cDNAs to produce synthetic RNA transcripts, wherein those RNA transcripts are transcribed from positive sense RNA of segments A and B, transfect a host cell with those synthetic RNA transcripts, incubate the host cell in a medium of culture, and isolate live, chimeric Birnavirus from the culture medium. The method according to claim 7, wherein the Birnavirus is the Infectious Bursal Disease Virus (IBDV). P774 9. A plasmid selected from the group consisting of pUC19FLADD78, pUC18FLA23 and pUC19FLBP2. 10. A vaccine comprising a live chimeric Birnavirus according to claim 7, wherein said live chimeric Birnavirus is inactivated prior to administration. 11. The vaccine according to claim 10, wherein the Birnavirus is the Infectious Bursal Disease Virus (IBDV). The method according to claim 1, wherein the host cells are poultry cells. The method according to claim 12, wherein the poultry cells are chicken or turkey cells. The method according to claim 13, wherein the poultry cells are chicken embryo fibroblast cells or chicken embryo kidney cells. P774