INFECTIOUS DENGUE 2 VIRUS PDK-53 AS QUADRA VALENT VACCINE

Field of the Invention

The invention relates to infectious cDNA clones for Dengue 2 virus, strain 16681, and its live, attenuated vaccine derivative, PDK-53 (DEN-2 PDK-53) . The invention also relates to infectious cDNA clones for chimeric viruses characterized as expressing structural genes of a Dengue 1, Dengue 3, or Dengue 4 attenuated virus in the context of the nonstructural genes of the Dengue 2 PDK-53 virus (DEN-2/1, DEN-2/3, DEN-2/4) . The invention further relates to genetic constructs encoding these cDNAs, and host cells containing these constructs. The invention moreover relates to quadravalent vaccines providing immunity against all four serotypes of dengue virus comprising DEN-2 PDK-53 infectious clone derivative, DEN- 2/1, DEN-2/3, or DEN-2/4 viruses, and related methods of immunization.

Background of the Invention

Arthropod-borne viruses (arboviruses) are a diverse group of viruses that have been lumped together on the basis of their ecological niche, which involves cycles of transmission between vertebrate hosts and arthropod vectors such as mosquitos and ticks. The prototype arbovirus is yellow fever virus, a flavivirus, which was isolated in 1927. In the 1950s, the Rockefeller

Foundation established a number of field stations in

various tropical countries for the purpose of isolating new viruses. The 1985 International Catalogue of Arboviruses Including Certain Other Viruses of Vertebrates contains registrations for 504 discrete arboviruses, 124 of which have caused disease in humans. Thirty-four viruses of the Flavivirus genus (family Flaviviridae) of arboviruses are human pathogens (Karabatsos, 1985) . (All publications cited hereunder are incorporated herein by reference. ) According to a 1992 World Health Organization (WHO) press release (Press Release WHO/74, November 24, 1992) , dengue hemorrhagic fever is one of the most important and increasing mosquito-transmitted infections in the world, with more than 85 countries in Asia, the Pacific Islands, Africa, Central America, and South America being threatened with dengue outbreaks. Dengue fever was known in the past as "breakbone fever" due to the severe muscular and joint pain that accompanied the high fever during this infection. Dengue is an under-reported disease: it is thought that millions of cases occur each year.

Dengue (DEN) viruses, which are flaviviruses, are classified antigenically into 4 serotypes (DEN-1, DEN-2, DEN-3, and DEN-4) . Multiple serotypes are now endemic in most countries in the tropics. DEN viruses are transmitted to humans principally by Aedes aegypti mosquitos throughout much of the tropical and subtropical region of the world. Viruses of all four serotypes infect humans and cause clinically inapparent infection or illness ranging from dengue fever to severe and often

fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) . DHF/DSS has been associated epidemiologically and experimentally with immune enhancement of virus replication by preexisting, subneutralizing levels of heterotypic antibody. About 90% or more of patients with DHF/DSS are children who are 14 years old or younger (Halstead, 1970; Halstead, 1988) . Case fatality rates in untreated individuals can be as high as 15-20%. Between 1956 and 1978, hospitalization of more than 350,000 dengue patients and about 12,000 deaths in Southeast Asia were reported to the WHO (Halstead, 1980) . More recent dengue epidemics in Asia, the Pacific islands, the Americas, and Africa indicate that the incidence, with up to 40 million cases annually, and geographic distribution of the disease is increasing in Aedes aegypti-infested areas of the world (Halstead, 1984; Gubler, 1988; Brandt, 1990) .

Since eradication of Aedes aegypti mosquitos appears to be practically infeasible, development of safe, effective vaccines against all four serotypes of DEN virus is a WHO priority (Gubler, 1988; Brandt, 1988; Brandt, 1990) . Since the level of DEN virus replication in certified cell cultures yields insufficient antigenic mass to produce effective inactivated vaccines, priorities are given to developing effective live, attenuated vaccine viruses and using a variety of expression systems such as recombinant vaccinia or avipox virus (live vaccine) , recombinant baculovirus (subunit vaccine) , and recombinant E. coli (subunit vaccine) to express certain genes of the DEN viral genome (Brandt, 1988; Brandt, 1990) .

Flaviviruses are enveloped RNA viruses 45 to 50 nm in diameter that contain a single-stranded, positive-sense capped RNA genome of approximately 11 kb. The RNA genome does not have a 3'-terminal poly(A) tail. Because the genetic molecule of flaviviruses is positive or messenger RNA (mRNA) -sense, naked genomic RNA injected, transfected, or electroporated into mammalian or invertebrate cells is capable of associating directly with the ribosomal protein synthetic machinery of the cell. All of the viral proteins are translated from the inserted viral genomic mRNA. These virus-specified proteins then replicate the viral genome, resulting in intracellular virus maturation and release of infectious virus from the transfected cell. The gene organization of the flavivirus mRNA genome, illustrated below, is 5' -noncoding region (5' -NC) -capsid- premembrane/membrane (prM/M) -envelope (E) -nonstructural protein 1 (NS1) -NS2A-NS2B-NS3-NS4A-NS4B-NS5-3 ' -noncoding region (3'-NC). The structural proteins capsid, prM/M, and E and nonstructural proteins are translated as a large precursor polyprotein molecule from a single long open reading frame in the mRNA genome. The individual mature viral proteins are processed from the polyprotein by both cell and virus specified proteases (Westaway et al . , 1985; Coia et al . , 1988; Speight and Westaway, 1989; Rice et al . , 1985 ) .

Genome Organization of Dengue Virus and Other Flaviviruses

C M E NS1 2A 2B NS3 4A 4B NS5 3'-NC

The structural proteins are those viral proteins that are incorporated into the mature virion. The virion consists of an icosahedral capsid (C) that packages the viral genomic mRNA (nucleocapsid) . The nucleocapsid is surrounded by a cell-derived lipid membrane into which the envelope (E) and mature membrane (M) proteins are imbedded. The virus-specific nonstructural genes, NS1- NS5, are expressed in the cytoplasm of the infected cell and are involved in the replication and maturation of the viral RNA genome and viral proteins.

The E glycoprotein of the virus is exposed to the environment and is involved in attachment and entry of the virus into the cell. The E protein is the primary viral immunogen against which the infected vertebrate host develops virus-specific neutralizing antibody. The E gene is the most common target for development of molecular systems to express the encoded E glycoprotein. However, immunization with various purified nonstructural genes of the virus have been shown to elicit protective immunity against challenge with wild-type virus, probably via cytotoxic T-cell mediated lysis of infected cells which express viral nonstructural proteins on the cell surface.

Vaccination can be one of the most cost effective ways to prevent dengue fever and DHF/DSS. Since 1979 the WHO has supported research on dengue vaccine development at the Mahidol University in Bangkok, Thailand (Press Release WHO/74, November 24, 1992) . Investigators at Mahidol University have developed four live, attenuated candidate vaccine viruses, one for each of the four serotypes, by serial passage of the virulent parent

viruses in primary dog kidney (PDK) or fetal rhesus lung (FRhL) cell culture (Yoksan et al . , 1986; Bhamarapravati et al . , 1987). Phase 1 and Phase 2 clinical trials in Thailand have demonstrated that the vaccine is both safe and immunogenic in humans. The vaccines now need to be tested for efficacy in large numbers of children (Press Release WHO/74, November 24, 1992) . To preclude the possible severe DHF/DSS immune enhancement phenomenon in vaccinees who might be infected naturally with a heterologous serotype of wild-type DEN virus following immunization with a single serotype of vaccine virus, it is essential that humans be vaccinated with a quadravalent vaccine to provide immunity against all four serotypes of the virus.

Summary of the Invention The invention provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a DEN-2 PDK-53 infectious clone-derived virus.

The invention also provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a chimeric DEN-2/1 virus.

The invention further provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a chimeric DEN-2/3 virus.

The invention moreover provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a chimeric DEN-2/4 virus.

The invention additionally provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising DEN-2 PDK-53 infectious clone-derived and chimeric DEN-2/1, DEN-2/3, and DEN-2/4 viruses.

In another aspect, the invention provides a method of immunization in which a desired immune response is produced against all four serotypes of dengue virus comprising the step of administering to a subject a quadravalent vaccine comprising DEN-2 PDK-53 infectious clone-derived and chimeric DEN-2/1, DEN-2/3, and DEN-2/4 viruses.

In yet another aspect, the invention provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681. The invention also provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus of a strain characterized as replicating to high titer in cell culture. The invention further provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, having the identifying characteristics of ATCC 69826.

In still another aspect, the invention provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, attenuated derivative, PDK-53.

The invention also provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus attenuated derivative,

characterized as replicating to high titer in cell culture.

The invention further provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, attenuated derivative, PDK-53, having the identifying characteristics Of ATCC 69825.

In another aspect, the invention provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2/1 virus, wherein the virus is characterized as expressing the prM and E genes of a DEN-1 attenuated virus in the context of the nonstructural genes of the DEN-2 PDK-53 virus. The DEN-1 attenuated virus may be DEN-1 PDK-13. The invention also provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2 virus, wherein the virus is characterized as expressing the antigenicity of a DEN-1 attenuated virus. In yet another aspect, the invention provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2/3 virus, wherein the virus is characterized as expressing the prM and E genes of a DEN-3 attenuated virus in the context of the nonstructural genes of the DEN-2 PDK-53 virus. The DEN-3 attenuated virus may be DEN-3 PGMK30/FRhL-3.

The invention also provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2 virus, wherein the virus is

characterized as expressing the antigenicity of a DEN-3 attenuated virus.

In still another aspect, the invention provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2/4 virus, wherein the virus is characterized as expressing the prM and E genes of a DEN-4 attenuated virus in the context of the nonstructural genes of the DEN-2 PDK-53 virus. The DEN-4 attenuated virus may be DEN-4 PDK-48. The invention also provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2 virus, wherein the virus is characterized as expressing the antigenicity of a DEN-4 attenuated virus. Additionally, the invention provides a genetic construct comprising a DNA sequence operably encoding the polyprotein of DEN-2 virus, strain 16681. The polyprotein may be the polyprotein encoded by the nucleotide sequence of SEQ ID NO:l. The invention also provides a genetic construct comprising a DNA sequence operably encoding at least one protein of DEN-2 virus, strain 16681. The protein may be a protein encoded by the nucleotide sequence of SEQ ID NO: 1. Further, the invention provides a genetic construct comprising a DNA sequence operably encoding the polyprotein of DEN-2 virus, strain 16681, attenuated derivative, PDK-53. The polyprotein may be the polyprotein encoded by the nucleotide sequence of SEQ ID NO:2.

The invention also provides a genetic construct comprising a DNA sequence operably encoding at least one protein of DEN-2 virus, strain 16681, attenuated derivative, PDK-53. The protein may be a protein encoded by the nucleotide sequence of SEQ ID NO: 2.

Moreover, the invention provides a genetic construct comprising a DNA sequence operably encoding at least one structural protein of DEN-1 PDK-13. The structural protein may be a structural protein encoded by the nucleotide sequence of SEQ ID NO: 124.

In another aspect, the invention provides a genetic construct comprising a DNA sequence operably encoding at least one structural protein of DEN-3 PGMK30/FRhL-3. The structural protein may be a structural protein encoded by the nucleotide sequence of SEQ ID NO: 125.

In still another aspect, the invention provides a genetic construct comprising a DNA sequence operably encoding at least one structural protein of DEN-4 PDK-48. The structural protein may be a structural protein encoded by the nucleotide sequence of SEQ ID NO: 126.

In yet another aspect, the invention includes a host cell comprising any of the above genetic constructs.

Brief Description of the Drawings

Figure 1: Strategy for construction of the full genome-length cDNA clone of DEN-2 virus. Using PCR technology, cDNA is amplified from the genomic RNA of the virus and cloned. Subclones are spliced together at unique, overlapping restriction enzyme sites to construct

the full genome-length clone. Numbered arrows upstream (right arrows) and downstream (left primers used to amplify the cDNA in PCR reactions.

Figure 2: Transcription of genomic mRNA from the full-length infectious cDNA clone of DEN-2 virus. The recombinant plasmid is linearized at the unique Xbal site at the 3 ' -end of the genomic cDNA. Bacteriophage T7 RNA polymerase recognizes the T7 promoter engineered at the 5 '-end of the cDNA and transcribes full-length viral mRNA from the cDNA template.

Figure 3 : Restriction enzyme sites identified in the nucleotide sequence of the RNA genome of DEN-2 16681 virus. Locations for the sites are indicated by the genome nucleotide numbers. Restriction enzymes that cleave the DEN-2 genomic cDNA at only a single location are listed vertically at the top of the figure. The resolution of the RENZ graph is 97.5 nucleotides per dot.

Figure 4: Growth curve of DEN-2 16681 virus in C6/36 mosquito cells. Figure 5: (A) Polaroid prints showing RT/PCR amplification of the entire mRNA genome of DEN-2 virus, strain 16681, in the form of 5 cDNA amplicons. The molecular weight marker (MW) consists of linear, double- stranded DNA markers of various base pair (bp) lengths. The top 2 gels show 5-μl aliquots of the original RT/PCR reactions. The bottom two gels show 10% of the yield following HMC agarose gel purification of the remaining 95-μl reaction aliquots. (B) Primers (amplimers) used in the RT/PCR reactions and the expected sizes of the resulting cDNA amplicons.

Figure 6: EcoRI restriction enzyme digests of F2, F2-Sal, Sal-F2, and F3 miniprep recombinant plasmid DNA. Plasmids from individual colonies resulting from transformation with independent ligated, recombinant plasmid molecules are numbered. The insert in the single F2-8 plasmid was too small and was discarded. The remaining recombinant plasmids contained cDNA inserts of expected size. As expected, F2-Sal cDNA contained two internal EcoRI sites; the Sal-F2 and F3 plasmids contained a single internal EcoRI site. EcoRI digestion of the recombinant plasmids regenerated linearized, wild-type 3.9-kb pCRII vector. For an undetermined reason, one of the EcoRI sites in plasmid F3-1 did not cut.

Figure 7: Schematic diagram showing the genomic locations of DEN-2 16681 virus-specific cDNA clones.

Clones indicated with asterisks were spliced together at the indicated restriction enzyme sites to construct the full genome-length cDNA clone. Black horizontal bars indicate clone regions that were sequenced. Light gray regions of horizontal bars indicate clone regions that were not sequenced.

Figure 8: (A) Effect of adding Taq extender reagent to PCR reactions. The 5.2-kbp amplicon of St. Louis encephalitis virus was readily obtained by extended PCR (+) but not by standard PCR (-) . (B) Agarose gel electropherogram showing DEN-2 PDK-53 Fl, F2, and F3 amplicons derived by extended PCR.

Figure 9: Schematic diagram showing the genome locations of errors identified in the cDNA clones of DEN-2 16681. Errors are indicated by short vertical tick marks.

Figure 10: Schematic diagram illustrating the approximate genome locations of the nucleotide discrepancies between the data of Applicants and those of Blok et al. (1992) for the sequence of the genome of DEN-2 virus, strain 16681.

Figure 11: Nucleotide sequence of the genome of DEN- 2 strain 16681 virus. Differences between the data determined by Blok eϋ al . (1992) (DEN-2-16681.BLOK) and those obtained by Applicants (DEN-2-16681.RK) . The genome nucleotide positions of the sequence differences are listed vertically. The solid squares indicate those nucleotide differences that also encode amino acid substitutions. The remaining nucleotide differences are either silent, encoding the same amino acid, or lie within the 5 ' -noncoding (5'-NC) or 3 ' -noncoding region (3 '-NO . Figure 12: Schematic diagram showing the DEN-2 PDK- 53 virus-specific cDNA clones and the approximate locations of cDNA errors (vertical tick marks) identified by nucleotide sequence analyses. Clones marked with an asterisk were used in the construction of the DEN-2 PDK-53 virus-specific full-length cDNA clone. Clone #19 had a 203-bp deletion (horizontal line) .

Figure 13: Schematic summary of the DEN-2 16681 vs. PDK-53 virus sequencing projects. Arrows indicate the nucleotide differences detected between the two genomes. Triangles indicate those nucleotide changes that resulted in amino acid substitutions.

Figure 14: Finalized nucleotide and amino acid sequence of the RNA genome of DEN-2 virus, strain 16681 (SEQ ID N0:1) . The nucleotide and amino acid mutations

that were determined to have occurred in DEN-2 virus, strain PDK-53, are indicated at the appropriate positions (SEQ ID NO:2) . The EcoRI, SstI, Mull,and T7 promoter sites that were engineered immediately preceding the 5 ' - terminal nucleotide of the virus-specific genomic cDNA are shown. The start positions of the viral genes and noncoding regions (5'-NC and 3 ' -NC) are shown. Potential sites of Asn-linked glycosylation (Asn-X-Ser or Thr, where X = any amino acid) in prM, E, and NS1 are indicated by asterisks. The deduced amino acid sequence is indicated in standard single-letter abbreviation: A = Ala, C = Cys, D = Asp, E = Glu, F = Phe, G = Gly, H = His, I = He, K = Lys, L = Leu, M = Met, N = Asn, P = Pro, Q = Gin, R = Arg, S = Ser, T = Thr, V = Val, W = Trp, Y = Tyr. Figure 15: Construction of intermediate clone F2 by ligating the F2-Sal Sphl/Hpal fragment and Sal-F2 Hpal/Kpnl fragment into pUClδ. The resulting F2 clone contained a nonsilent cDNA error at genome nucleotide position 1730. Figure 16: Correction of the intermediate F2 clone. A new PCR amplicon was cloned and sequenced. The Sphl/Hpal fragment of this clone was spliced into F2 to construct F2-C having the correct nucleotide at genome position 1730. Figure 17: Construction of the intermediate Fl/3/4/5 cDNA clone for DEN-2 16681 virus. The thick solid black bars indicate DEN-2 virus-specific cDNA, illustrated with the RENZ sites of the MCS of the plasmid. The RENZ sites used in each step of the splicing strategy are indicated in underlined, bold characters. The top half of the

figure shows construction of Fl/3/4/5-pUC18. The bottom portion of the figure illustrates the making of Fl/3/4/5- pUC19. The final step in the construction of the full genome-length cDNA clone involved the ligation of the F2-C Sphl/KpnI cDNA fragment into plasmid containing cDNA

Fl/3/4/5 and cut with RENZs Sphl/KpnI. Although F2-C cDNA could not be cloned into Fl/3/4/5-pUC18, it was readily cloned into Fl/3/4/5-pUC19. The pUC18 plasmid containing a small insert of cDNA made for Venezuelan equine encephalitis (VEE) virus was used simply to move Fl and F4/5 into pUC18 in a 3-molecule ligation reaction. The VEE virus-specific cDNA was spliced out during this process. Arrowheads under cDNA bars indicate orientation of mRNA-sense cDNA strand. Figure 18: Orientation specific cloning of full genome-length cDNA of DEN-2 16681 virus into the multiple cloning site of pUC19. Although the full-length cDNA was readily cloned in pUC19, multiple attempts to insert the cDNA into pUC18 failed. Presumably, interaction of the cDNA with pUC18-specific gene transcripts, translation of a toxic DEN-2 polypeptide, or translation of a toxic pUC18/DEN-2 fusion polypeptide produced deleterious effects in E. coli . Large arrows indicate orientation of mRNA-sense cDNA strands in the pUC plasmid backbone. Smaller arrows indicate orientations of the lac Z and ampicillin genes as well as the origin of replication. DEN-2 insert is indicated by a thick solid black line. Figure 19: Insertion of the MCS of plasmid pUC19 into pBR322 in both orientations to construct pBRUC-138 and pBRUC-139. The pUC18 Hindlll (blunt-ended = BL) /EcoRI

MCS fragment was ligated into pBR322 cut with Aval (BL) /EcoRI to construct pBRUC-138. The pUClδ EcoRI (BL) /Hindlll MCS fragment was ligated into pBR322 cut with Aval (BL) /Hindlll to make pBRUC-139. In both cases, the tetracycline gene of pBR322 was removed. pBRUC-138 = 2992-bp (61-bp MCS + 2931-bp pBR322 deletion vector) . pBRUC-139 = 3022-bp (61-bp MCS + 2961-bp pBR322 deletion vector) . Orientations of ORI, ROP, and the Amp gene are indicated. Figure 20: Construction of pD2/IC-30P, the full genome-length cDNA clone of DEN-2 16681 virus, in plasmid pBR322 (pBRUC-139 (SphI-) derivative) . The F3/4/5 clone cDNA was ligated into pBRUC-139 first (Top of Figure) , followed by Fl-E and F2-C. Viable, infectious DEN-2 virus was successfully obtained from viral mRNA transcribed from this clone.

Figure 21: Construction of pD2/IC-130V, the full genome-length cDNA clone of DEN-2 PDK-53 virus. A nonsilent error in cDNA clone F3-3C was corrected by splicing in a correct BstBI/Nhel fragment from clone F3.5- 6 (Top) . The resulting corrected clone F3-3CC was spliced into the 16681 F345-F clone in pBRUC-139. cDNA fragments F1-79B, F2-16B, and the recombinant F3/4/5 vector DNA were spliced together in a single ligation reaction to produce pD2/IC-130V. The Nhel site occurs at genome nucleotide position 6646. Therefore, the PDK-53 virus-specific full- length cDNA clone contains the parental 16681 virus- specific nucleotide at position 8571. This nucleotide difference is silent; it does not encode an amino acid change. Other than the 8571 position, DEN-2 16681 and

PDK-53 viruses are identical in nucleotide sequence from nucleotide position 6646 to the 3' terminus of the genome.

Figure 22: Agarose gel electropherogram of viral genomic mRNA extracted from gradient-purified, wild-type DEN-2 16681 virus and Venezuelan equine encephalitis (VEE) virus. The quantity of RNA loaded onto the gel ranged from 22 ng to 383 ng. The stock RNA was quantitated spectrophotometrically at 260 nm. The genome-length RNA band is clearly visible between the 4153-bp and 6788-bp MW marker bands. Bands were visualized by incorporating 200 ng/ml of ethidium bromide stain in the gel and electrophoresis buffer.

Figure 23: Transcription of RNA from pVE/IC-92 (VEE virus clone) and pD2/IC-20 (DEN-2 16681 virus clone) . Transcription reaction conditions (100 ng linearized DNA template, 12.5 mM DTT, 2.7 u/μl RNasin, 0.15 mM NTPs, 3.3 U/μl T7 RNA polymerase (Stratagene) in commercial buffer (Stratagene) ) yielded high quantity and quality of infectious mRNA transcripts from the pVE/IC-92 clone and 3 ' -end truncation products of that clone. However, these reaction conditions failed to permit transcription of RNA from the pD2/IC-20 clone or two of its 3 ' -end transcription products (clone linearized at the Nsil or Mrol site instead of at the 3' -terminal Xbal site). pVE/lC-92 plasmid linearized at the Mlul (3 ' -terminal) , SphI, Tthllll, Hindlll, Sail, and StuI sites in the cDNA clone yielded RNA transcripts of 11447, 11377, 7541, 2407, 1620, and 674 base length, respectively (the more intense, prominent bands in these gel lanes) .

Figure 24: Transcription of RNA from the DEN-2 16681 cDNA clone pD2/IC-20. (A) Transcription of RNA using different quantities of linearized plasmid template (a,b) . The cap analog m7G(5 ' )ppp(5 ' )A was not included in the reaction. (B) Transcription of 5 '-capped RNA with inclusion of cap analog in the reaction. Transcription was accomplished with the Ampliscribe transcription kit from Epicentre Technologies. T7 pol = bacteriophage T7 RNA polymerase. Figure 25: Transcription of full genome-length, infectious viral mRNA from Xbal-linearized DEN-2 16681 plasmid pD2/IC-30P (A and D replicate clones resulting from independent bacterial colonies transformed with the recombinant pBRUC/DEN-2 plasmid) and PDK-53 plasmid pD2/IC-130V (F and J replicates) . Genomic "viral RNA" extracted from gradient-purified wild-type DEN-2 16681 virus was electrophoresed in lanes 2 and 10. Aliquots of transcription reactions sampled before (T7 RNA polymerase "-") and after (T7 Pol "+") addition of T7 RNA polymerase are shown. Only the linearized plasmid DNA template is observed in the absence of the polymerase.

Figure 26: Transcription of RNA from pD2/IC-20, pD2/IC-30P, and pD2/IC-130V in the presence or absence of T7 RNA polymerase or cap analog in the transcription reaction. All lanes shown are on a single gel. Transcription was performed with the Ampliscribe transcription kit.

Figure 27: Derivation tree for the construction of the DEN-2 16681 and PDK-53 virus-specific full genome- length cDNA clones pD2/IC-30P and pD2/IC-130V,

respectively, and chimeric 16681/PDK-53 clones derived from the two prototype clones.

Figure 28: Genotype maps of DEN-2 16681 and PDK-53 virus-specific full genome-length cDNAs and their chimeric derivatives. The scale at the top indicates relative genome nucleotide position in thousands. The graph resolution is 119.1444 bp/dot. cDNA regions contributed by the parental DEN-2 16681 virus are indicated by solid black bars. Regions derived from the DEN-2 PDK-53 vaccine virus are indicated by stippled bars. The 8 mutations identified by sequence analyses of the genomes of the 16681 and PDK-53 viruses are indicated. The virus- specific 5-noncoding nucleotides are indicated in lower case characters. The amino acids encoded by the virus- specific nucleotide mutations in the protein coding region of the genome are indicated in upper case, single-letter amino acid abbreviation.

Figure 29: Results of spot-sequencing PCR amplicons amplified from seed stocks of viruses derived from full genome-length cDNA clones. Dots indicate nucleotide sequence identity to the DEN-2 16681 virus. The expected virus-specific nucleotides for the genotype of each virus are shown. Those nucleotide positions that have actually been confirmed by sequence analysis are indicated by underlined nucleotide base characters. The actual genome nucleotide positions are indicated at the bottom of the Figure.

Figure 30: Recombinant full-length pD2/IC-30P-A and pD2/IC-130V-F plasmids extracted from 1-ml aliquots of E. coli TB-1 cultures submitted to ATCC.

Figure 31: Partial nucleotide sequences of candidate vaccine viruses:

DEN-1 16007 PDK-13 (Dl.VAC) (SEQ ID NO: 124)

DEN-2 16681 PDK-53 (D2.VAC) (≤e_e_ SEQ ID NO: 2) DEN-3 16562 PGMK-30/FRhL-3 (D3.VAC) (SEQ ID NO: 125) DEN-4 1036 PDK-48 (D4.VAC) (SEQ ID NO: 126) aligned with the nucleotide and deduced amino acid sequences of DEN-2 16681 virus (see SEQ ID NO:l) . Dots in the DEN-1, DEN-3, and DEN-4 sequences signify identity with the DEN-2 sequence.

Figure 32: Partial amino acid sequences of candidate vaccine viruses:

DEN-1 16007 PDK-13 (Dl.VAC) (SEQ ID NO: 124)

DEN-2 16681 PDK-53 (D2.VAC) (≤£e_ SEQ ID NO: 2) DEN-3 16562 PGMK-30/FRhL-3 (D3.VAC) (SEQ ID NO: 125) DEN-4 1036 PDK-48 (D4.VAC) (SEQ ID NO: 126) aligned with the deduced amino acid sequence of DEN-2 16681 virus (£££ SEQ ID NO:l) . Dots in the DEN-1, DEN-3, and DEN-4 sequences signify identity with the DEN-2 sequence.

Figure 33: Mutagenesis analysis of the 5' end of the prM gene. The 447-452 sequence ("AACCAC" in DEN-2) can be mutated to "CTCGAG" in all four DEN viruses to create a Xhol site for cassette splicing. This modification results in conservative Thr-Thr to Ser-Ser substitutions at amino acid positions prM 4-5 in DEN-2 virus. By creating this Xhol site, all four viruses will contain the sequence FHLSSR at amino acid positions prM 1-6 (see Figure 32) . Nucleotide mutations that are necessary to create the Xhol site are indicated by bold, underlined

characters in the nucleotide sequences of D2.VAC, Dl.VAC, D3.VAC, and D4.VAC and their respective primers designed for amplification in PCR.

Figure 34: Mutagenesis analysis of the 3' end of the E gene. The 2344-2349 sequence ("TCACGC" in DEN-2) can be mutated to "TCTAGA" in all four DEN viruses to create a Xbal site for cassette splicing. This modification results in no amino acid change in DEN-2 at this site, but substitutions do occur in the other three viruses. By creating this Xhol site, all four viruses will contain the sequence SRS at amino acid positions E 470-472 (see Figure 32) . Nucleotide mutations that are necessary to create the Xbal site are indicated by bold, underlined characters in the nucleotide sequences of D2.VAC, Dl.VAC, D3.VAC, and D4.VAC and their respective primers designed for amplification in PCR.

Figure 35: Construction of DEN-2 PDK-53 cassette plasmids pFl-Xho and pF2-Xba. (A) pFl-Xho: Clone PCR cDNA amplicons Fl-prM5' and Fl-prM3' into TA-vector. Sequence and splice correct clones together at the SphI site in the TA-vector to ^' construct pFl-prM53 (not shown) . Subclone the prM53 cDNA into Sstl/Sphl-cut pFl-E (see Figure 20) to construct pFl-Xho. (B) pF2-Xba: Clone PCR cDNA amplicons F2-E5' and F2-E3 ' into TA-vector. Splice correct clones together at the Xbal site in the TA-vector to construct pF2-E53 (not shown) . Subclone the Sphl/Hpal E53 cDNA fragment into pF2-16B (see Figure 21) , which itself is subcloned into pBRUC-139 between the Sphl/KpnI sites (not shown) , to construct pF2-Xho. PCR amplimer designations are underlined. Solid black bars indicate newly

synthesized and sequence-characterized cDNA. Stippled bar indicates previously synthesized cDNA. Graph resolution = 64.1857 nucleotides/dot.

Figure 36: Construction of chimeric plasmids containing the prM and E genes (Xhol-Xbal cDNA fragment) of DEN-1, DEN-3, or DEN-4 candidate vaccine virus within the genetic background of DEN-2 PDK-53 virus. pD2V-CAS12 was constructed by ligating the Sstl/SphI fragment of pFl- Xho and Sphl/KpnI fragment of pF2-Xba (see Figure 33) into a truncated form of pD2/IC-130V (see Figure 21) . pD2/IC- 130V was truncated by restricting the full-length clone at the NsiI-4696 and 3 ' -end Xbal sites, blunt-ending with T4 DNA polymerase, and religating. This procedure removed genome nucleotides 4696-10723, thereby removing the Xhol- 5426 and 3 ' -end Xbal sites, which would otherwise interfere with construction of chimeric plasmid cassettes using Xhol and Xbal sites. The cassette strategy employs PCR amplification of DEN-1, DEN-3, and DEN-4 cDNAs containing the prM and E genes; cutting the amplicons with Xhol/Xbal; cloning resulting fragments into pD2V-CAS12 to construct pDlV-CAS12, pD3V-CAS12, and pD4V-CAS12 chimeric cassettes; confirming the chimeric Xhol/Xbal insert by nucleotide sequence analysis; and then subcloning the Sstl/Kpnl fragment of the chimeric cassette into pD2/IC- 130V to construct the chimeric full genome-length cDNA clones from which chimeric DEN-2/1, -2/3, and -2/4 viruses are derived. The genetic background of DEN-2 PDK-53 virus is illustrated by the solid black bars. The heterologous DEN-1, DEN-3, and DEN-4 cDNA inserts are indicated by the stippled bars. The pBRUC-139 plasmid backbone is not

illustrated for pDlV-CAS12, pD3V-CAS12, or pD4V-CAS12 chimeric plasmid. Resolution = 110.5464 bp/dot.

Detailed Description of the Invention We developed a quadravalent vaccine by initially constructing a full genome-length infectious cDNA clone for DEN-2 virus. We chose serotype 2 of DEN virus because virus strains of this serotype generally replicate to high titer in cell culture. We chose to develop an infectious clone for the 16681 strain of DEN-2 virus because the candidate vaccine viruses developed by Mahidol University are currently the best live, attenuated vaccine virus candidates in terms of immunogenic efficacy and lack of reactogenicity in vaccinees. We developed an infectious cDNA clone of the 16681 strain, which is the parent to the DEN-2 PDK-53 candidate vaccine virus developed at Mahidol University, to permit engineering of second and later generation live, attenuated DEN vaccine viruses.

The infectious clone strategy was initiated with the virulent parental 16681 strain obtained from the Division of Vector-Borne Infectious Diseases (DVBID) of the Centers for Disease Control and Prevention (CDC) virus collection. We synthesized cDNA from the DEN-2 16681 viral RNA. The immediate objective was to obtain an accurate full genome- length infectious cDNA clone of the 16681 strain of DEN-2 virus, since it was essential to develop a reliable experimental system to permit routine genetic engineering of the cDNA and recovery of virus. Our approach involved using polymerase chain reaction (PCR) technology to create cDNA clones that could be spliced together to construct a

single full genome-length clone (Figure 1) from which full-length, infectious DEN-2 genomic mRNA could be transcribed (Figure 2) .

The first full-length sequence-characterized cDNA clone, designated pD2/IC-20, was constructed in the high copy number pUC19 plasmid vector. Successful transcription of genome-length DEN-2 16681 viral RNA from pD2/IC-20 was clearly demonstrated by agarose gel electrophoresis of the transcription reaction product. However, RNA transcribed from this particular clone failed to yield infectious virus. It was determined that cDNA errors had occurred during the clone manipulations. We then decided to reconstruct the full-length clone in the low copy number pBR322 plasmid. The full-length cDNA of DEN-2 16681 virus was successfully moved into pBR322 to construct pD2/lC-30P. Full-length, infectious DEN-2 16681 genomic RNA was subsequently transcribed from pD2/IC-30P.

The DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PGMK-30/FRhL-3, and DEN-4 PDK-48 vaccine viruses were obtained from Mahidol University. Our goal involved replacement of the entire genomic cDNA backbone of the DEN-2 16681 full- length clone with the cognate cDNA cloned from the genome of the DEN-2 PDK-53 candidate vaccine virus. The prM and E genes of the DEN-2 PDK-53 virus are then replaced with the prM and E genes of the DEN-1 PDK-13, DEN-3

PGMK30/FRhL-3, and DEN-4 PDK-48 candidate vaccine viruses to construct chimeric DEN-2/1, DEN-2/3, and DEN-2/4 viruses containing the nonstructural genes of the DEN-2 PDK-53 virus and the prM and E genes of the heterologous DEN viruses.

DEN-2 PDK-53 Infectious cDNA Clone Backbone c M E NS1 2A 2B NS3 4A 4B NS5 3'-NC

prM E DEN-1 PDK-13

prM E DEN-3 PGMK30/FRhL-3

prM E DEN-4 PDK-48

It is contemplated that chimeric, infectious clone- derived DEN-2/1, DEN-2/3, and DEN-2/4 viruses will result in immediate improvement in the efficacy of a quadravalent vaccine. Our preliminary data from Mahidol University indicate that very small amounts of the DEN-2 PDK-53 vaccine virus were required to infect and immunize humans. However, the DEN-1, DEN-3, and DEN-4 vaccine virus candidates had approximately 30-fold to 2000-fold lower infectivity for humans. The low infective efficacies of the DEN-1, DEN-3, and DEN-4 viruses create significant problems in terms of vaccine efficacy in eliciting seroconversion in vaccinees, as well as problems of vaccine production for mass vaccination programs, since a large volume, up to 1 ml, of undiluted cell culture- derived vaccine virus must be administered to achieve even minimal levels of infectivity for these viruses. Since the increased infectivity of the DEN-2 PDK-53 vaccine virus is likely due to more efficient virus replication, and since this replicative efficacy is controlled by the nonstructural proteins of the virus, then chimeric vaccine viruses that express the relevant immunogenic structural proteins of DEN-1, DEN-3, or DEN-4 virus in the context of replication control by the nonstructural gene products of

the DEN-2 PDK-53 virus should replicate better and be more infective and immunogenic in human vaccinees than the original DEN-1, DEN-3, and DEN-4 vaccine viruses containing nonchimeric genotypes.

A quadravalent vaccine is obtained upon completion of the following steps:

(1) A full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, is constructed.

(2) A full genome-length infectious cDNA clone for a DEN2-16681 attenuated derivative, PDK-53, is constructed, preferably by substituting the genomic cDNA backbone of the DEN2-16681 full length clone with the corresponding cDNA cloned from the genome of the DEN-2 PDK-53 candidate vaccine virus.

(3) The candidate DEN-1, DEN-3, and DEN-4 vaccine viruses are subjected to PCR amplification of cDNA from extracted genomic RNA, and chimeric infectious cDNA clones expressing the prM and E genes of DEN-1, DEN-3, and DEN-4 viruses, respectively, in the context of the nonstructural genes of the DEN-2 PDK-53 virus are constructed.

(4) The infectious clone-derived chimeric DEN-2/1, DEN-2/3, and DEN-2/4 vaccine viruses are tested to ensure that they:

(a) Are viable;

(b) Express appropriate virus-specific immunogens;

(c) Replicate to sufficient titer in cell culture;

(d) Are infectious and immunogenic for humans; and

(e) Retain phenotypic markers of attenuation.

There is no good animal model for investigating dengue pathogenesis. DEN viruses are naturally transmitted between mosquitos and humans. Although lower primates can be infected with these viruses, they do not develop the clinical profiles that occur in humans. Infectious clone-derived viruses can be compared to their more virulent parental strains using certain in vi tro and in vivo markers:

In Vi tro Markers:

Plaque size in cell culture;

Temperature sensitivity;

Cytopathic effects (CPE) in LLC-MK ₂ cells; and

Replication in macrophages.

In Vivo Markers:

Virulence by intracranial route in mice; Viremia in monkeys; Virulence by intracranial route in monkeys; and

Elicitation of neutralizing antibodies in animals.

Infectious cDNA clones are expressed, the resulting RNA transcripts are transfected into permissible cells, and the live, attenuated viruses are formulated into vaccines.

Additionally, the DEN-2 PDK-53 and chimeric DEN- 2/1, DEN-2/3, and DEN-2/4 infectious cDNA clones can by themselves confer immunity by DNA immunization, a form of gene therapy involving the direct inoculation of naked DNA into the host such that its expression produces an immune response (e.g., Ulmer et al . , 1993 (DNA immunization protected against influenza); Cox et al . , 1993 (DNA immunization protected against herpesvirus); Xiang et al . , 1994 (DNA immunization protected against rabies) ; Sedegah et al . , 1994 (DNA immunization protected against malaria) ) .

Moreover, infectious cDNA clones are exquisite tools for studying the molecular biology of virus structure, function, and replication. This has been amply demonstrated for many RNA viruses in the literature, including Venezuelan equine encephalitis virus as reported by Kinney et al . (1989). A successful infectious cDNA clone of DEN-2 virus permits important investigations of

dengue virus replication, pathogenesis, and antigenic structure. In ectious clone cDNA templates permit the directed engineering of virus vaccines. Directed site- specific, nonrandom mutations can readily be made in infectious cDNA clones, and therefore in clone-derived viruses, using a wide variety of DNA modification enzymes, restriction endonucleases, and in vi tro mutagenesis methods. DNA is easier to manipulate than RNA, and the 10" ⁹ error rate of DNA replication is much lower than the 10 ^~3 - 10' ⁴ error rate produced by RNA polymerases. Infectious cDNA clones permit direct analyses of the phenotypic effects of individual and cumulative mutations in the viral genome. An infectious cDNA clone provides a "gold standard" reference sequence for a vaccine.

Particular aspects of the invention may be more readily understood by reference to the following examples, which are intended to exemplify the invention, without limiting its scope to the particular exemplified embodiments.

EXAMPLES

Information:

Most of the background, protocols, and recipes used in recombinant DNA work can be found in Molecular Cloning: A Laboratory Manual (Sambrook et al . , 1989) , and Current Protocols in Molecular Biology (Ausubel et al . , 1989) .

Viruses:

The virulent parental DEN-2 16681 strain was immediately available in the DVBID collection of viruses. We received the DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PGMK- 30/FRhL-3, and DEN-4 PDK-48 vaccine viruses from Mahidol University. The DEN vaccine viruses were passaged in primary dog kidney (PDK) cells because this cell culture is included among those cell types that are certified for human use by the Bureau of Biologies, US Food and Drug Administration (Yoksan et al . , 1986) . The virus strain designations are shown below:

Vaccine

Parent Derivative

Virus Strain Strain

DEN-1 16007 PDK-13

DEN-2 16681 PDK-53

DEN-3 16562 PGMK-30/FRhL-3

DEN-4 1036 PDK-48

PDK = primary dog kidney cells

FRhL = fetal rhesus lung cells

PGMK = primary green monkey kidney cells

DEN-l 16007 Parent

► Recovered from serum of a patient with hemorrhagic fever and shock in Thailand in 1964 ► Passaged 3X in BS-C-1 cells, IX in LLC-MK ₂ cells

* ^■ Passaged 2X in Toxorhynchi tes amboinensis mosquitos

► PDK-1

1 PDK-43 Vaccine

DEN-2 16681 Parent

► Recovered from serum of a patient with hemorrhagic fever and shock in Thailand in 1964

► Passaged 3X in BS-C-1 cells, IX in LLC-MK ₂ cells >• Passaged 2X in Toxorhynchi tes amboinensis mosquitos

► PDK-1 i PDK-53 Vaccine

DEN-3 16562 Parent

► Recovered from serum of a patient with hemorrhagic fever and shock in the Philippines in 1964

► Passaged 3X in BS-C-1 cells, IX in LLC-MK ₂ cells

►• Passaged 2X in Toxorhynchi tes amboinensis mosquitos ► PGMK-1

J PGMK-30 DEN-3 virus grown in PGMK cells I replicated to very low titer in PDK FRhL-3 Vaccine cells (Yoksan et al . , 1986)

DEN-4 1036 Parent

*■ Recovered from serum of a patient with dengue fever in

Indonesia in 1976 ► Passed 4X in Aedes aegypti mosquitos ► PDK-1

1

PDK-48

The DEN-2 full-length cDNA clone was derived from the DVBID seed of DEN-2 16681 virus, which had the passage history:

Human

3X BS-C-1 cells

2X LLC-MK ₂ cells

2X T. amboinensis mosquitos

4X C6/36 cells (Aedes albopictus)

Complementary DNA (cDNA) was amplified by RT/PCR directly, without further cell culture passage, from virus present in vaccine vials of the DEN-1 PDK-43, DEN-2 PDK- 53, DEN-3 PGMK-30/FRhL-3, and DEN-4 PDK-48 viruses.

Stock virus seed was prepared from virus-infected cells grown in 75 or 150 cm ² plastic tissue culture flasks. The culture medium was clarified by centrifugation for 30

min at 10,000 rpm in a Sorvall GSA rotor, bringing the final concentration of fetal bovine serum (FBS) to 10% (v/v) , and then freezing the clarified virus suspension in aliquots of 0.5 - 1.0 ml at -70°C. Gradient purified DEN- 2 16681 virus was prepared according to the method of Obijeski et al . (1976) as reported by Kinney et al . (1983) .

Cell Lines:

Infectious virus was derived from the infectious cDNA clones by electroporation of BHK-21-15 (baby hamster kidney-21, clone 15) cells with transcribed viral RNA. Viruses were also grown in LLC-MK ₂ monkey kidney cells, Vero African green monkey kidney cells, and C6/36 mosquito cells (Aedes albopictus C6 cells, clone 36, Igarashi (1978)) . All four cell lines were grown in Eagle's minimal essential medium (MEM) supplemented with 10% (v/v) heat-inactivated (56°C for 30 min) FBS, 1.25 g/L of sodium bicarbonate, 100 units/ml of penicillin G, and 100 μg/ml of streptomycin sulfate. Confluent cell monolayers grown in plastic tissue culture flasks were infected by decanting the growth medium, permitting the virus inoculum to adsorb for 1.5 h at 37°C, and then adding MEM containing 5% FBS. For plaque titration of viruses, confluent cell monolayers in plastic 6-well trays were inoculated with 200 μl of the appropriate dilution of virus. Virus was adsorbed to the cell monolayer for 1.5 h at 37 °C. The cells were then overlaid with 3 ml of 1% (w/v) Noble agar (maintained at 40°C) in MEM lacking

phenol red pH indicator and containing 2% FBS and 0.01% (w/v) DEAE-dextran. Following incubation for 6 days at 37 °C in a 5% C0 ₂ atmosphere, a second 1-ml agar overlay containing 50 μg/ml of neutral red vital stain was added. Viral plaques were counted 2-5 days later.

E. coli:

The E. coli K-12 strains used in this project included XLl-Blue, MC-1061, SURE, JM101, and TB-1.

Recombinant plasmid containing full genome-length cDNA of DEN-2 virus was successfully replicated in E. coli XLl- Blue, MC-1061, and TB-1. Flavivirus cDNA, particularly the gene region encoding the envelope glycoprotein, is troublesome in E. coli . Bacteria hosting the recombinant plasmid containing the full-length cDNA clone grew slowly and were often difficult to streak for isolation on agar plates containing selective antibiotic. Transformation efficiencies were sometimes improved somewhat by incubation of agar plates at 30°C or ambient temperature rather than at 37°C. Bacterial stocks were stored frozen at -70°C in 10% (v/v) glycerol.

Precautions for Working with RNA:

RNA is a fragile molecule that is very readily degraded by the many ubiquitous RNases present in the environment. Many of these RNases are resistant to treatment with detergents and heat, including autoclaving. All reagents and materials that contacted the viral RNA in

this project were RNase-free to avoid degradation of the viral RNA by these ubiquitous, very stable enzymes. The investigator wore tight-fitting gloves, maintained all reagents on ice, used a plastic tool to open the lids of microtubes, used individually packaged pipets, preferably plastic for aqueous solutions, disposable plasticware which is generally RNase-free before opening, and used "For RNA Only" microtubes, Gilson micropipetors (P-10, P- 20, P-100, P-200, P-1000) and tips with aerosol barriers. Use of recycled glassware was avoided. Weigh boats, magnetic stirrers, and pH meters were not used. Chemicals were weighed in sterile, RNase-free disposable plastic 50- ml centrifuge tubes, and solutions were adjusted to the appropriate pH by aliquoting a small volume of the solution onto pH paper. Whenever possible, commercially prepared, guaranteed RNase-free reagents were purchased. Otherwise, newly-opened chemicals were reserved "For RNA Only". Water and stock salt solutions, except for those containing Tris, were treated overnight with 0.1% (v/v) diethylpyrocarbonate (DEPC) to inactivate RNases via alkylation and then autoclaved for 20 min. It is advisable to use the best sterile technique when working with RNA.

Extraction of Viral Genomic RNA from Virus Seed:

Virus seeds containing at least 10 ⁶ PFU (plaque forming units) /ml of virus are ideal for providing appropriate yields of RNA. Seed with virus titer of 10 ⁴ or lower can be problematic in terms of yielding sufficient

RNA. For these low-titer seeds it is best to pool the yields of several extracted seed aliquots.

RNA extraction involved the addition of 200 μl of cold RNA lysis buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% (w/v) sarkosyl, and 100 mM beta-mercaptoethanol) , and 30 μl of 3 M sodium acetate, pH 5.2, to an empty RNase-free 1.5-ml microtube on ice. In a biosafety cabinet, 200 μl of DEN virus seed was added to the microtube and mixed vigorously for 30 sec with a mechanical mixer. The tube was centrifuged briefly to pellet the liquid; then 400 μl of cold phenol (commercially supplied by AMRESCO) equilibrated to pH 4.5 and 80 μl of cold chloroform were added. The tube was mixed vigorously for 30 sec, placed on ice for 15 min, mixed again, then centrifuged for 1 min at maximum speed in a refrigerated microcentrifuge to separate the aqueous and organic phases. The top aqueous phase containing the extracted RNA was transferred to a fresh 1.5-ml microtube on ice, 400 μl of cold isopropanol was added, and the tube was incubated for at least 1 h or overnight at -20°C. The RNA was precipitated by centrifugation for 10 min at maximum speed at 4°C. The supernatant was removed with a pipet rather than by decantation and rinsed with 500 μl of 75% (v/v) ethanol. After spinning again for 10 min, the ethanol was removed with a pipet . The tube was centrifuged again briefly and the residual liquid was removed with a micropipet. The RNA pellet was air dried briefly, resuspended in 50 μl of cold RNase-free dH ₂ 0, and stored frozen. For seeds containing low virus titer, the

RNA pellets in 3-6 microtubes were pooled in a total volume of 50 μl.

RT/PCR Synthesis of Dengue Virus-Specific cDNA Fragments

Full-length genomic mRNA was extracted directly from 200 μl of DEN virus seed. The standard reverse transcriptase/poly erase chain reaction (RT/PCR) was performed in a 100-μl reaction solution containing 5-18 μl of the extracted viral RNA, 1 μl each of 100 μM stock solutions (stored frozen in dH ₂ 0) of the upstream mRNA- sense primer-amplimer and downstream complementary-sense primer-amplimer, 10 μl of 10X standard PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.5, 15 mM MgCl ₂ and 0.1% (w/v) gelatin), 8.0 μl of 2.5 mM dNTPS (2.5 mM each of dATP, dCTP, dGTP, and dTTP; Pharmacia-LKB) , 0.5 μl of 1 M dithiothreitol (DTT) , 0.5 μl of RNase inhibitor (RNasin, 40 U/μl, Boehringer-Mannheim) , 0.5 μl of Taq DNA polymerase (5 U/μl, Perkin-Elmer), and 0.5 μl of RAV-2 reverse transcriptase (18 U/μl, Takara) . The reaction solution was made as two components:

► PCR Reaction Mix: 10 0 μl lOX Standard PCR Buffer

8 0 μl 2.5 mM dNTPs

0 5 μl 1 M DTT

0 5 μl RNasin (40 U/μl)

0 5 μl Taq DNA Polymerase (5

U/μl)

0 5 μl RAV-2 RT (18 U/μl)

60 0 μl RNase-Free dH-,0

80 0 μi Reaction Mix for 1 reaction. Make more than needed for all reaction tubes. Store excess at -70°C for reuse.

► Template/Primer Mix: 18.0 μl DEN-2 RNA Template 1.0 μl 100 μM Up-Amplimer 1.0 μl 100 μM Down-Amplimer

20.0 μl

► Reaction Solution: 80.0 μl PCR Reaction Mix

20.0 μl Template/Primer Mix

100.0 μl In a thin-walled, 200- μl microtube.

The RT/PCR reactions in thin-wall 200-μl microtubes (Phenix Research Products) were incubated without oil overlay in a Perkin-Elmer Model 9600 thermocyder according to the following program:

50 °C for 60 min = First strand cDNA synthesis by reverse transcriptase

94°C for 4 min

50°C for 1 min

72°C for 5 min

30 Cycles

Following completion of the RT/PCR reactions, 5-μl aliquots of each of the 100-μl reactions were analyzed by agarose gel electrophoresis. The DNA bands in the agarose gel were stained in ethidium bromide (500 ng/ml) solution and visualized on an ultraviolet light box. Since extraneous non-target cDNA bands are often amplified in addition to the target cDNA molecules, the remaining 95 μl of each RT/PCR reaction was electrophoresed in a larger, preparative agarose gel, and the target cDNA was stained briefly, excised with a razor blade, and physically extracted from the agarose slice.

High-Melt-Crush (HMC) Extraction of DNA from Agarose:

An agarose gel slice containing DNA was placed in a 1.5-ml microtube and crushed thoroughly with a spatula or pestle. The volume of the crushed agarose was brought to 400-500 μl with TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM disodium EDTA) and 400 μl of phenol (supplied by AMRESCO) , pH 8, was added. The agarose suspension was mixed vigorously using a mechanical mixer, frozen, thawed and mixed, frozen, thawed and mixed, and then centrifuged for 10 min at maximum speed at 4°C. The top aqueous phase was transferred to a fresh microtube, extracted with 400 μl of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged for 2 min. The top aqueous phase was transferred to a fresh tube and extracted with 700 μl of diethyl ether or chloroform. If chloroform was used, the top phase was again transferred to a fresh tube after a brief spin to separate phases. The DNA was precipitated

for at least 30 min at -70°C or overnight at -20°C following addition of 2.5 volumes (essentially filling the microtube) of 95% ethanol containing 300 mM ammonium acetate and 10 mM MgCl ₂ . The DNA was pelleted at 4°C by centrifugation for 20 min at maximum speed. The liquid was decanted, and the DNA pellet was rinsed with 500 μl of 75% ethanol, air-dried briefly, dissolved in 30 μl of TE buffer, and stored frozen or in the refrigerator. A 3-μl aliquot of the extracted DNA was analyzed for purity and quantity by agarose gel electrophoresis. Generally,

20-80% of the DNA loaded onto a gel can be recovered from the gel by this method.

Agarose Gels:

DNA was analyzed by electrophoresis in 1% (w/v) agarose gels run in TBE buffer (100 mM Tris-HCl, pH 8, 91 mM boric acid, and 20 mM disodium EDTA) . DNA bands were visualized by staining the gel in water containing 500 ng/ml of ethidium bromide and exposure to ultraviolet light. Gels used for analyzing RNA transcripts were made with RNase-free reagents. Ethidium bromide stain was incorporated in the gel and running buffer so that the RNA bands could be visualized immediately. To obtain gel- purified DNA fragments, DNA was electrophoresed in 0.7% (w/v) agarose gels made with genetic technology grade Seakem agarose (FMC) or with biotechnology grade agarose (3:1 high resolution blend, AMRESCO) .

Cloning of Dengue Virus-Specific cDNA Fragments:

Some DNA polymerases add an extra "A" nucleotide overhang at the 3 ' -end of synthesized DNA strands. The Taq DNA polymerase does this. To enable the cloning of DNA molecules synthesized using Taq DNA polymerase, TA- cloning vectors have been engineered (Marchuk et al . , 1991) . These vectors generally have a single "T" overhang engineered at the 3 ' -terminus of EcoRV-cut, blunt-ended, linearized plasmid vector. The EcoRV site occurs within the multiple cloning site (MCS) of the plasmid. The MCS is a series of contiguous, unique restriction enzyme (RENZ) sites engineered into a vector plasmid to permit subcloning of exogenous DNA fragments following restriction with a variety of RENZs. The HMC-purified DEN cDNA amplicons were cloned into the 3900-bp pCRII (Invitrogen) , the 2887-bp pT7Blue (R) (pT7Blue, Novagen) , or the 3003-bp pGEM-5Zf (Promega) TA-vector plasmid. The RENZ sites available in the MCS region of these TA- vectors, as well as the RENZ sites of the MCS of the general purpose cloning plasmids, pUClδ and pUC19, used in this project are shown below.

pUC18 pUC19 pT7Blue ΌCRTT pGEM-5Zf

T7 SP6 T7

EcoRI Hindlll Hindlll Nsil Apal

SstI SphI BspMI Hindlll AatH

Kpnl PstI SphI Kpnl SphI

Smal Sail PstI SstI Ncol

BamHI Xbal Sse8387I BamHI SstH

Xbal BamHI Sail Spel EcoRV

Sail Smal AceI BstXl Spel

PstI Kpnl Hindi EcoRI NotI

SphI SstI Xbal EcoRV PstI

Hindlll EcoRI Spel EcoRI Sail

Ndel PstI Ndel

EcoRV BstXl Sad

BamHI NotI BstXl

Aval Aval Nsil

Smal SphI SP6

Kpnl Nsil

Sa Xbal

Banll Apal

EcoRI T7

The pUC18/19 plasmids possess identical MCS sites in reverse orientation in the plasmid backbone. Their purpose is to permit cloning of DNA in either orientation into the plasmid using the same pair of RENZs - this

reversibility was exploited in this project. The TA- vectors used here all possessed T7 and/or SP6 bacteriophage RNA promoters to enable RNA transcription from cloned DNA. These promoters were not used in this project. All of the plasmids contain the gene for ampicillin resistance. They also contained the lac Z portion of the E. coli lac operon. This permits color discrimination between bacterial colonies that receive a recombinant or a wild-type plasmid. In the presence of IPTG and X-gal, bacterial colonies that are transformed with a wild-type plasmid lacking a cDNA insert develop a blue color, whereas cells that receive a recombinant plasmid with cDNA cloned into the MCS of the plasmid are white. Agar plates contained 800 μg of IPTG and 800 μg of X-gal.

Fifty to 100 ng of HMC-purified amplicon was ligated to 50 ng of the pCRII vector using the TA-vector cloning kit supplied by Invitrogen exactly as specified by the instructions supplied with the kit. Frozen, transformation competent £ _ coli INVαF' cells, supplied with the Invitrogen kit and stored at -70°C, were transformed with the ligated DNA as described in the kit instructions. The transformed cells were plated on YTA ₅₀ agar plates (8 g of DIFCO tryptone, 5 g of DIFCO yeast extract, 5 g of NaCl, and 15 g of BACTO agar per liter of dH ₂ 0) containing 50 μg/ml of ampicillin. Only bacterial cells transformed with the pCRII plasmid, which contains an ampicillin resistance gene, grow on this medium. The agar plates were incubated at 37 ^C C overnight.

Similarly, cDNA was ligated to the other TA-vectors or to pUC18/l9 cut with the appropriate RENZ(s) . Ligations were performed at room temperature or at 12°C. E. coli XLl-Blue, SURE, TB-1, or MC-1061 cells were transformed by electroporation and plated on YTA ₅₀ plates. Electroporation was performed according to Dower eϋ al . (1988) using cuvettes with a 2-cm electrode gap in a Bio¬ Rad Gene Pulser set at 2.5 kV voltage, 25 μF capacitance, and 200 ohms resistance. Electroporation-competent cells were prepared by growing a fresh bacterial culture to an optical density of 0.5-0.7 at 600 nm. The cells from 1.5 - 3 L of culture were pelleted by centrifugation for 10 min at 4°C and 5000 rmp in a Sorvall GSA rotor, pooled, washed twice in 1 mM Hepes buffer, and resuspended in 2 ml of 10% (v/v) sterile glycerol per L of original culture. The concentrated cells in glycerol were stored at -70°C.

Bacterial colonies were transferred to 2 ml of 2XYT- Amp ₅₀ broth (16 g of tryptone, 20 g of yeast extract, and 5 g of NaCl per liter of dH ₂ 0) and incubated overnight with shaking at 300 rpm at 37 ^C C in a floor model incubator - shaker (model Innova 4300, New Brunswick) . Recombinant plasmid was extracted from these 2-ml minicultures and analyzed by agarose gel electrophoresis for the presence of cDNA insert. Recombinant plasmids are larger than wild type vector plasmid because of the cDNA insert, and they migrate more slowly than wild type plasmid in agarose gels.

All of the DEN-2 16681 virus-specific cDNA amplicons were cloned into the pCRII TA-vector. Aliquots of insert- positive miniprep plasmids were digested with the

restriction enzyme EcoRI. Since the pCRII MCS contains two EcoRI recognition sites (palindromic hexameric sequence GAATTC) on either side of the EcoRV cDNA cloning site, this RENZ cleaved the cDNA insert from the plasmid vector and cleaved any EcoRI sites that were present within the cDNA itself. The EcoRI-restricted DNA was analyzed by agarose gel electrophoresis to determine that the cloned cDNA was of appropriate size. In our experience, cloning of PCR-derived cDNA amplicons 2000 bp or smaller in size into the TA-vector is efficient.

Cloning amplicons larger than 3500 bp into the TA-vector can be very difficult.

After screening, certain of the miniprep plasmids were selected for further analysis. Their corresponding bacterial minicultures were streaked for isolation on YTA ₅₀ plates, and an isolated colony was inoculated into 50-200 ml of YTA ₅₀ broth to grow up a preparative amount of recombinant plasmid. The preparative scale for the extraction of the plasmid was essentially identical to that for minipreps except for scaled up volumes.

Extraction of Plasmid DNA from Minicultures of E. coli :

White colonies containing recombinant plasmid were picked with a sterile toothpick and shaken overnight at 300 rpm in 2 ml of 2X-YTA ₅₀ broth. Each miniculture was decanted into a 1.5-ml microtube, and the cells were pelleted by centrifugation at 6000 rpm for 2 min. The supernatant was aspirated, and the cell pellet was resuspended gently by up/down micropipeting in 200 μl of

GTE buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 25 mM disodium EDTA) and then mixed with 300 μl of lysis buffer (0.2 N NaOH, 1% (w/v) sodium dodecylsulfate (SDS)) . After incubation on ice for 5 min, 300 μl of cold potassium acetate solution (3 M potassium acetate, 7 M acetic acid, pH 4.8) was added, and the solution was chilled for 5 min on ice and then centrifuged at maximum speed for 10 min at 4°C. The supernatant was poured into a fresh microtube, RNase A was added to 20 μg/ml, and the mixture was incubated at 37°C for 30 min. The sample was extracted twice with 600 μl of chloroform and centrifuged for 1 min at maximum speed at room temperature. The DNA pellet was dissolved in 32 μl of dH ₂ 0. Eight μl of 4M NaCl and 40 μl of 13% (w/v) PEG-8000 was added, and the mixed solution was incubated for 5 min on ice. The sample was centrifuged for 15 min at maximum speed at 4°C, the liquid was aspirated with a micropipet , and the pellet was rinsed with 500 μl of 75% ethanol. The air dried pellet was dissolved in 30 μl of dH ₂ 0 and stored frozen until used.

Extraction of Plasmid DNA from Large Cultures of E. coli :

Preparative-scale plasmid extraction was performed by inoculating 100 ml of 2X-YTA ₅₀ broth with 2 ml of an overnight culture of E. coli . The culture was shaken overnight at 300 rpm and 37°C. The cells were pelleted by centrifugation for 10 min at 5000 rpm in a Sorvall GSA rotor and resuspended in 6 ml of cold GTE buffer. Nine ml of a freshly made solution of 0.2 N NaOH and 1% (w/v) SDS

was added. The sample was incubated for 5 min on ice, then 9 ml of cold 3 M potassium acetate solution was added. After another 5-min incubation on ice, the tube was centrifuged for 20 min at 10,000 rpm at room temperature and the supernatant was transferred to a fresh 30-ml glass tube. RNase A was added to 20 μg/ml, and the sample was incubated for 30 min at 37°C and then extracted twice with 6 ml of chloroform. Twelve ml of room- temperature isopropanol was added and the tube was centrifuged immediately for 20 min at 10,000 rpm at room temperature. The supernatant was decanted, and the DNA pellet was rinsed with 1 ml of 75% ethanol, air dried briefly, and resuspended in 480 μl of dH ₂ 0. The DNA was precipitated by addition of 120 μl of 4 M NaCl and 600 μl of 13% PEG-8000, incubation for 5 min on ice, and centrifugation for 15 min at maximum speed at 4°C. The DNA pellet was rinsed with 500 μl of 75% ethanol, air dried briefly, rehydrated in TE buffer, and stored frozen.

Nucleotide Sequence Analysis of the Dengue cDNA Clones:

Nucleotide sequence analyses of DEN-2 16681 cDNA clones #1-#15 were performed by cloning EcoRI restriction fragments of each clone into the single-stranded bacteriophage M13mpl8 or M13mpl9. Since this is not the current method of choice for sequencing, the method will be described only briefly here. The procedure used for the extraction of plasmid DNA from bacterial cells was also used to extract the intracellular double-stranded replicative form (RF) DNA of M13 from bacteriophage-

infected J57. coli JM101 cells. The RF DNA was linearized at the EcoRI site of the MCS and ligated to the DEN-2 HMC- purified EcoRI cDNA restriction fragments. Electroporation-competent E. coli JM101 cells were transformed by electroporation and plated onto H-agar plates (10 g of DIFCO tryptone, 5 g of NaCl, 15 g of BACTO agar, and 1% (w/v) thiamine per liter of dH ₂ 0) containing 800 μg each of isopropyl-β-D-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG or X-gal) . The electroporated cells were mixed with 300 μl of a fresh logarithmic culture of JM101 cells and 3 ml of warm (51°C) top H-agar containing 9 g/L of agar and then poured onto the H-agar plates. Cells that were transfected with recombinant DNA supported replication of recombinant M13 virus, resulting in the formation of bacteriophage plaques in the JM101 cell lawn on the agar plate. The IPTG/BCIG histochemistry of the system permitted identification of white plaques containing recombinant bacteriophage into which cDNA had been ligated into the EcoRI site of the MCS, whereas wild-type nonrecombinant M13 bacteriophage produced blue plaques . Isolated plaques were picked, inoculated into 3 ml of a fresh, pre-logarithmic phase culture of JM101, and shaken at 37°C for 8-16 h. The minicultures were clarified by centrifugation in 1.5-ml microtubes, the bacteriophage particles were precipitated with PEG-8000, and the single- stranded, circular bacteriophage DNA was isolated from the virions by phenol extraction. The recombinant, circular, single-stranded bacteriophage DNA was sequenced by the dideoxynucleotide termination method. Sequencing kits can

be purchased from various commercial vendors. Radioactive ³² P-dCTP or ³⁵ S-dCTP was incorporated into the strands synthesized in the sequencing reactions. Sequencing was accomplished with many DEN-2 virus-specific primers designed to sequence the entire genome. The sequence reactions were electro-phoresed in 6% (w/v) polyacrylamide gels, which were dried onto filter paper and overlaid with X-ray film. The DNA bands of the autoradiographs were read by the investigator, and the data was entered into a sequence project data spreadsheet. This sequencing method has been used extensively in the past ( e . g. , Kinney et al . , 1986; Johnson et al . , 1986; Deubel et al . , 1986; Deubel et al . , 1988; Kinney eϋ al . , 1989; Trent et al . , 1987) . Nucleotide sequencing was also performed by the current method of direct sequencing of double-stranded plasmid DNA by the dideoxynucleotide termination method using the Applied Biosystems Taq DyeDeoxy Terminator Cycle Sequencing Kit, cycle sequencing in the Model 9600 thermocyder according to the instruction manual supplied with the kit, and analyzing the DNA sequence on an ABI Model 373A DNA sequencing apparatus. Sequencing reactions in 200-μl thin-walled microtubes contained 9.5 μl of reaction mix (buffer, the four dideoxynuleotides, and Taq polymerase supplied in the kit) , 7.0 μl of double or single-stranded template DNA (150 pg/bp) , and 3.2 μl of 10 μM sequencing primer (32 pmol) . After mixing, the reactions were placed in a Perkin-Elmer Model 9600 thermocyder, and programmed cycle sequencing was performed for 25 cycles of incubation at 96°C for 15 sec,

50°C for 15 sec, and 60 ^C C for 4 min. Strand extension was performed at 60°C rather than 72°C because the fluorescent dye-labeled dideoxynucleotide terminators are heat sensitive. The reaction was then applied to a Centrisep gel column (Princeton Separations) to remove unincorporated dye-labeled dideoxynucleotides according to the instructions supplied with the columns. The eluted DNA was vacuum dried for 1 h using a Savant Speed Vac Concentrator and stored at -70°C. The DNA was hydrated with 5 μl of deionized formamide and 1 μl of 50 mM disodium EDTA, then heated in an aluminum block for 2 min at 90°C. A 3-μl aliquot of the denatured DNA sample was applied to one of 24 wells of a polyacrylamide-urea gel in an Applied Biosystems 373A DNA sequencer. The color-coded sequence chromatograph was read by visual inspection, and the resulting nucleotide sequence was entered into a computer-maintained sequence data spreadsheet. The sequencing kit incorporates dideoxynucleotide terminators that are each labeled with a unique fluorescent dye that permits laser detection of all four terminators in a single polyacrylamide gel lane in the Model 373 sequencer. The data was recorded in the form of colored chromatograms that are easily read by the investigator. Single-stranded recombinant M13 DNA can also be sequenced in this manner.

Extraction of M13 Single-Stranded DNA for Sequencin :

White bacteriophage plaques containing recombinant M13 DNA were picked with sterile toothpicks and placed into 2-ml slightly turbid (less than 0.15 A ₆₀₀ ) cultures of

E. coli JM101. The cultures were shaken at 300 rmp and 37°C overnight and then clarified by centrifugation in microtubes at maximum speed for 10 min at room temperature. One ml of the supernatant was transferred to a fresh 1.5-ml microtube containing 200 μl of sterile 20% (w/v) PEG-8000 in 250 mM NaCl. The tubes were mixed by inversion, incubated for 15 min at room temperature, and centrifuged at maximum speed for 5 min at room ^• temperature. The PEG supernatant was removed completely, and the DNA pellet was resuspended in 300 μl of TE buffer. An equal volume of pH 8-buffered phenol was added, and the solution was mixed vigorously several times during a period of 20 min at room-temperature. The tube was centrifuged for 5 min at room temperature, and the top aqueous phase was transferred to a fresh 1.5-ml microtube. After sequential extraction with phenol:chloroform:isoamyl alcohol and chloroform, the DNA was precipitated by adding 2.5 volumes of 95% ethanol containing 300 mM ammonium acetate and 10 mM MgCl ₂ and incubating at -20°C overnight. The tube was centrifuged at maximum speed for 15 min at 4°C, and the supernatant was decanted. Following a rinse with 500 μl of 75% ethanol, the DNA was air dried briefly, resuspended in 60 μl of TE buffer, and stored at 4°C.

Primers:

Primer design was based on the sequence of DEN-2 virus, strain 16681, published by Blok et al . (1992) , and DEN-2 virus, Jamaican strain 1409, as reported by Deubel et al . (1986) and Deubel et al . (1988) .

Primers were synthesized by the Biotechnology Core Facility at the CDC in Atlanta, Georgia. We received the dried primers via mail and adjusted them to a concentration of 100 μM in dH ₂ 0. The designations and sequences of all of the primers-amplimers used in this project are listed in Appendix A.

To amplify the 3 ' -end of the DEN-2 virus genome, a downstream amplimer was designed that was complementary to the published sequence of the 3 ' terminus of the genome. A unique Xbal restriction enzyme site was incorporated at the 5 ' end of this amplimer to provide a unique site to permit linearization of the recombinant plasmid containing the full-length cDNA clone at the 3' terminus of the cloned genomic cDNA. This linearization was necessary to obtain appropriately terminated DEN virus-specific run-off RNA transcripts from the cDNA clone in transcription reactions with bacteriophage T7 RNA polymerase. Linearization at this 3 '-terminal Xbal site resulted in the incorporation of a 5-nucleotide TCTAG extension to the 3' terminus of the genomic mRNA transcribed from the full- length cDNA clone of DEN-2 16681 virus, and a 4-nucleotide CTAG extension to the 3 ' terminus of RNA transcribed from the DEN-2 PDK-53 cDNA clone. The difference between the two cDNA clones in the length of the extraneous 3 ' -terminal extension was due to the differently designed 3 ' -terminal amplimers used to obtain the 3 ' end genomic cDNA amplicon. Amplimer CD2-10687.XBA or CD2-10687.X2 was used to amplify and clone the 3 '-terminal portion of DEN-2 16681 or PDK-53 virus, respectively.

The promoter for the bacteriophage T7 RNA polymerase was engineered at the 5 ' terminus of the cloned genomic cDNA by incorporating the recognition sequence of the T7 RNA polymerase into the sequence of the 5 '-terminal upstream, mRNA-sense amplicon D2-SMT71 immediately preceding the 5 '-terminal nucleotide of the DEN-2 viral genome. This design ensured that the T7 RNA polymerase initiated RNA transcription at the 5 '-terminal nucleotide of the DEN-2 virus-specific cDNA (Milligan et al . , 1987) . Amplimers for PCR reactions were designed to take advantage of RENZ sites identified within the nucleotide sequence of the genome of DEN-2 16681 virus. cDNA molecules were amplified to permit ligation or splicing together of overlapping contiguous cDNA clones at shared, overlapping, unique RENZ sites (Figure 3) .

Transcription of Genomic mRNA from DEN Virus-Specific Full-Length cDNA Clones:

The recombinant plasmid containing the full-length cDNA clone was prepared for RNA transcription by linearization at the unique Xbal site located at the 3 ' terminus of the cloned genomic cDNA. The restriction reaction containing the Xbal-restricted plasmid was extracted sequentially with phenol:chloroform:isoamyl alcohol and chloroform and then precipitated. The DNA was redissolved in 50 μl of TE buffer and digested with proteinase K at a concentration of 1 mg/ml for 1 h at 37°C to hydrolyze contaminating RNases. The sample was then extracted twice with "For RNA Only" phenol:chloroform:isoamyl alcohol buffered to pH 8,

extracted twice with chloroform to remove traces of phenol, and precipitated by adding one-tenth volume of RNase-free 3 M sodium acetate, pH 5.2, and 2.5 volumes of ethanol and incubating for at least 1 h at -70°C or overnight at -20°C.

DEN-2 virus-specific genomic RNA was transcribed from the linearized cDNA template using a commercial T7 transcription kit (Ampliscribe T7 transcription kit, Epicentre Technologies) . Transcription reactions were performed for 2 h at 37°C in RNase-free 1.5-ml microtubes in 20-μl reactions containing 100-1000 ng of linearized DNA template, 7.5 mM each of CTP, GTP, and UTP, 0.75 mM ATP, 2.7 mM m ⁷ GpppA cap analog, 6.7 mM DTT, 2.0 μl of a 10X concentration of a proprietary buffer supplied with the commercial kit, and 2.0 μl of the proprietary Ampliscribe enzyme solution supplied with the kit. Reaction solutions were used directly and without further treatment to transfect BHK-21 cells.

Transfection of BHK-21 Cells with Genomic RNA Transcripts:

BHK-21 clone 15 cells were transfected with RNA transcripts by electroporation (Liljestrδm et al . , 1991) . Fresh cultures of BHK-21 cells were grown to 90% confluency, rinsed twice with cold RNase-free phosphate buffered saline (PBS) , and released from the plastic by incubation with 3 ml of commercial trypsin-EDTA solution (GIBCO-BRL) . The cells were pelleted by low-speed centrifugation at 1200 rpm for 5 min in a Beckman GPKR centrifuge. The cells were washed twice with cold PBS,

resuspended in cold PBS and kept on ice. The cells were counted using a hemacytometer and microscope, and the cell concentration was adjusted to 10 ⁷ cells/ml. One-half ml of the washed, adjusted cells were mixed with each transcription reaction solution in 1.5-ml microtubes on ice. The mixture was transferred to a cold electroporation cuvette with 0.2-cm electrode gap, which was placed in the cuvette holder of the Bio-Rad Gene Pulser. The cells were shocked twice using settings of 1.5 kV voltage, 25 μFD of capacitance, and resistance set to infinity. The shocked cells were incubated for 10 min at room temperature and then added to 75 cm ² tissue flasks containing 20 ml of MEM containing 10% FBS. Transfected cell cultures were incubated at 37°C for 5-8 days until CPE was evident in the cell monolayer and/or expression of DEN virus-specific antigens was identified in an aliquot of the cell monolayer scraped from the flask using DEN virus-specific mouse hyperimmune ascitic fluid or monoclonal antibodies in indirect immunofluorescence tests.

RESULTS

Replication of DEN-2 16681 Virus:

DEN-2 16681 virus replicates to high titer in cell culture. The CDC virus seed used in this study contained 2.0 X 10 ⁷ plaque forming units (PFU) /ml. This titer was determined by plaque titration of the seed virus in monolayer cultures of Vero cells. This seed titered 1.3 X 10 ⁴ PFU/ml in LLC-MK ₂ cells. A growth curve for this virus

was determined in C6/36 Aedes albopictus cell culture (Figure 4) . This level of replication is quite high for a flavivirus. The DEN-2 16681 virus is eminently suitable to serve as the parent to an infectious cDNA clone of DEN virus.

The DEN-2 PDK-53 vaccine virus, taken directly from a vaccine vial obtained from Mahidol University, contained 3.4 X 10 ⁴ PFU/ml of virus, as titrated in Vero cell monolayers, and 1.5 X 10 ⁴ PFU/ml as titrated in LLC-MK ₂ cell monolayers.

RT/PCR Amplification and Cloning of DEN-2 16681 cDNA:

The entire genome of DEN-2 virus, parental strain 16681, was amplified from genomic RNA in the form of 5 cDNA clones of various sizes (T7-F1, F2, F3, F4, and F5) . PCR amplification with 5 sets of upstream and downstream amplimers yielded the predicted amplicon sizes in PCR reactions. Figure 5 shows the migration of these cDNA fragments in agarose gels.

Recombinant plasmids, obtained by ligating the cDNA amplicons into the pCRII TA-vector, were extracted from minicultures derived from transformed E. coli XLl-Blue colonies. Uncut plasmids were screened for the presence of cDNA insert by comparing their mobility in agarose gels with the mobility of uncut wild-type pCRII vector plasmid. Selected plasmids were then restricted with the restriction enzyme EcoRI to confirm the size of the inserted cDNA fragment. EcoRI digests of F2-Sal, Sal-F2,

and F3 plasmids derived from independent transformed bacterial colonies are shown in Figure 6.

The following 15 DEN-2 16681 virus-specific cDNA clones, shown schematically in Figure 7, were selected for nucleotide sequence analysis:

RT/PCR

Clone Amplicon

1 Fl - A8

2 Fl - A21

3 Fl - A25

4 Fl - A26

5 F2- Sal AA2-4

6 F2- Sal AA2-8

7 Sal .-F2 AA3-3

8 Sal .-F2 AA3-4

9 F3 - AA4-4

10 F3 - AA4-6

11 F4 - 10

12 F4 - 12

13 F5 - AA6-1

14 F5 - AA6-2

15 F5 - AA6-4

RT/PCR Amplification and Cloning of DEN-2 PDK-53 cDNA:

The entire genome of DEN-2 virus, vaccine strain PDK- 53, was amplified from genomic RNA in the form of 23 cDNA clones of various sizes. Even though the PDK-53 vaccine contained only about 10 ⁴ PFU/ml of virus, we were able to routinely amplify cDNA from RNA that was extracted directly from this seed virus. To accomplish this, we routinely use the "extended PCR method", incorporating the Taq extender reagent (Stratagene) in the PCR reactions. We had previously shown that the Taq extender

significantly enhanced yields of large molecular weight amplicons in the PCR amplification of the nonstructural genes of the flavivirus, St. Louis encephalitis virus (Figure 8A) . For extended PCR reactions, reaction mixtures were made as for standard PCR reactions, but the standard PCR buffer was replaced with the Taq extender buffer and 1 unit of AmpliTaq DNA polymerase (Perkin- Elmer) and 1 unit of the Taq extender enzyme per kbp of expected amplicon size was included in the reaction. Figure 8B shows the correct agarose gel migration of large cDNA amplicons Fl (containing the T7 RNA polymerase promoter at the 5' end of the mRNA-sense strand of the amplicon), F2, and F3 obtained by PCR amplification using DEN-2 PDK-53 viral genomic RNA as template. The standard PCR reaction also worked for a number of DEN-2 PDK-53 amplifications.

The PDK-53 PCR products were cloned into the pGEM-5Zf TA-vector (Promega) or the pT7Blue(R) TA-vector (Novagen) . Although we seemed to have the best cloning efficiency of PCR amplicons in the pCRII TA-vector, the other vector kits were less expensive and worked well. The cloning efficiency of PCR products into the TA-vector decreased rapidly as amplicon size increased beyond 2000 bp.

The following 23 DEN-2 PDK-53 virus-specific cDNA clones were selected for nucleotide sequence analysis :

Expected

RT/PCR Amplicon

CLONE AMPLICON Lenσth Up-Am limer Down-Amp1imer

1 F-5 1552-bp D2-SMT71 CD2-1510

2 Fl-7 II fl

3 Fl-9 II II

4 F1-75A II IT

5 F1-79B II II

6 F2-14 3355-bp D2-1261 CD2-4615

7 F2-16B II II

8 F3-33 2676-bp D2-4257 CD2-6932

19 F3-3C tl II

10 F4-9 2373-bp D2-6493 CD2-8865

11 F4.9-22 2937-bp D2-6493 CD2-9429

12 F4.9-53 II II

13 F4.5-1 1897-bp D2-8440 CD2-10337

14 F4.5-2 II II

15 F4.5-6 II tl

16 F4.5-7 II II

17 F5-72 1914-bp D2-8773 CD2-10687.X2

18 F5-77 tl tt

19 F5-78 II tl

20 F3.5-4 1375-bp D2-6046 CD2-7420

21 F3.5-6 tl II

22 F3.5-19 II It

23 F3-3K 2676-bp D2-4257 CD2-6932

Nucleotide Sequence Analyses of DEN-2 16681 cDNA Clones:

EcoRI fragments of the 15 DEN-2 16681 virus-specific cDNA clones were subcloned into the single-stranded bacteriophage M13mpl8 or M13mpl9 for sequencing. Sequencing of the entire viral genome was performed manually using radioisotopic labeling and exposure,

development, and reading of autoradiographs. The data was read from the films and entered by hand into a sequence data spreadsheet.

The locations of observed cDNA artifacts or "errors" dictated the splicing strategy of subclones to construct the full genome-length clone. If the nucleotide at a particular position of one cDNA clone differed from the nucleotides at that same position in 2 or more independent clones, then the nucleotide in the first clone was deemed to be an error. If only 2 cDNA clones were sequenced for a given region of the genome and they differed in sequence at a particular position, then if one of the cDNA clones agreed with the sequence data of Blok et al. (1992) , then the clone containing the nucleotide that was in agreement with the latter investigators was deemed to be correct.

The approximate locations of the cDNA errors identified in the 16681 clones are illustrated in Figure 9.

The full genome-length cDNA clone of DEN-2 16681 virus was first constructed in pUC19. Unfortunately, RNA transcribed from this clone was not infectious. When over 90% of the full-length cDNA in the clone was resequenced, it was determined that several mutations had occurred during splicing and cloning manipulations of the subclones in E. coli . One of these mutations was a base deletion in the NS4B gene. This deletion would cause a frameshift of the amino acid sequence, resulting in ribosomal translation of a nonsense polypeptide downstream of the mutation point. This fatal deletion, by itself, would explain the noninfectious nature of the RNA transcribed from the first full-length clone in pUC19.

The final, correct cDNA subclones (Fl-E, F2-E, F3/4/5-F) that were incorporated into the full-length, successfully- infectious clone of 16681 virus were reanalyzed by direct sequencing of the double-stranded plasmid DNA via the thermocycling method using the Taq DyeDeoxy Terminator Cycle Sequencing Kit. Sequence analysis was performed using the automated 373A DNA sequencing machine. The color-coded sequence chromatograms were read by the investigator and the data was entered manually into a computer-based spreadsheet.

We independently confirmed the sequence of the 5 ' - terminal 32 nucleotides of the DEN-2 16681 viral genome. A 5' -end RNA-cDNA hybrid molecule, made with primer cD2- 996 and reverse transcriptase, was 3 ' -tailed with dCTP and annealed to dGTP-tailed, Pstl-cut M13mpl9 RF DNA. One of the resulting M13 clones had a cDNA run-off product containing the 5" -terminal end of the genome. The 5 '-end sequence was identical to that published for DEN-2 1409 (Deubel et al . , 1988) and DEN-2 16681 (Blok et al . , 1992) . We have not independently confirmed the sequence of the

3' -terminal 36 nucleotides of DEN-2 16681 virus or the 5'- or 3' -terminal nucleotides of DEN-2 PDK-53 virus.

We sequenced uncloned, PCR-derived amplicon cDNA fragments directly for the following regions of the DEN-2 16681 viral genome: nucleotides 70-260, 330-870, 890-

1690, 1890-3720, 3770-4050, 4080-4320, and the 3 ' -terminal 9990-10686. Unlike the sequencing of cloned DNA, direct analysis of PCR amplicons provides sequence information for the majority population of amplified cDNA molecules,

and therefore for the majority population of template RNA molecules.

We observed very early in the project that the nucleotide sequence of DEN-2 16681 virus that we determined at the CDC laboratory differed significantly from the sequence of DEN-2 16681 virus as published by Blok et al . (1992) . Our nucleotide sequence, differed from that published by Blok et al . (1992) at 60 nucleotide positions, which were located throughout the genome. Amino acid substitutions were encoded by 26 of these nucleotide differences. The approximate genomic locations of the nucleotide differences are illustrated in the schematic diagram in Figure 10. The exact nucleotide positions of the discrepancies are shown in Figure 11.

Nucleotide Sequence Analyses of DEN-2 PDK-53 cDNA Clones:

The DEN-2 PDK-53 virus-specific cDNA clones were analyzed by direct sequencing of the double-stranded plasmid DNA by the thermocycling method using the Taq DyeDeoxy Terminator Cycle Sequencing Kit. The 3 ' -end sequence from nucleotide position 10290-10686 was also determined by direct sequencing of PCR-derived amplicon cDNA. Sequence analysis was performed using the automated 373A DNA sequencing machine. The color-coded sequence chromatograms were read by the investigator and the data was entered manually into a computer-based spreadsheet. The approximate locations of the cDNA errors identified in the PDK-53 cDNA clones are illustrated in Figure 12.

Our determination of the nucleotide sequence of DEN-2 PDK-53 virus differed significantly from the PDK-53 genomic sequence published by Blok et al . (1992) . The latter investigators reported a total of 53 nucleotide differences that encoded 27 amino acid mutations between the nucleotide sequences of the genome of DEN-2 16681 virus and that of its vaccine derivative, PDK-53 virus. They reported the following nonsilent mutations : 1 in the capsid, 2 in prM, 1 in M, 3 in E, 3 in NS1, 3 in NS2A, 2 in NS2B, 3 in NS3, 3 in NS4A, 3 in NS4B, and 3 in NS5. We detected only 8 nucleotide mutations between the genomes of these two virus strains . One mutation occurred in the 5 ' -NC region of the genome, while 7 nucleotide mutations, 4 of which encoded amino acid substitutions, occurred in the coding region of the genome as shown in Figure 13 and the following table.

Table: Summary of nucleotide differences between the genomes of DEN-2 16681 virus and its vaccine derivative virus, strain PDK-53.

Genome

Genome Nucleotide Amino Acid

Position Gene 16681 PDK-53 16681 PDK-53

57 5'-NC C T - -

524 ^a prM-29 A T Asp Val

2055 ^a E-373 C T Phe Phe

2579 ^a NS1-53 G A Gly Asp

4018 NS2A-151 C T Leu Phe

5547 NS3-342 T C Arg Arg

6599 ^a NS4A-75 G C Gly Ala

8571 ^a NS5-334 C T Val Val

^a 16681 vs. PDK-53 difference agrees with Blok et al (1992)

The few nucleotide positions where our data and those of Blok et al. (1992) agreed, in terms of sequence differences between the 16681 and PDK-53 viral genomes, were distributed throughout the genome. The entire genome of DEN-2 16681 virus was cloned and sequenced before we received the PDK-53 vaccine virus at our laboratory.

Except for the 3 ' -terminal cDNA clones #17-#19, every PDK- 53 virus-specific cDNA clone constructed in our laboratory contained at least one nucleotide position of 16681/PDK-53 sequence difference confirmed by both ourselves and Blok et al . (1992) . Therefore, our PDK-53 virus-specific cDNA clones did not result from contamination of PDK-53- specific PCR reactions with 16681 virus-specific cDNA template. Our PDK-53 virus-specific cDNA clones, which also contained the many sequence discrepancies between our data and those of Blok et al. (1992) , encoded the nucleotide sequence from the 5 ' terminus to nucleotide position 10337 of the genome of PDK-53 virus. The 3'- terminal 387 nucleotides (10337-10723) of DEN-2 PDK-53 virus were identical to those of the parental 16681 virus. Since none of the PDK-53 virus-specific cDNA clones covering this region of the genome contained a point of confirmed 16681/PDK-53 sequence difference, we repeated the PCR amplification of the 3' terminus of the PDK-53 virus genome. This was done to ensure that the 3 ' - terminal cDNA clones #17-#19 did not result from PCR reactions contaminated by 16681 virus-specific DNA template. The PCR reaction components were pipetted in a room in which DEN cloning had not been performed previously, using new micropipetors, newly opened pipet tips with aerosol barrier, and freshly made stock reagents. Direct sequencing of the resulting double- stranded PCR cDNA amplicon confirmed that the 3 '-387 nucleotides of DEN-2 PDK-53 virus was indeed identical to the 3' terminus of the 16681 parent.

The finalized nucleotide sequence of DEN-2 virus, strain 16681, including the nucleotide and amino acid mutations identified for DEN-2 PDK-53 virus, is shown in Figure 14.

Construction of DEN-16681 Full-Length Clone in pUC19:

For the construction of the full genome-length cDNA clone of DEN-2 16681 virus, 5 of the sequence- characterized PCR-amplified cDNA subclones were selected for splicing. However, clone #5 contained a cDNA "error" that was not readily spliced out with the existing clones. This error, which was a C-to-T mutation at nucleotide position 1730 and encoded a nonsilent Thr-to-lie amino acid substitution at E-265, was incorporated into the F2 construct. The intermediate F2 construct was the result of splicing the F2-Sal clone (#5) Sphl/Hpal fragment to the Sal-F2 clone (#7) Hpal/Kpnl fragment in the MCS of plasmid pUC18 (Figure 15) . To correct the error, a new PCR amplicon was made using primers D2-1261 and CD2-2955. Resulting clones in the TA-vector were sequenced, and the correct Sphl/Hpal fragment of a new clone was substituted for the faulty Sphl/Hpal fragment of the original F2 construct (Figure 16) . The corrected F2 clone was designated F2-C.

The relevant cDNA clones of DEN-2 16681 virus were spliced together via a series of intermediate ligation products in the MCS of pUC18 to yield Fl/3/4/5, which contained all of the genome except for the Sphl-Kpnl 1380- 4493 region present in clone F2-C. Multiple attempts to

ligate the F2-C Sphl/KpnI cDNA fragment into Fl/3/4/5 in pUC18 failed. The cDNA insert of Fl/3/4/5-pUC18 was then transferred to the MCS of pUC19, resulting in Fl/3/4/5- pUC19. This operation simply reversed the orientation of the cDNA insert within the context of the pUC plasmid. Ligation of Sphl/KpnI-cut Fl/3/4/5-pUC19 and F2-C Sphl/KpnI insert readily yielded transformants in E. coli Xll-Blue that contained the full-length cDNA clone Fl/2/3/4/5-pUC19, which was designated pD2/IC-20. The detailed splicing procedures for pD2/IC-20 are illustrated in Figure 17. The orientation-specific cloning of the full genome-length cDNA in pUC19 rather than pUC18 is diagrammed in Figure 18.

The full genome-length cDNA of DEN-2 16681 virus was cloned into the MCS of pUC19. Apparent full genome-length viral mRNA was transcribed from linearized pD2/IC-20. This transcribed product failed to yield infectious virus following electroporation of BHK-21 cells. Most of the cDNA in the pD2/IC-20 clone was resequenced, and several cloning artifacts, including a fatal single-nucleotide deletion, were identified. Original subunit intermediate cDNA constructs in pUC18 were resequenced to confirm that they possessed the correct sequence and corrected where necessary. The corrected primary cDNA clones Fl, F2-C, and F3/4/5 were then ligated into the low-copy plasmid pBR322, rather than the high copy-number pUC18 plasmid. It was envisioned that the cDNA would be more stable in a slower-replicating plasmid in E. coli .

To enable more straightforward cloning into pBR322, the MCS of pUC19 was spliced into the pBR322 plasmid

(Figure 19) . This resulted in plasmids pBRUC-138 and pBRUC-139 containing the pUC MCS in both orientations within the pBR322 plasmid backbone. The SphI site was removed from both pBRUC plasmids by cutting with SphI, blunt ending of the cut ends using T4 DNA polymerase, and then ligating the ends back together. This was necessary for the construction of the full-length cDNA clone because SphI is one of the cDNA restriction/splicing sites for the clone. The F3/4/5-F cDNA clone of DEN-2 16681 virus, which had been verified by sequence analysis, was cloned into pBRUC-139 (SphI ^" ) (Figure 20) . Following this ligation, the Fl-E and F2-C cDNA clone fragments were also moved into the pBR322 backbone to construct the full genome- length cDNA clone, pD2/IC-30P (Figure 20) . This recombinant plasmid was replicated successfully in both TB-1 and MC-1061 strains of E. coli .

Construction of DEN-2 PDK-53 Infectious cDNA Clone;

The full-length infectious clone of DEN-2 16681 virus was used in the construction of the infectious clone for PDK-53 virus. Since the 3 ' -noncoding regions of the genomes of both viruses are identical, and the amino acid sequences of the translated precursor polyproteins encoded by genome nucleotide positions 6646-10269 are identical in both viruses, the infectious clone of PDK-53 virus was constructed using the 16681 3 ' -end cDNA from the Nhel site at nucleotide position 6646 to the 3 ' terminus of the genome (Figure 21) . After correcting a cDNA error in the

PDK-53 F3-3C subunit clone, this fragment and the F2-16B cDNA fragment were ligated into the infectious clone backbone to construct the DEN-2 PDK-53 virus-specific full-length cDNA clone, pD2/IC-130V (Figure 21) .

Transcription of Viral mRNA from DEN-2 Infectious cDNA Clones:

Viral genomic RNA extracted from gradient-purified virions was analyzed by nondenaturing RNA agarose gel electrophoresis to observe the level of RNA degradation and the limits of detectability by ethidium bromide staining. Figure 22 shows an agarose gel electropherogram for 22-383 ng of viral genomic RNA obtained from purified preparations of wild-type DEN-2 16681 virus and wild-type Venezuelan equine encephalitis (VEE) virus, strain Trinidad donkey. Although degradation of the RNA is visible as a spectrum of smaller molecular weight nucleic acid (smear in Figure 22) , definite full-genome length RNA bands are clearly visible. This smear of nucleic acid is probably also due, in part, to multiple conformations of the single-stranded RNA molecules which migrate through the gel at different rates. The relative gel migration of the single-stranded RNA does not correlate directly with the sizes of the double-stranded molecular weight marker

DNA bands (MW, Figure 22) ; the VEE and DEN-2 viral genomes are 11,447 and 10,723 nucleotides in length, respectively. BHK-21 and C6/36 cells were transfected successfully by electroporation with 2000, 500, 100, 10, 1, and 0.1 ng of viral genomic RNA extracted from purified VEE or DEN-2

16681 virus, as indicated by development of CPE, expression of viral proteins detected by indirect immunofluorescence tests using virus-specific antibody, and/or by plaque titration of infectious virus from the transfected-cell culture medium. RNA quantities of 1 ng or less were essentially undetectable in the ethidium bromide-stained agarose gel system we used. Therefore, authentic RNA transcripts derived from full genome-length cDNA and visualized in agarose gel electropherograms of transcription reactions should be infectious for BHK-21 cells by electroporation.

Investigators previously constructed an infectious cDNA clone for VEE virus as reported by Kinney et al . (1989) . RNA transcription reaction conditions that yielded high quantity and quality of infectious mRNA transcripts from the pVE/IC-92 infectious clone of VEE virus failed in multiple attempts to transcribe RNA from the pD2/IC-20 clone of DEN-2 16681 virus. Figure 23 shows an agarose gel electropherogram that demonstrates successful transcription of RNA from the VEE clone, but not pD2/IC-20.

In an attempt to improve RNA transcription from the DEN-2 clone, commercial transcription kits were purchased. The Megascript transcription kit supplied by Ambion also failed to transcribe RNA from the DEN clone. However, the Ampliscribe kit obtained from Epicentre Technologies enabled efficient transcrip-tion of RNA from the DEN-2 clone (Figure 24) .

The success of the Ampliscribe kit apparently was due to the high concentration of ribonucleotides and a very

high, but proprietary, concentration of T7 RNA polymerase. The RNA transcribed from pD2/IC-20 was not infectious. However, viral mRNA transcribed from DEN-2 16681 clone pD2/2-IC30P and PDK-53 clone pD2/IC-130V was infectious (Figure 25) .

Viral mRNA transcripts from both replicates of pD2/IC-30P (A and D) and pD2/IC-130V (F and J) were infectious, producing viable infectious virus in electroporated BHK-21 cells. Figure 26 shows RNA transcripts from pD2/IC-20, pD2/IC-30P, and pD2/IC-130V.

Construction of DEN-2 16681/PDK-53 Chimeric cDNA Clones:

Several chimeric full-length cDNA clones were derived from the pD2/IC-30P and pD2/IC-130V clones. All clones were constructed in the pBRUC-139 derivative of the pBR322 plasmid vector. E. coli strains XLl-Blue, MC-1061, and

TB-1 were successfully transformed with ligated recombinant plasmids containing full genome-length cDNA. Viable virus was derived from all of the indicated clones.

The evolutionary tree for the chimeric viruses is diagrammed in Figure 27.

Details concerning the splicing strategies for the chimeric clones are shown in Figure 28. Appropriate cDNA fragments were cut and ligated together at the internal

Sail, SphI, Kpnl, and Nhel sites as well as at the 5 '-SstI and 3 ' -Xbal sites.

Viable prototype and chimeric viruses were derived from each of the clones indicated in Figure 28 by electroporation of BHK-21 cells with viral genome-length

mRNA transcribed from linearized plasmids. Seed stocks of these viruses were prepared by centrifuge-clarification of the cell culture medium, adjustment of the FBS concentration to 10%, and freezing of seed aliquots at -70°C. Virus concentrations were determined by plaque titration of the virus seeds in monolayer cultures of Vero cells. The results of these virus titrations are shown in the following table.

Table. Plaque titration of DEN-2 16681 and PDK-53 stock seed viruses and chimeric viruses recovered from BHK-21 cells transfected with infectious clone-derived viral mRNA transcripts.

Virus (PFU/ml) Genotype ³

DEN-2 16681 8.0 X 10 ⁷ c D F G L R G V

DEN-2 PDK-53 5.1 X 10 ³ t V . D F . A .

D2/IC-30P-A 3.6 X 10 ⁵ D2/IC-30P-A2 1.7 X 10 ⁵

D2/IC-130V-F 4.0 X 10 ⁵ t V . , D F . A D2/IC-130V-J 2.2 X 10 ⁵ t V .. D F . A

D2/IC-130V2-1 2.8 X 10 ⁵ t V . . . . . A D2/IC-130V2-7 8.8 X 10" t V . . A

D2/IC-31-12 2.1 X 10 ⁵ t V . . .

D2/IC-31-15 3.2 X 10 ^s t V . . .

D2/IC-32-A 1.4 X 10 ⁶ . . . D F

D2/IC-32-G 1.2 X 10 ⁶ . . . D F

D2/IC-33-C 9.6 X 10 ⁴ A D2/IC-33-P 1.9 X 10 ⁵ A

D2/IC-321-L 1.1 X 10 ⁶ t V . D F . .

D2/IC-321-N 7.6 X 105 t V . D F . . D2/IC-323-B 7.2 X 10 ⁵ . . . D F . A

D2/IC-323-I 8.8 X 10 ⁵ . . . D F . A

D2/IC-31-57-5 2.4 X 10 ⁵ t D2/IC31-524-D 3.2 X 10 ⁴ c V ^a Genotype is designated in small case for the virus-specific 5 ' -noncoding nucleotide and in upper case single-letter amino acid abbreviation for amino acids encoded by virus -specific nucleotide mutations. Dots represent nucleotide or amino acid sequence identity with DEN-2 16681 virus.

To establish the validity of the clone-derived chimeric viruses, relevant genomic cDNA fragments were amplified directly from seed viruses by PCR and spot- sequenced. The results are shown in Figure 29. This validation process is ongoing. Except for D2/IC-31-524 virus, appropriate cDNA insert regions in chimeric viruses have been confirmed by sequence analysis. Except for D2/IC-30P, D2/IC-130V, and D2/IC-31-57, which have been fully confirmed, clone-derived chimeric viruses have yet to be spot-sequenced in a recipient clone-derived cDNA region to definitely establish the chimeric nature of the virus. The recipient clone is the recombinant plasmid backbone into which a cDNA fragment, the insert fragment, from a heterologous donor clone is spliced. Where duplicate clone-derived viruses were obtained, both viruses of a given genotype were spot-sequenced, and both gave the same result, which is shown in Figure 29.

Submission of pD2/IC-30P and pD2/IC-130V to ATCC:

Patent deposits of the full genome-length cDNA clones of DEN-2 16681 and PDK-53 viruses were submitted to the American Type Culture Collection (ATCC) , Rockville, Maryland, U.S.A. Both pD2/IC-30P-A and pD2/IC-130V-F were grown overnight in E. coli TB-1 cells. Six cryogenic vials containing 1 ml each of frozen cell culture in 10% glycerol were submitted by dry ice shipment. Prior to shipment, plasmid was extracted from a 1 ml aliquot of each virus-specific culture. The recombinant full-length

plasmid was recovered from the cells as shown in Figure 30.

The pD2/IC-30P-A deposit with the ATCC was assigned accession number ATCC 69826, and the pD2/IC-130V-F deposit with the ATCC was assigned accession number ATCC 69825. Date of deposit was May 25, 1995.

Construction of Chimeric DEN-2/1. -2/3. and -2/4 Infectious Clones: We contemplate deriving chimeric DEN-2/1, DEN-2/3, and DEN-2/4 viruses from recombinant full genome-length cDNA clones containing the genetic background of DEN-2 PDK-53 virus and the prM and E genes of the DEN-1, DEN-3, and DEN-4 candidate vaccine viruses, respectively. To accomplish this, the prM and E genes of the vaccine viruses were amplified by PCR. Because our laboratory has been establishing a sequence database to analyze the molecular epidemiology of several flaviviruses, including all of the serotypes of dengue virus, the primers used for cDNA amplification in the PCR were readily available at our laboratory. The amplified cDNA molecules were sequenced directly, thus providing the sequence of the population of virions in the virus seed. The amplified cDNA amplicons for the DEN-1, DEN-3, and DEN-4 vaccine viruses have all been cloned into the pGEM-5Zf TA-vector. The cloned cDNA has not been analyzed by sequencing, since it will be necessary to rederive the cDNA amplicons by PCR to incorporate appropriate RENZ cleavage sites within the amplicon for splicing into the full-length cDNA backbone of DEN-2 PDK-53 virus. The partial nucleotide sequences

of the genomes of the DEN-1, DEN-3, and DEN-4 vaccine viruses were aligned with the DEN-2 PDK-53 sequence. All four sequences are aligned with the nucleotide sequence of DEN-2 16681 virus and its deduced amino acid sequence in Figure 31. The deduced amino acid sequences of the DEN viruses are aligned in Figure 32.

It is readily evident from the aligned nucleotide sequence data that useful restriction enzyme sites in the DEN-2 virus-specific cDNA are not conserved in the DEN-1, DEN-3, and DEN-4 viruses. Therefore, splicing sites must be engineered into the cDNA to enable the splicing of heterotypic DEN-1, DEN-3, and DEN-4 prM and E genes into the DEN-2 backbone. It is not yet clear precisely how the nonstructural proteins of flaviviruses interact with the structural proteins during intracellular maturation of the virus. Furthermore, the interaction of the capsid protein with the genomic mRNA molecule in the nucleocapsid of the virion has not been defined. However, coexpression of the E and prM proteins has been more successful than expression of E alone in expression systems in vi tro . The DEN-2 nonstructural proteins are involved in all virus- specific intracellular polyprotein processing and replication of viral mRNA, and the predominant portion of the mRNA genome interacting with the capsid protein is presumably, but not necessarily, DEN-2 virus-specific. For these reasons, our strategy is to splice in the prM and E genes of DEN-1, DEN-3, and DEN-4 viruses very precisely, while maintaining the DEN-2 context of the bracketing capsid and NS1 protein regions.

The strategies for creating Xhol and Xbal splice sites at the 5 ' end of the prM gene and near the 3 ' end of the E gene are illustrated in detail in Figures 33 and 34, respectively. Briefly, mutagenic primers containing the appropriate RENZ site are utilized in PCR reactions to synthesize new cDNA for the prM and E genes of all four viruses. A DEN-2 PDK-53 virus-specific cDNA cassette plasmid, designated pD2V-CAS12, containing the genome region from the 5 ' terminus through nucleotide position 4696 is constructed via intermediate plasmid constructs pFl-Xho and pF2-Xba as illustrated in Figures 35 and 36. The Xhol/Xbal cDNA fragments cut directly from DEN-1, DEN- 3, and DEN-4 virus-specific amplicons synthesized by PCR using the mutagenic primers are ligated into the pD2V- CAS12 cassette plasmid to create subclone chimeras. The Sstl/Kpnl fragment of the resulting pDlV-CAS12, pD3V- CAS12, and pD4V-CAS12 cassettes are moved into pD2/lC-130V restricted with Sstl/Kpnl to create the chimeric full genome-length cDNA clones (Figure 36) .

Discussion:

Infectious cDNA clones permit the directed engineering of viral genomes. Depending on their viability in terms of ability to replicate in cell culture, infectious clone-derived viruses can be modified by incorporating point mutations, multiple mutations, deletions, gene regions of related or heterologous viruses, or nonviral genes. Infectious cDNA clones have been developed for many RNA viruses, including flaviviruses DEN-4 (Lai et al. , 1991) , yellow fever (Rice

et al., 1989), Kunjin (Khromykh and Westaway, 1994), Japanese encephalitis (Sumiyoshi et al. , 1992), and TBE (unpublished data) . We describe herein the development of infectious cDNA clones for DEN-2 16681 virus and its candidate vaccine derivative, strain PDK-53. We also describe the construction of chimeric viruses, incorporating the prM and E genes of candidate DEN-1, DEN- 3, and DEN-4 vaccine viruses within the genetic background of the DEN-2 PDK-53 vaccine virus. Although the candidate vaccine viruses developed at Mahidol University are currently the best live DEN virus vaccine candidates in terms of immunogenicity and safety in adult humans, the DEN-1, DEN-3, and DEN-4 vaccine viruses replicate poorly in cell culture and possess low infectivity in humans, requiring up to 2000-fold more PFU of virus to infect and immunize humans than is needed for the DEN-2 PDK-53 vaccine virus. The low infectivities of these viruses have significant implications for vaccine production in cell culture, potentially decreased immunogenic efficacy, and more rapid inactivation under conditions of a poorly maintained cold chain in tropical countries where dengue viruses are endemic.

The purpose of engineering chimeric DEN vaccine viruses is to enhance the replicative ability and immunogenicity of the DEN-1, DEN-3, and DEN-4 vaccine viruses. A primary assumption has been that the attenuated DEN-2 PDK-53 vaccine virus replicates to appropriate levels in cell culture. In fact, it does appear that the genome of DEN-2 PDK-53 virus is eminently suited to serve as the genetic backbone for chimeric

viruses containing the prM and E genes of DEN-1, DEN-3, and DEN-4 vaccine viruses. We have recently completed growth curves for DEN-2 16681 virus, DEN-2 PDK-53 virus, and their infectious clone derivative viruses in LLC-MK ₂ cells.

The viruses were titrated in Vero cell monolayers. These data are shown in the following table:

Maximum Maximum

Titer Titer

Virus (PFU/ml ) at Day

DEN-2 16681 2 . 6 x 10 ⁸ 10

D2/IC-30P-A 1 . 7 X 10 ⁷ 8

D2/IC-30P-A2 6 . 6 X 10 ⁷ 7

DEN- 2 PDK- 53 3 . 8 X 10 ⁷ 9

D2/IC-130V-F 2 . 9 X 10 ⁷ 7

D2/IC-130V-J 1 . 7 X 10 ⁷ 7

The DEN-2 PDK-53 virus and its infectious clone derivative viruses grow to approximately 10 ⁷ PFU/ml in LLC-MK ₂ cells, about as well as the DEN-2 16681 virus.

A second assumption is that the chimeric DEN viruses will be viable and the DEN-2 PDK-53 virus-specific replication machinery will significantly increase replication of the chimeric viruses in cell culture and increase their infectivity and immunogenicity in humans relative to the wild-type vaccine viruses. The high degree of conservation of amino acid sequences among the polyproteins of the four DEN viruses should ensure that the chimeric viruses will be viable. The level of

replication attained by the chimeric DEN viruses is determined empirically, as was determined for the DEN-2 PDK-53 infectious clone derivative virus.

Bray et al. (1991) constructed chimeric DEN-4/1 and DEN-4/2 viruses that appeared to appropriately express

DEN-1 and DEN-2 structural protein antigens in the genetic background of DEN-4 virus. These investigators spliced much of the 5 ' -noncoding region, and the capsid, prM and E genes of DEN-1 or DEN-2 virus into the full-length cDNA clone of DEN-4 virus. The near 3 ' -terminal splice site they chose in the E gene is very close to that proposed by us in our project. These chimeric viruses replicated very slowly relative to the wild-type viruses. The authors attributed this slow replication to possible suboptimal gene expression, assembly, and/or maturation due to incompatibility of heterotypic genes or RNA packaging in the nucleocapsid. Another possibility is that cDNA errors may have been incorporated into their constructs. In contrast, Pletnev et al. (1993) engineered chimeric viruses between DEN-4 virus and tick-borne encephalitis (TBE) virus, which is a very distant flavivirus relative of DEN viruses. Thus, DEN virus chimeras may be derived that are viable.

A third assumption is that our chimeric DEN viruses will express the appropriate structural protein antigens of DEN-1, DEN-3, and DEN-4 viruses, and that vaccinees will respond with development of appropriate serum titers of DEN-1, DEN-3, and DEN-4 neutralizing antibodies following immunization with the chimeric viruses . We describe the insertion of the prM and E genes of DEN-1,

DEN-3, and DEN-4 viruses into the DEN-2 clone. Thr-to-Ser amino acid substitutions near the amino terminus of the prM protein in DEN-2, DEN-2/1, DEN-2/3, and DEN-2/4 viruses resulting from mutagenesis to create the Xhol site of the cassettes should be conservative in nature and affect the phenotype of derived viruses minimally, if at all. Alternatively, a unique Mlul site (ACGCGT) could be created via a single, silent A-to-G point mutation at nucleotide position 453 in the DEN-2 clone. The Mlul site immediately preceding the T7 promoter could easily be eliminated by cutting the clone with Mlul, blunt-ending, and religation. The clone-derived DEN-2 and chimeric viruses would then have the prM amino-terminal sequence "FHLTTR." The carboxyl-terminal 24 amino acids of the E glycoprotein of all of the infectious clone-derived viruses will be those of the DEN-2 PDK-53 virus. Therefore, the E protein of all of the chimeric viruses will have amino acid mutations in this region. Yet, the carboxyl-terminal 39 amino acids of the DEN virus E protein comprise membrane-spanning, transmembrane domains. In all enveloped viruses, the transmembrane domains of the integral viral proteins of related viruses are quite variable in amino acid sequence. It has often been noted that the important conserved feature of amino acids in this domain lies in their hydrophobic, "lipid-loving" nature rather than in the absolute sequence. Creation of a Mrol site (TCCGGA) or a unique Agel site (ACCGGT) at nucleotide positions 2281-2286 in the DEN-2 clone would

result in amino acids "SG" or "TG", respectively, at positions E-449 and E-450 in the clone-derived viruses. The E protein of all flaviviruses share a similar gross tertiary structure that is indicated by the absolute conservation of the 6 Cys residues in the prM protein and in the 12 Cys residues in the ectodomain (the .region located on environment side of the viral lipid envelope) of the E protein of DEN, Japanese encephalitis, West Nile, Murray Valley encephalitis, St. Louis encephalitis, Kunjin, yellow fever, TBE, Langat, and Powasson flaviviruses (data not shown) . Cys residues are involved in intrachain Cys-Cys disulfide bonds that determine the overall structure of the protein. We fully expect the DEN-2/1, DEN-2/3, and DEN-2/4 chimeric viruses to be viable and to replicate more efficiently than the wild- type DEN-1, DEN-3, and DEN-4 vaccine viruses, respectively. Furthermore, chimeric recombinants involving the genetic backbone of one flavivirus and the structural genes of a variety of different flaviviruses may also be viable, as has been demonstrated for DEN-4/TBE virus recombinants (Pictnev et al., 1993). Such recombinant viruses offer the potential opportunity to engineer chimeric vaccine viruses for a number of flavivirus-associated diseases within the genetic background of a single flavivirus. The X-ray crystallographic structure of the E glycoprotein of TBE flavivirus has recently been published (Rey et al. , 1995) . This development has significant implications for the future design of flavi irus molecular vaccines.

A fourth assumption is that the chimeric DEN viruses will retain the attenuated phenotype of the wild-type DEN- 1, DEN-3, and DEN-4 vaccine viruses, despite enhanced replicative efficacy provided by the more efficient nonstructural genes and 5 ' and 3 ' noncoding regions of the DEN-2 PDK-53 virus. This presupposes that DEN-2 PDK-53 virus has attenuating mutations in the noncoding regions or in the nonstructural genes and/or that attenuating mutations occur in the prM/E region of the genomes of DEN- 1, DEN-3, and DEN-4 viruses. Mutations in essentially any region of the viral genome may be capable of attenuating a virulent virus. This has been demonstrated for a number of viruses including polio virus, VEE virus, and Theiler's virus. Noncoding as well as protein coding regions may be involved in attenuation. Attenuating mutations in the envelope proteins of enveloped viruses are common (Barrett et al. , 1990) .

The nucleotide mutations in DEN-2 PDK-53 virus at genome nucleotide positions 57 (5 ' -noncoding region) , 524 (prM) , 2579 (NS1) , 4018 (NS2A) , and 6599 (NS4A) may be involved in attenuation of the virus. Unless the prM amino acid mutation is the only mutation affecting virulence of the virus, the DEN-2 PDK-53 genetic background, within which the structural genes from heterologous viruses will be expressed, does itself possess genotypic markers of attenuation. We can determine the genetic loci involved in the attenuation of the DEN-2 PDK-53 virus by analyzing DEN-2 16681/PDK-53 recombinant viruses derived from chimeric 16681/PDK-53

full-length clones. The E gene of DEN-2 PDK-53 virus contains no attenuating mutations.

Although investigators have sequenced the structural genes of numerous DEN-3 virus strains ( e . g. , Lanciotti et al., 1994), none have sequenced the DEN-3 16562 virus, parent to the DEN-3 PCMK-30/FRhL-3 vaccine virus. After determining the sequences of the prM and E genes of this virus, we can establish if any amino acid mutations have occurred within these genes in the DEN-3 vaccine virus. By comparison, nucleotide sequence information for the parental DEN-1 and DEN-4 viruses have been determined (unpublished data (parental DEN-1 virus) ; Lanciotti et al., submitted for publication (parental DEN-4 virus)). The nucleotide sequences of the E gene of DEN-4 1036 virus and both prM and E genes of DEN-1 16007 virus have been determined. The following amino acid mutations were identified:

Amino Acid

E Protein -

Virus Amino Acid Parent Vaccine type Position Strain Strain

DEN-1 E-130 Val Ala

E-203 Glu Lys

E-204 Arg Lys

E-225 Ser Leu

E-384 Ala Glu

E-477 Met Val

DEN-4 E-345 Glu Lys

E-364 Val Ala

There were six amino acid mutations in the E protein of DEN-1 16007 PDK-13 virus and 2 mutations in that of DEN-4 1036 PDK-48 virus. There were no amino acid substitutions in the prM protein of the DEN-1 vaccine virus. Glu-to-Lys and Lys-to-Glu amino acid substitutions, as occur at DEN-1 E-203 and DEN-4 E-345, are common motifs in sequence comparisons between parent viruses and their vaccine derivatives. It is likely that the heterologous prM/E cDNA inserts in recombinant full-length cDNA clones will transport genetic loci of attenuation into the chimeric DEN-2/1, DEN-2/3, and DEN-2/4 virus derivatives. The optimum scenario for the chimeric viruses involves increased replication ability in the presence of genetic loci of attenuation in the heterologous DEN-1, DEN-3, and

DEN-4 structural gene inserts within the genetic background of the DEN-2 PDK-53 virus.

Nucleotide sequence analysis of expressed genes is essential. The error rate in the original RT/PCR derived cDNA clones of DEN-2 16681 virus was 8.2 x 10" ⁴ , that is 1 cDNA error for every 1227 nucleotides of cloned, sequenced cDNA. In a previous sequencing project involving VEE virus and employing classical, non-PCR cDNA synthesis methodology, the error rate was calculated to be 3.9 x 10 ^~4 or 1 error for every 2543 nucleotides of cloned, sequenced cDNA. These errors are due to nucleotide incorporation errors by reverse transcriptase during first strand cDNA synthesis and perhaps to the cloning of individual variants within the original population of virions. Unlike many DNA polymerases, RNA polymerases and reverse transcriptase have no editing function. Incorrect nucleotides incorporated during strand elongation are not detected or removed before continuing. The Taq DNA polymerase is also known to incorporate errors into PCR amplicons. Thus, at least 4-8 cDNA "errors" can be expected to occur in 10 kb of cloned cDNA. We have observed the incorporation of spurious in-frame termination codons (TAA, TAG, TGA) in cDNA clones derived from both VEE and DEN viruses. Premature termination of amino acid translation would result in a truncated protein and would undoubtedly be a lethal mutation for a candidate infectious clone. Much of the utility of genes expressed in vi tro is compromised when those genes are not characterized by sequence analysis. If cDNA errors occur in candidate infectious cDNA clones, it may be difficult

to determine if phenotypic effects of directed mutations are due to the engineered mutation, to cDNA errors, or to synergistic action or compensation between errors and engineered mutations. Wiktor et al. (1984) reported that two cDNA errors caused spurious amino acid substitutions in rabies virus glycoprotein expressed in recombinant vaccinia virus and resulted in expression of non-authentic rabies glycoprotein. After sequence analysis and correction of the cDNA, expression of authentic rabies glycoprotein was obtained. A faulty cDNA clone may behave as expected in one circumstantial context, yet behave very inappropriately and be highly misleading in a different context. A faulty structural gene cDNA clone of the virulent VEE Trinidad donkey (TRD) virus that was expressed in recombinant vaccinia virus was essentially authentic by monoclonal antibody analysis of expressed VEE virus-specific proteins and by protection of immunized mice from challenge with virulent VEE virus (Kinney et al. , 1988a; Kinney et al. , 1988b) . However, incorporation of this cDNA clone into an infectious cDNA clone of VEE virus completely abrogated the virulence of the clone- derived virus, whereas the corrected cDNA fragment resulted in derivation of virulent virus (Kinney et al. , 1993) .

Although Lai et al. (1991) originally derived their infectious clone of DEN-4 virus from sequence characterized subunit cDNA clones (Zhao at el., 1986; Mackow et al. , 1987) , the original full-length clone was not infectious (Lai et al. , 1991) . While these

investigators indicated that they sequenced both strands of much of the cloned genomic cDNA, they did not indicate that they sequenced more than a single clone for a given cDNA region. Nucleotides encoding cDNA errors will be confirmed on both cDNA strands, but will not be identified as errors unless the sequences of two or more independent cDNA clones covering the same region of the genome are sequenced. The functional full-length clone of DEN-4 virus was obtained by repeated splicing of large new cDNA fragments into the full-length clone until a functional clone was obtained. The authors did not indicate that the newly cloned regions were characterized by nucleotide sequence analysis (Lai et al., 1991) . It is probable that the slowed replication of the DEN-4/1 and DEN-4/2 chimeric viruses relative to wild-type viruses reported by Bray et al. (1991) is due to the presence of cDNA artifacts within the full-length cDNA clone. The critical importance of accurate nucleotide sequence characterization of genes expressed in vi tro, particularly when those genes are expressed in the form of infectious cDNA clones, is still not widely appreciated by many in the molecular biology field.

Although putative nucleotide sequences for the genomes of DEN-2 16681 and DEN-2 PDK-53 viruses have been reported in the literature (Blok et al. , 1992) , our sequence results indicate that the published data is highly flawed. Blok et al. (1992) reported 53 nucleotide mutations between the two viruses; we determined only 8 mutations. We analyzed at least two independent cDNA clones for regions covering the entire genomes of both

viruses. The DEN-16681 sequencing project was completed prior to receiving the DEN-2 PDK-53 virus in our laboratory, and the nucleotide sequence of the PDK-53 virus was determined from cDNA amplified directly from virus present in vaccine vials.

There are now only two classes of infectious clones developed for vaccine flaviviruses that have themselves been administered to humans : the infectious clone of yellow fever virus, vaccine strain 17D (Rice et al. , 1989; Hahn et al. , 1987; Rice et al. , 1985) , and the DEN-1, DEN- 2, DEN-3, and DEN-4 vaccine derivative infectious clones described herein. Both classes of infectious clones have the important advantage of being derived from vaccine viruses that have been tested for efficacy and safety in humans. The yellow fever 17D virus vaccine has long been one of the most effective human vaccines developed; immunization with this virus provides lifelong immunity. In the case of DEN virus, it is essential that vaccines provide immunity against infection by all four serotypes of the virus. DEN-1, DEN-2, DEN-3, and DEN-4 vaccine viruses have been developed at Mahidol University, Bangkok, Thailand. All four vaccine viruses have been tested in humans and have been demonstrated to be immunogenic and safe for human adults. Replicating vaccines in the form of live, attenuated viruses offer distinct advantages in terms of immunogenic efficacy due to replicative amplification of viral antigens (antigenic mass) in the vaccinees and replication in appropriate target tissues. Inactivated or subunit antigens usually suffer from a lack of sufficient

antigenic mass and subsequent failure to stimulate an effective immune response. Expression of proteins in recombinant vaccinia virus, which replicates primarily at the site of inoculation, may provide protection against parenteral challenge with virulent virus, but may not protect against an aerosol challenge. This was demonstrated for VEE virus when it was shown .that recombinant vaccinia virus expressing the structural proteins of VEE virus protected mice from intraperitoneal challenge, but not intranasal challenge, with virulent VEE virus (Kinney et al., 1988b). Immunization with the live, attenuated VEE TC-83 vaccine virus, on the other hand, provided immunity against both parenteral challenge (immunity provided by circulating serum IgG antibody) and intranasal challenge (mucosal, IgA-base immunity) with virulent VEE virus. Furthermore, the level of immunity, as measured by titers of VEE virus-specific neutralizing antibody, were considerably higher in TC-83 virus- immunized mice and horses (the natural epidemic host for VEE virus) than in animals immunized with recombinant vaccinia/VEE virus (Kinney et al., 1988b; Bowen et al. , 1992) . Similar results have been reported for vaccinia/influenza A virus recombinants in rodents (Smith et al. , 1986). Furthermore, a replicating vaccine virus provides the appropriate T-cell epitopes to stimulate cell-mediated immunity as well as humoral immunity. T- cell epitopes may be lacking in subunit vaccines. In short, vaccination with a safe live, attenuated vaccine virus provides the optimal immunization of a natural infection in terms of the type and level of immunity

elicited and the repertoire of viral antigens involved in generating the immune response.

To use the DEN viruses described herein as vaccine candidates, it is necessary to rederive the viruses by transfection of a cell line, such as primary dog kidney, certified for human use under conditions of good laboratory practice and management to ensure the avoidance of potential adventitious agents that might be present in uncertified cell lines. Although the cDNA-derived viruses originate from candidate vaccine viruses that have undergone testing in humans, they require recertification by analysis for possible in vi tro phenotypic markers of attenuation and by safety testing in small animals and probably nonhuman primates. All investigative studies involving the pathogenesis of DEN virus are hampered by the unavailability of a suitable animal model. Certain in vi tro characteristics are apparently associated with attenuation of DEN viruses, but the only definitive test is vaccine trial in human volunteers. Vaccine trails would presumably follow those of the original wild-type vaccine viruses developed at Mahidol University. The protocol includes titration of the individual vaccine virus candidates in adult human volunteers to determine the minimal infectious/immunogenic dose for each virus. This is followed by immunization trials with different bivalent and trivalent combinations of vaccine virus. The final test is the quadravalent vaccine composed of appropriate doses of all four vaccine viruses. If the preliminary trials are successful, larger trials are scheduled, and the vaccine viruses are tested in children,

who are the primary target for vaccine delivery.

We describe herein a preferred method to develop an infectious cDNA clone for a flavivirus. Optimally, a wild-type vaccine virus serves as the template for the clone construction. Large cDNA fragments are amplified from the genomic mRNA by PCR using virus-specific primers and directly cloned into a TA-vector or into the MCS of a low-copy number plasmid following restriction of the amplicon cDNA. The low-copy pBRUC-139 vector contains the MCS of pUC19 to permit convenient cloning of cDNA using a variety of RENZ sites. Other low-copy plasmids are available. The bacteriophage T7 or SP6 promoter is usually engineered into the 5'-terminal mRNA-sense amplimer, and a unique RENZ site for linearization of the recombinant plasmid containing the full-length cDNA must be engineered into the 3-terminal complementary (negative) -sense amplimer. Exhaustive nucleotide analysis of the cDNA clones is desirable.

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APPENDIX A

PRIMERS DESIGNED FOR DEN-2 CLONING/SEQUENCING PROJECT;

SEQ.

ID

NO: PRIMER M MEERR//SENSE SEQUENCE

3 pUC/M13-P5 25/+ 5' -CCCAGTCACGACGTTGTAAAACGAC-3 ^•

4 pUC/M13-P5B 27/+ 5' -GGATGTGCTGCAAGGCGATTAAGTTGG-3 '

5 pUC/M13-P3 25/+ 5 ^■ -TGAGCGGATAACAATTTCACACAGG-3 '

6 pUC/M13-P3B 27/- 5' -GGCTTTACACTTTATGCTTCCGGCTCG-3 '

7 D2-1-ECO.T7 75/+ 5' -GCGGATATTG/GAATTC/TCTAGA/

AATTTAATACGACTCACTATA/ AGTTGTTAGTCTACGTGGACCGACAAAGACAG-3 '

(5' -Fill /EcoRI /Xbal/T7 Promoter/ 5"-end of DEN-2)

D2-SMT71 77/+ 5' -CCAGT/GAATTC/GAGCTC/ACGCGT/ AAATTTAATACGACTCACTATA/ AGTTGTTAGTCTACGTGGACCGACAAAGACAG-3 '

(5' -Fill/EcoRI/SstI/MluI/T7 Promoter/ 5'-end of DEN-2)

9 D2-1 24/+ 5' -AGTTGTTAGTCTACGTGGACCGAC-3 '

10 D2-28 34/+ 5' -GACAGATTCTTTGAGGGAGCTGAGCTCAACGTAG-3 '

11 D2-134 28/+ 5 ^■ -TCAATATGCTGAAACGCGAGAGAAACCG-3 '

12 CD2-250 26/- 5' -GGGATTGTTAGGAAACGAAGGAACGC-3 '

13 D2-274 32/+ 5'-CCACCAACAGCAGGGATACTGAAAAGATGGGG-3 '

14 CD2-378 25/- 5'-TGCAGATCTGCGTCTCCTATTCAAG-3 '

15 D2-528 25/+ 5' -CGTGAACATGTGTACCCTCATGGCC-3 '

16 CD2-616 26/- 5'-TTGCACCAACAGTCAATGTCTTCAGG-3 '

17 D2-G16 25/+ 5 ' -ACCAGAAGACATAGATTGTTGGTGC-3 '

18 CD2-618 25/- 5' -GCACCAACAGTCTATGTCTTCTGGC-3 '

19 CD2-771 25/- 5'-ATGTTTCCAGGCCCCTTCTGATGAC-3 '

20 D2-847 25/+ 5' -GCAGCAATCCTGGCATACACCATAG-3 '

21 D2-996 27/+ 5 ' -GGTTGACATAGTCTTAGAACATGGAAG-3 '

22 CD2-996 27/- 5' -CTTCCATGTTCTAAGACTATGTCAACC-3 '

ID

NO : PRIMER MER/SENSE SEQUENCE

23 D2- 1005 35/+ -GTCTTAGAACATGGAAGTTGTGTGACGACGATGGC-3 '

24 D2 - 1141 25/+ -ACAACAGAATCTCGCTGCCCAACAC-3 '

25 D2-1211 25/+ -GCAAACACTCCATGGTAGACAGAGG-3 '

26 CD2 -1211 25/- -CCTCTGTCTACCATGGAGTGTTTGC-3 '

27 CD2 - 1227 27/- -CCACATCCATTTCCCCATCCTCTGTCT-3 '

28 D2-1261 30/+ -GGAAAGGGAGGCATTGTGACCTGTGCTATG-3 '

29 D2 -1416 28/+ -GGAAATCAAAATAACACCACAGAGTTCC-3 '

30 CD2 - 1503 34/- -CTGCAGCAACACCATCTCATTGAAGTCGAGGCCC-3 '

31 D2-1510 25/+ -GACTTCAATGAGATGGTGCTGCTGC-3 '

32 CD2- 1510 25/+ -GCAGCAGCACCATCTCATTGAAGTC-3 '

33 D2 - 1546 28/+ -AAGCTTGGCTGGTGCACAGGCAATGGTT-3'

34 CD2 -1567 27/- -TGGTAACGGCAGGTCTAGGAACCATTG-3 '

35 D2-1777 23/+ -GGACATCTCAAGTGCAGGCTGAG-3 •

36 CD2-1777 23/+ -CTCAGCCTGCACTTGAGATGTCC-3 '

37 D2- 1863 27/+ -GAAGGAAATAGCAGAAACACAACATGG-3 '

38 CD2- 1888 33/- -CCCTTCATATTGTACTCTGATAACTATTGTTCC-3 '

39 D2 -2047 32/+ -CCTCCATTCGGAGACAGCTACATCATCATAGG-3 '

40 CD2 -2047 32/- -CCTATGATGATGTAGCTGTCTCCGAATGGAGG-3 '

41 D2-2170 29/+ -ATGGCCATTTTAGGTGACACAGCCTGGGA-3 '

42 CD2-2200 27/ - -TGTAAACACTCCTCCCAGGGATCCAAA-3'

43 D2 -2308 29/+ -CTCATAGGAGTCATTATCACATGGATAGG-3 '

44 CD2 -2504 35/- -GGGGATTCTGGTTGGAACTTATATTGTTCTGTCC-3'

45 CD2 -2622 30/- -TGATTCAATTCTGGTGTTATTTGTTTCCAC-3 '

46 D2- 2702 25/+ -AAGGAATCATGCAGGCAGGAAAACG-3 '

47 CD2 -2864 22/- -ACTTCCASCGAGTTCCAAGCTC-3 ' A A

48 D2 -2992 25/+ -AACAGAGCCGTCCATGCCGATATGG-3 '

49 CD2 -3105 22/- -TCCATTGCTCCAAAGGGTGTGT-3' G

50 D2-3236 25/π -AGCTTGAGATGGACTTTGATTTCTG-3 '

ID

NO: PRIMER MER/SENSE SEQUENCE

51 CD2-3410 22/- 5' -GGTCTGATTTCCATCCCGTACC-3 '

52 D2-3621 23/+ 5' -GTCCTTTAGAGACCTGGGAAGAG-3 '

53 CD2-3739 25/- 5' -GITTTCTCAAGAGTAGTCCAGCTGC-3 • C

54 D2-3905 25/+ 5' -ATCAATTGGCAGTGACTATCATGGC-3 '

55 CD2-4002 25/- 5' -TGTTAAGAG.CAGTGGAGAAACGGAC-3 ' A G

56 CD2-4060 25/- 5' -GATTGAGACCTTTGATCGTCAACGC-3 '

57 D2-4214 25/+ 5' -TGACAGGACCATTAGTGGCTGGAGG-3'

58 D2-4257 34/+ 5' -CGTGCTCACTGGACGATCGGCCGATTTGGAACTG-3 '

59 CD2-4323 24/- 5' -GGGCTGCTTCCTGATAT1TCTGCC-3 ' C

60 D2-4497 25/+ 5 ¹ -CCTGTGGGAAGTGAAGAAACAACGG-3 '

61 CD2-4557 30/- 5' -GCTCCATCTTCCAGTTCAGCCTTTCCCATG-3 '

62 CD2-4615 25/- 5' -CTCCGGCTCCAATCTGAGAGTATCC-3 ' G G A

63 D2-4746 25/+ 5' -CCTAATATCATATGGAGGAGGCTGG-3 '

64 D2-4792 25/+ 5' -GAAGGAGAAGAAGTCCAGGTATTGG-3 '

65 CD2-4922 25/- 5' -£TGTCGACAATTGGAGATCCTGACG-3 ' T T

66 D2- -4994 25/+ 5' -GTGGAGCATATGTGAGTGCTATAGC-3 •

67 D2- -5124 25/+ 5 ¹ -TCTGACTATGGCCGGAAGGTATCTC-3 '

68 D2- -5173 25/+ 5' -ACATTAATCTTGGCCCCCACTAGAG-3 '

69 cD2- -5272 19/- 5' -CGATCTCCCGCCCGGTGTG-3 ' A

70 cD2- -5318 25/- 5" -CTAACTGGTGATAGCAGCCTCATGG-3 ^■

71 cD2- -5656 27/- 5' -CCTACTGAGTTGTATCACTTTCTTTCC-3 '

72 cD2 -5891 26/- 5' -TGGATTTCTTCCTATTCTCCCTCTTC-3 '

73 D2- -5770 25/+ 5' -TTCAAGGCTGAGAGGGTTATAGACC-3 '

74 D2 -6152 25/+ 5' -TCTGGTTGGCCTACAGAGTGGCAGC-3 '

75 cD2- -6252 27/- 5' -CCTTCTTTTGTCCAGATTTCCACTTCC-3 '

A

SEQ. 103

ID

NO: PRIMER MER/SENSE SEQUENCE

76 D2-6493 35/+ 5' -GCGTACAACCATGCTCTCAGTGAACTGCCGGAGAC-3 '

77 CD2-6605 24/- 5' -TTCCCAGGGTCATCTTCCCTATAC-3 '

G

78 CD2-6624 31/- 5' -GATGCTAGCCGTGATTATGCAGCACATTCCC-3 '

79 D2-6748 25/+ 5' -AAACAGAGAACACCCCAAGACAACC-3 '

80 CD2-6932 21/- 5' -CGGCATACAGCGTCCATGCTG-3 '

81 D2-7055 25/+ 5' -GTCTCGGGAAAGGATGGCCATTGTC-3 '

82 CD2-7195 25/- 5' -CTCTGGTTGCTTTTGCTTGAAGTCC-3 ' A G G

83 CD2-7217 27/- 5' -CCGCCGCTGCTCTTTTCTGAGCTTCTC-3 '

84 D2-7378 25/+ 5' -AGGACTACATGGGCTCTGTGTGAGG-3 '

85 CD2-7515 19/- 5' -GAGAAGTCCAGCTCCGGCC-3 '

86 D2-7769 25/+ 5' -AGAGAAACATGGTCACACCAGAAGG-3 '

87 CD2-7885 22/- 5' -GTTCTTCGTGTCCTGGTCCTCC-3 '

88 D2-8165 25/+ 5' -GGAAATATGGAGGAGCCTAGTGAGG-3 '

89 CD2-8210 22/- 5' -ACCCAGTACATCTCATGTGTGG-3 '

90 D2-8428 28/+ 5' -GAGCATGAAACATCATGGCACTATGACC-3 '

91 D2-8440 25/+ 5' -TCATGGCACTATGACCAAGACCACC-3 '

92 CD2-8529 22/- 5' -CAGlCTGA£CACTCCGTTCACC-3 ' C A G

93 D2-8773 25/+ 5' -AAGGTGAGAAGCAATGCAGCCTTGG-3 '

94 D2-8798 29/+ 5' -GGGCCATATTCACTGATGAGAACAAGTGG-3 ^•

95 CD2-8865 22/- 5' -2CTTTCCCTGTCAACCAGCTCC-3' C T

96 D2-9046 25/+ 5' -AATGAAGATCACTGGTTCTCCAGAG-3 '

97 D2-9131 25/+ 5' -ACGTGAGCAAGAAAGAGGGAGGAGC-3 '

98 CD2-9166 22/- 5' -TGTCCCATCCTGCTGTGTCATC-3 ' A G

99 CD2-9234 30/- 5' -GCTAGTTTCTTGTGTTCTCCTTCCATGTGG-3 '

100 D2-9344 25/+ 5' -TCATATCGAGAAGAGACCAAAGAGG-3 '

101 CD2-9429 24/- 5' -ACTCCTTCTCCCTCCATCTGTCTG-3 '

ID

NO: PRIMER MER/ SEQUENCE

102 CD2-9438 27/- 5' -ATGCTTTTGAAGATTCCTTCTCCCTCC-3 ' A C

103 CD2-9468 32/- 5' -GCACAGCGATTTCTTCTGTGATTGTTAGGTGC-3 '

104 D2-9645 25/+ 5 ^■ -ACAATGGGAACCTTCAAGAGGATGG-3 '

105 D2-9656.BAM 45/+ 5'-TTATCACATT/GGATCC/TTCAAGAGGATGGA ATGATTGGACACAAG-3 '

(5'-Fill/BamHI/DEN-2 Sequence)

106 CD2-9668 28/- 5' -CAGAAGGGCACTTGTGTCCAATCATTCC-3 '

107 CD2-9779 21/- 5'-CTCCCTGGGAAATTCGGGCTC-3 ' T G

108 CD2-9796 28/- 5' -CCGTCTCCCGCAAAGACCACCCTGCTCC-3 '

109 CD2-9796.XBA 44/- 5 ^■ -TTATCACCTA/TCTAGA/CCGTCTCCC GCAAAGACCACCCTGCTCC-3 '

110 CD2-9913 26/- 5'-GTTGGAACCCAATGTGATGGTACTGC-3 '

111 D2-9937 25/+ 5' -ACAAGTCGAACAACCTGGTCCATAC-3 ^•

112 CD2-9977 21/- 5' -GCATGTCTTCCGTCGTCATCC-3 ' T

113 CD2-10003 25/- 5' -CTTGAATCCACACCCTGTTCCAGAC-3 '

114 D2-10203 25/+ 5 ^■ -ATACACAGATTACATGCCATCCATG-3 '

115 CD2-10261 21/- 5 ⁱ -TTTTGC£TTCTACCACAGGAC-3 ' T A

116 D2-10289 25/- 5' -GAAACAAGGCTAGAAGTCAGGTCGG-3 '

117 CD2-10337 23/- 5' -GACGGGGCTCACAGGTAGCATAG-3 '

118 D2-10418 25/+ 5' -GCCTGTAGCTCCACCTGAGAAGGTG-3 '

119 D2-10470 25/+ 5' -GGAAGCTGTACGCATGGCGTAGTGG-3 '

120 CD2-10530 19/- 5' -GGGCCCCCGTTGTTGCTGC-3 ' A

121 CD2-10687 59/- 5'-AGAACCTGTTGATTCAACAGCACCATTCCATTTTCTG-3 '

122 CD2-10687.XBA 59/- 5' -TTATCACCTA/GCATGC/TCTAGA/

AGAACCTGTTGATTCAACAGCACCATTCCATTTTCTG-3 '

(5' -Fill/SphI/Xbal/ 3 '-End DEN-2 Sequence)

123 CD2-10687 .X2 52/- 5'-TTATCACCTA/TCTAGA/

GAACCTGTTGATTCAACAGCACCATTCCATTTTCTG-3 '

(5'-Fill/XbaI/ 3 ' -End DEN-2 Sequence)

While particular embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that these embodiments are exemplary rather than limiting, and the true scope of the invention is that defined within the attached claims.

SEQUENCE LISTING

(1 ) GENERAL INFORMATION

(i) APPLICANT: MAHIDOL UNIVERSITY Bangkok, Thailand

The United States of

America, as represented by the Secretary,

Department of Health and Human Services c/o Centers for Disease Control and

Prevention

Technology Transfer Office

Mail Stop E-67

1600 Clifton Road

Atlanta, Georgia 30333

(ii) TITLE OF THE INVENTION: INFECTIOUS CDNA CLONES FOR DENGUE 2 VIRUS ...

(iii) NUMBER OF SEQUENCES: 137

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: NEEDLE & ROSENBERG, P.C.

(B) STREET: Suite 1200, 127 Peachtree Street, NE

(C) CITY: Atlanta

(D) STATE: GA

(E) COUNTRY: USA

(F) ZIP: 30303

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 1.5

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: U.S. Serial No. 08/483,292

(B) FILING DATE: 7 Jun 1995

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Spratt, Gwendolyn D.

(B) REGISTRATION NUMBER: 36,016

(C) REFERENCE/DOCKET NUMBER: 14114.0179/P

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 404-688-0770

(B) TELEFAX: 404-688-9880

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10723 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 97...10269 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

AGTTGTTAGT CTACGTGGAC CGACAAAGAC AGATTCTTTG AGGGAGCTAA GCTCAACGTA 60 GTTCTAACAG TTTTTTAATT AGAGAGCAGA TCTCTG ATG AAT AAC CAA CGG AAA 114

Met Asn Asn Gin Arg Lys 1 5

AAG GCG AAA AAC ACG CCT TTC AAT ATG CTG AAA CGC GAG AGA AAC CGC 162 Lys Ala Lys Asn Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn Arg 10 15 20

GTG TCG ACT GTG CAA CAG CTG ACA AAG AGA TTC TCA CTT GGA ATG CTG 210 Val Ser Thr Val Gin Gin Leu Thr Lys Arg Phe Ser Leu Gly Met Leu 25 30 35

CAG GGA CGA GGA CCA TTA AAA CTG TTC ATG GCC CTG GTG GCG TTC CTT 258 Gin Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe Leu 40 45 50

CGT TTC CTA ACA ATC CCA CCA ACA GCA GGG ATA TTG AAG AGA TGG GGA 306 Arg Phe Leu Thr lie Pro Pro Thr Ala Gly lie Leu Lys Arg Trp Gly 55 60 65 70

ACA ATT AAA AAA TCA AAA GCT ATT AAT GTT TTG AGA GGG TTC AGG AAA 354 Thr lie Lys Lys Ser Lys Ala lie Asn Val Leu Arg Gly Phe Arg Lys 75 80 85

GAG ATT GGA AGG ATG CTG AAC ATC TTG AAT AGG AGA CGC AGA TCT GCA 402 Glu lie Gly Arg Met Leu Asn lie Leu Asn Arg Arg Arg Arg Ser Ala 90 95 100

GGC ATG ATC ATT ATG CTG ATT CCA ACA GTG ATG GCG TTC CAT TTA ACC 450 Gly Met lie He Met Leu He Pro Thr Val Met Ala Phe His Leu Thr 105 110 115

ACA CGT AAC GGA GAA CCA CAC ATG ATC GTC AGC AGA CAA GAG AAA GGG 498 Thr Arg Asn Gly Glu Pro His Met He Val Ser Arg Gin Glu Lys Gly 120 125 130

AAA AGT CTT CTG TTT AAA ACA GAG GAT GGC GTG AAC ATG TGT ACC CTC 546 Lys Ser Leu Leu Phe Lys Thr Glu Asp Gly Val Asn Met Cys Thr Leu 135 140 145 150

ATG GCC ATG GAC CTT GGT GAA TTG TGT GAA GAC ACA ATC ACG TAC AAG 594 Met Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr He Thr Tyr Lys 155 160 165

TGT CCC CTT CTC AGG CAG AAT GAG CCA GAA GAC ATA GAC TGT TGG TGC 642 Cys Pro Leu Leu Arg Gin Asn Glu Pro Glu Asp He Asp Cys Trp Cys 170 175 180

AAC TCT ACG TCC ACG TGG GTA ACT TAT GGG ACG TGT ACC ACC ATG GGA 690 Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Met Gly 185 190 195

GAA CAT AGA AGA GAA AAA AGA TCA GTG GCA CTC GTT CCA CAT GTG GGA 738 Glu His Arg Arg Glu Lys Arg Ser Val Ala Leu Val Pro His Val Gly 200 205 210

ATG GGA CTG GAG ACA CGA ACT GAA ACA TGG ATG TCA TCA GAA GGG GCC 786 Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala 215 220 225 230

TGG AAA CAT GTC CAG AGA ATT GAA ACT TGG ATC TTG AGA CAT CCA GGC 834 Trp Lys His Val Gin Arg He Glu Thr Trp He Leu Arg His Pro Gly 235 240 245

TTC ACC ATG ATG GCA GCA ATC CTG GCA TAC ACC ATA GGA ACG ACA CAT 882 Phe Thr Met Met Ala Ala He Leu Ala Tyr Thr He Gly Thr Thr His 250 255 260

TTC CAA AGA GCC CTG ATT TTC ATC TTA CTG ACA GCT GTC ACT CCT TCA 930 Phe Gin Arg Ala Leu He Phe He Leu Leu Thr Ala Val Thr Pro Ser 265 270 275

ATG ACA ATG CGT TGC ATA GGA ATG TCA AAT AGA GAC TTT GTG GAA GGG 978 Met Thr Met Arg Cys He Gly Met Ser Asn Arg Asp Phe Val Glu Gly 280 285 290

GTT TCA GGA GGA AGC TGG GTT GAC ATA GTC TTA GAA CAT GGA AGC TGT 1026 Val Ser Gly Gly Ser Trp Val Asp He Val Leu Glu His Gly Ser Cys 295 300 305 310

GTG ACG ACG ATG GCA AAA AAC AAA CCA ACA TTG GAT TTT GAA CTG ATA 1074 Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Glu Leu He 315 320 325

AAA ACA GAA GCC AAA CAG CCT GCC ACC CTA AGG AAG TAC TGT ATA GAG 1122 Lys Thr Glu Ala Lys Gin Pro Ala Thr Leu Arg Lys Tyr Cys He Glu 330 335 340

GCA AAG CTA ACC AAC ACA ACA ACA GAA TCT CGC TGC CCA ACA CAA GGG 1170 Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser Arg Cys Pro Thr Gin Gly 345 350 355

GAA CCC AGC CTA AAT GAA GAG CAG GAC AAA AGG TTC GTC TGC AAA CAC 1218 Glu Pro Ser Leu Asn Glu Glu Gin Asp Lys Arg Phe Val Cys Lys His 360 365 370

TCC ATG GTA GAC AGA GGA TGG GGA AAT GGA TGT GGA CTA TTT GGA AAG 1266 Ser Met Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 375 380 385 390

GGA GGC ATT GTG ACC TGT GCT ATG TTC AGA TGC AAA AAG AAC ATG GAA 1314 Gly Gly He Val Thr Cys Ala Met Phe Arg Cys Lys Lys Asn Met Glu 395 400 405

GGA AAA GTT GTG CAA CCA GAA AAC TTG GAA TAC ACC ATT GTG ATA ACA 1362 Gly Lys Val Val Gin Pro Glu Asn Leu Glu Tyr Thr He Val He Thr 410 415 420

CCT CAC TCA GGG GAA GAG CAT GCA GTC GGA AAT GAC ACA GGA AAA CAT 1410 Pro His Ser Gly Glu Glu His Ala Val Gly Asn Asp Thr Gly Lys His 425 430 435

GGC AAG GAA ATC AAA ATA ACA CCA CAG AGT TCC ATC ACA GAA GCA GAA 1458 Gly Lys Glu He Lys He Thr Pro Gin Ser Ser He Thr Glu Ala Glu 440 445 450

TTG ACA GGT TAT GGC ACT GTC ACA ATG GAG TGC TCT CCA AGA ACG GGC 1506 Leu Thr Gly Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Arg Thr Gly 455 460 465 470

CTC GAC TTC AAT GAG ATG GTG TTG CTG CAG ATG GAA AAT AAA GCT TGG 1554 Leu Asp Phe Asn Glu Met Val Leu Leu Gin Met Glu Asn Lys Ala Trp 475 480 485

CTG GTG CAC AGG CAA TGG TTC CTA GAC CTG CCG TTA CCA TGG TTG CCC 1602 Leu Val His Arg Gin Trp Phe Leu Asp Leu Pro Leu Pro Trp Leu Pro 490 495 500

GGA GCG GAC ACA CAA GGG TCA AAT TGG ATA CAG AAA GAG ACA TTG GTC 1650 Gly Ala Asp Thr Gin Gly Ser Asn Trp He Gin Lys Glu Thr Leu Val 505 510 515

ACT TTC AAA AAT CCC CAT GCG AAG AAA CAG GAT GTT GTT GTT TTA GGA 1698 Thr Phe Lys Asn Pro His Ala Lys Lys Gin Asp Val Val Val Leu Gly 520 525 5.30

TCC CAA GAA GGG GCC ATG CAC ACA GCA CTT ACA GGG GCC ACA GAA ATC 1746 Ser Gin Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu He 535 540 545 550

CAA ATG TCA TCA GGA AAC TTA CTC TTC ACA GGA CAT CTC AAG TGC AGG 1794 Gin Met Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys Arg 555 560 565

CTG AGA ATG GAC AAG CTA CAG CTC AAA GGA ATG TCA TAC TCT ATG TGC 1842 Leu Arg Met Asp Lys Leu Gin Leu Lys Gly Met Ser Tyr Ser Met Cys 570 575 580

ACA GGA AAG TTT AAA GTT GTG AAG GAA ATA GCA GAA ACA CAA CAT GGA 1890 Thr Gly Lys Phe Lys Val Val Lys Glu He Ala Glu Thr Gin His Gly 585 590 595

ACA ATA GTT ATC AGA GTG CAA TAT GAA GGG GAC GGC TCT CCA TGC AAG 1938 Thr He Val He Arg Val Gin Tyr Glu Gly Asp Gly Ser Pro Cys Lys 600 605 610

ATC CCT TTT GAG ATA ATG GAT TTG GAA AAA AGA CAT GTC TTA GGT CGC 1986 He Pro Phe Glu He Met Asp Leu Glu Lys Arg His Val Leu Gly Arg 615 620 625 630

CTG ATT ACA GTC AAC CCA ATT GTG ACA GAA AAA GAT AGC CCA GTC AAC 2034 Leu He Thr Val Asn Pro He Val Thr Glu Lys Asp Ser Pro Val Asn 635 640 645

ATA GAA GCA GAA CCT CCA TTC GGA GAC AGC TAC ATC ATC ATA GGA GTA 2082 He Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr He He He Gly Val 650 655 660

GAG CCG GGA CAA CTG AAG CTC AAC TGG TTT AAG AAA GGA AGT TCT ATC 2130 Glu Pro Gly Gin Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser He 665 670 675

GGC CAA ATG TTT GAG ACA ACA ATG AGG GGG GCG AAG AGA ATG GCC ATT 2178 Gly Gin Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala He 680 685 690

TTA GGT GAC ACA GCC TGG GAT TTT GGA TCC TTG GGA GGA GTG TTT ACA 2226 Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly Val Phe Thr 695 700 705 710

TCT ATA GGA AAG GCT CTC CAC CAA GTC TTT GGA GCA ATC TAT GGA GCT 2274 Ser He Gly Lys Ala Leu His Gin Val Phe Gly Ala He Tyr Gly Ala 715 720 725

GCC TTC AGT GGG GTT TCA TGG ACT ATG AAA ATC CTC ATA GGA GTC ATT 2322 Ala Phe Ser Gly Val Ser Trp Thr Met Lys He Leu He Gly Val He 730 735 740

ATC ACA TGG ATA GGA ATG AAT TCA CGC AGC ACC TCA CTG TCT GTG ACA 2370 He Thr Trp He Gly Met Asn Ser Arg Ser Thr Ser Leu Ser Val Thr 745 750 755

CTA GTA TTG GTG GGA ATT GTG ACA CTG TAT TTG GGA GTC ATG GTG CAG 2418 Leu Val Leu Val Gly He Val Thr Leu Tyr Leu Gly Val Met Val Gin 760 765 770

GCC GAT AGT GGT TGC GTT GTG AGC TGG AAA AAC AAA GAA CTG AAA TGT 2466 Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys 775 780 785 790

GGC AGT GGG ATT TTC ATC ACA GAC AAC GTG CAC ACA TGG ACA GAA CAA 2514 Gly Ser Gly He Phe He Thr Asp Asn Val His Thr Trp Thr Glu Gin 795 800 805

TAC AAG TTC CAA CCA GAA TCC CCT TCA AAA CTA GCT TCA GCT ATC CAG 2562 Tyr Lys Phe Gin Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala He Gin 810 815 820

AAA GCC CAT GAA GAG GGC ATT TGT GGA ATC CGC TCA GTA ACA AGA CTG 2610 Lys Ala His Glu Glu Gly He Cys Gly He Arg Ser Val Thr Arg Leu 825 830 835

GAG AAT CTG ATG TGG AAA CAA ATA ACA CCA GAA TTG AAT CAC ATT CTA 2658

Glu Asn Leu Met Trp Lys Gin He Thr Pro Glu Leu Asn His He Leu

840 845 850

TCA GAA AAT GAG GTG AAG TTA ACT ATT ATG ACA GGA GAC ATC AAA GGA 2706

Ser Glu Asn Glu Val Lys Leu Thr He Met Thr Gly Asp lie Lys Gly

855 860 865 870

ATC ATG CAG GCA GGA AAA CGA TCT CTG CGG CCT CAG CCC ACT GAG CTG 2754

He Met Gin Ala Gly Lys Arg Ser Leu Arg Pro Gin Pro Thr Glu Leu

875 880 885

AAG TAT TCA TGG AAA ACA TGG GGC AAA GCA AAA ATG CTC TCT ACA GAG 2802

Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu

890 895 900

TCT CAT AAC CAG ACC TTT CTC ATT GAT GGC CCC GAA ACA GCA GAA TGC 2850

Ser His Asn Gin Thr Phe Leu He Asp Gly Pro Glu Thr Ala Glu Cys

905 910 915

CCC AAC ACA AAT AGA GCT TGG AAT TCG TTG GAA GTT GAA GAC TAT GGC 2898

Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly

920 925 930

TTT GGA GTA TTC ACC ACC AAT ATA TGG CTA AAA TTG AAA GAA AAA CAG 2946

Phe Gly Val Phe Thr Thr Asn He Trp Leu Lys Leu Lys Glu Lys Gin

935 940 945 950

GAT GTA TTC TGC GAC TCA AAA CTC ATG TCA GCG GCC ATA AAA GAC AAC 2994

Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala He Lys Asp Asn

955 960 965

AGA GCC GTC CAT GCC GAT ATG GGT TAT TGG ATA GAA AGT GCA CTC AAT 3042

Arg Ala Val His Ala Asp Met Gly Tyr Trp He Glu Ser Ala Leu Asn

970 975 980

GAC ACA TGG AAG ATA GAG AAA GCC TCT TTC ATT GAA GTT AAA AAC TGC 3090

Asp Thr Trp Lys He Glu Lys Ala Ser Phe He Glu Val Lys Asn Cys

985 990 995

CAC TGG CCA AAA TCA CAC ACC CTC TGG AGC AAT GGA GTG CTA GAA AGT 3138

His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser

1000 1005 1010

GAG ATG ATA ATT CCA AAG AAT CTC GCT GGA CCA GTG TCT CAA CAC AAC 3186

Glu Met He He Pro Lys Asn Leu Ala Gly Pro Val Ser Gin His Asn

1015 1020 1025 1030

TAT AGA CCA GGC TAC CAT ACA CAA ATA ACA GGA CCA TGG CAT CTA GGT 3234

Tyr Arg Pro Gly Tyr His Thr Gin He Thr Gly Pro Trp His Leu Gly

1035 1040 1045

AAG CTT GAG ATG GAC TTT GAT TTC TGT GAT GGA ACA ACA GTG GTA GTG 3282

Lys Leu Glu Met Asp Phe Asp Phe Cys Asp Gly Thr Thr Val Val Val

1050 1055 1060

ACT GAG GAC TGC GGA AAT AGA GGA CCC TCT TTG AGA ACA ACC ACT GCC 3330

Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala

1065 1070 1075

TCT GGA AAA CTC ATA ACA GAA TGG TGC TGC CGA TCT TGC ACA TTA CCA 3378 Ser Gly Lys Leu He Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro 1080 1085 1090

CCG CTA AGA TAC AGA GGT GAG GAT GGG TGC TGG TAC GGG ATG GAA ATC 3426 Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu He 1095 1100 1105 1110

AGA CCA TTG AAG GAG AAA GAA GAG AAT TTG GTC AAC TCC TTG GTC ACA 3474 Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr 1115 1120 1125

GCT GGA CAT GGG CAG GTC GAC AAC TTT TCA CTA GGA GTC TTG GGA ATG 3522 Ala Gly His Gly Gin Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met 1130 1135 1140

GCA TTG TTC CTG GAG GAA ATG CTT AGG ACC CGA GTA GGA ACG AAA CAT 3570 Ala Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His 1145 1150 1155

GCA ATA CTA CTA GTT GCA GTT TCT TTT GTG ACA TTG ATC ACA GGG AAC 3618 Ala He Leu Leu Val Ala Val Ser Phe Val Thr Leu He Thr Gly Asn 1160 1165 1170

ATG TCC TTT AGA GAC CTG GGA AGA GTG ATG GTT ATG GTA GGC GCC ACT 3666 Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly Ala Thr 1175 1180 1185 1190

ATG ACG GAT GAC ATA GGT ATG GGC GTG ACT TAT CTT GCC CTA CTA GCA 3714 Met Thr Asp Asp He Gly Met Gly Val Thr Tyr Leu Ala Leu Leu Ala 1195 1200 1205

GCC TTC AAA GTC AGA CCA ACT TTT GCA GCT GGA CTA CTC TTG AGA AAG 3762 Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu Leu Leu Arg Lys 1210 1215 1220

CTG ACC TCC AAG GAA TTG ATG ATG ACT ACT ATA GGA ATT GTA CTC CTC 3810 Leu Thr Ser Lys Glu Leu Met Met Thr Thr He Gly He Val Leu Leu 1225 1230 1235

TCC CAG AGC ACC ATA CCA GAG ACC ATT CTT GAG TTG ACT GAT GCG TTA 3858 Ser Gin Ser Thr He Pro Glu Thr He Leu Glu Leu Thr Asp Ala Leu 1240 1245 1250

GCC TTA GGC ATG ATG GTC CTC AAA ATG GTG AGA AAT ATG GAA AAG TAT 3906 Ala Leu Gly Met Met Val Leu Lys Met Val Arg Asn Met Glu Lys Tyr 1255 1260 1265 1270

CAA TTG GCA GTG ACT ATC ATG GCT ATC TTG TGC GTC CCA AAC GCA GTG 3954 Gin Leu Ala Val Thr He Met Ala He Leu Cys Val Pro Asn Ala Val 1275 1280 1285

ATA TTA CAA AAC GCA TGG AAA GTG AGT TGC ACA ATA TTG GCA GTG GTG 4002 He Leu Gin Asn Ala Trp Lys Val Ser Cys Thr He Leu Ala Val Val 1290 1295 1300

TCC GTT TCC CCA CTG CTC TTA ACA TCC TCA CAG CAA AAA ACA GAT TGG 4050 Ser Val Ser Pro Leu Leu Leu Thr Ser Ser Gin Gin Lys Thr Asp Trp 1305 1310 1315

ATA CCA TTA GCA TTG ACG ATC AAA GGT CTC AAT CCA ACA GCT ATT TTT 4098 He Pro Leu Ala Leu Thr He Lys Gly Leu Asn Pro Thr Ala He Phe 1320 1325 1330

CTA ACA ACC CTC TCA AGA ACC AGC AAG AAA AGG AGC TGG CCA TTA AAT 4146 Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn 1335 1340 1345 1350

GAG GCT ATC ATG GCA GTC GGG ATG GTG AGC ATT TTA GCC AGT TCT CTC 4194 Glu Ala He Met Ala Val Gly Met Val Ser He Leu Ala Ser Ser Leu 1355 1360 1365

CTA AAA AAT GAT ATT CCC ATG ACA GGA CCA TTA GTG GCT GGA GGG CTC 4242 Leu Lys Asn Asp He Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu 1370 1375 1380

CTC ACT GTG TGC TAC GTG CTC ACT GGA CGA TCG GCC GAT TTG GAA CTG 4290 Leu Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu 1385 1390 1395

GAG AGA GCA GCC GAT GTC AAA TGG GAA GAC CAG GCA GAG ATA TCA GGA 4338 Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gin Ala Glu He Ser Gly 1400 1405 1410

AGC AGT CCA ATC CTG TCA ATA ACA ATA TCA GAA GAT GGT AGC ATG TCG 4386 Ser Ser Pro He Leu Ser He Thr He Ser Glu Asp Gly Ser Met Ser 1415 1420 1425 1430

ATA AAA AAT GAA GAG GAA GAA CAA ACA CTG ACC ATA CTC ATT AGA ACA 4434 He Lys Asn Glu Glu Glu Glu Gin Thr Leu Thr He Leu He Arg Thr 1435 1440 1445

GGA TTG CTG GTG ATC TCA GGA CTT TTT CCT GTA TCA ATA CCA ATC ACG 4482 Gly Leu Leu Val He Ser Gly Leu Phe Pro Val Ser He Pro He Thr 1450 1455 1460

GCA GCA GCA TGG TAC CTG TGG GAA GTG AAG AAA CAA CGG GCC GGA GTA 4530 Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gin Arg Ala Gly Val 1465 1470 1475

TTG TGG GAT GTT CCT TCA CCC CCA CCC ATG GGA AAG GCT GAA CTG GAA 4578 Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys Ala Glu Leu Glu 1480 1485 1490

GAT GGA GCC TAT AGA ATT AAG CAA AAA GGG ATT CTT GGA TAT TCC CAG 4626 Asp Gly Ala Tyr Arg He Lys Gin Lys Gly He Leu Gly Tyr Ser Gin 1495 1500 1505 1510

ATC GGA GCC GGA GTT TAC AAA GAA GGA ACA TTC CAT ACA ATG TGG CAT 4674 He Gly Ala Gly Val Tyr Lys Glu Gly Thr Phe His Thr Met Trp His 1515 1520 1525

GTC ACA CGT GGC GCT GTT CTA ATG CAT AAA GGA AAG AGG ATT GAA CCA 4722 Val Thr Arg Gly Ala Val Leu Met His Lys Gly Lys Arg He Glu Pro 1530 1535 1540

TCA TGG GCG GAC GTC AAG AAA GAC CTA ATA TCA TAT GGA GGA GGC TGG 4770 Ser Trp Ala Asp Val Lys Lys Asp Leu He Ser Tyr Gly Gly Gly Trp 1545 1550 1555

AAG TTA GAA GGA GAA TGG AAG GAA GGA GAA GAA GTC CAG GTA TTG GCA 4818 Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu Glu Val Gin Val Leu Ala 1560 1565 1570

CTG GAG CCT GGA AAA AAT CCA AGA GCC GTC CAA ACG AAA CCT GGT CTT 4866 Leu Glu Pro Gly Lys Asn Pro Arg Ala Val Gin Thr Lys Pro Gly Leu 1575 1580 1585 1590

TTC AAA ACC AAC GCC GGA ACA ATA GGT GCT GTA TCT CTG GAC TTT TCT 4914 Phe Lys Thr Asn Ala Gly Thr He Gly Ala Val Ser Leu Asp Phe Ser 1595 1600 1605

CCT GGA ACG TCA GGA TCT CCA ATT ATC GAC AAA AAA GGA AAA GTT GTG 4962 Pro Gly Thr Ser Gly Ser Pro He He Asp Lys Lys Gly Lys Val Val 1610 1615 1620

GGT CTT TAT GGT AAT GGT GTT GTT ACA AGG AGT GGA GCA TAT GTG AGT 5010 Gly Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser 1625 1630 1635

GCT ATA GCC CAG ACT GAA AAA AGC ATT GAA GAC AAC CCA GAG ATC GAA 5058 Ala He Ala Gin Thr Glu Lys Ser He Glu Asp Asn Pro Glu He Glu 1640 1645 1650

GAT GAC ATT TTC CGA AAG AGA AGA CTG ACC ATC ATG GAC CTC CAC CCA 5106 Asp Asp He Phe Arg Lys Arg Arg Leu Thr He Met Asp Leu His Pro 1655 1660 1665 1670

GGA GCG GGA AAG ACG AAG AGA TAC CTT CCG GCC ATA GTC AGA GAA GCT 5154 Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala He Val Arg Glu Ala 1675 1680 1685

ATA AAA CGG GGT TTG AGA ACA TTA ATC TTG GCC CCC ACT AGA GTT GTG 5202 He Lys Arg Gly Leu Arg Thr Leu He Leu Ala Pro Thr Arg Val Val 1690 1695 1700

GCA GCT GAA ATG GAG GAA GCC CTT AGA GGA CTT CCA ATA AGA TAC CAG 5250 Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro He Arg Tyr Gin 1705 1710 1715

ACC CCA GCC ATC AGA GCT GAG CAC ACC GGG CGG GAG ATT GTG GAC CTA 5298 Thr Pro Ala He Arg Ala Glu His Thr Gly Arg Glu He Val Asp Leu 1720 1725 1730

ATG TGT CAT GCC ACA TTT ACC ATG AGG CTG CTA TCA CCA GTT AGA GTG 5346 Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser Pro Val Arg Val 1735 1740 1745 1750

CCA AAC TAC AAC CTG ATT ATC ATG GAC GAA GCC CAT TTC ACA GAC CCA 5394 Pro Asn Tyr Asn Leu He He Met Asp Glu Ala His Phe Thr Asp Pro 1755 1760 1765

GCA AGT ATA GCA GCT AGA GGA TAC ATC TCA ACT CGA GTG GAG ATG GGT 5442 Ala Ser He Ala Ala Arg Gly Tyr He Ser Thr Arg Val Glu Met Gly 1770 1775 1780

GAG GCA GCT GGG ATT TTT ATG ACA GCC ACT CCC CCG GGA AGC AGA GAC 5490 Glu Ala Ala Gly He Phe Met Thr Ala Thr Pro Pro Gly Ser Arg Asp 1785 1790 1795

CCA TTT CCT CAG AGC AAT GCA CCA ATC ATA GAT GAA GAA AGA GAA ATC 5538 Pro Phe Pro Gin Ser Asn Ala Pro He He Asp Glu Glu Arg Glu He 1800 1805 1810

CCT GAA CGC TCG TGG AAT TCC GGA CAT GAA TGG GTC ACG GAT TTT AAA 5586

Pro Glu Arg Ser Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys

1815 1820 1825 1830

GGG AAG ACT GTT TGG TTC GTT CCA AGT ATA AAA GCA GGA AAT GAT ATA 5634 Gly Lys Thr Val Trp Phe Val Pro Ser He Lys Ala Gly Asn Asp He 1835 1840 1845

GCA GCT TGC CTG AGG AAA AAT GGA AAG AAA GTG ATA CAA CTC AGT AGG 5682 Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys Val He Gin Leu Ser Arg 1850 1855 1860

AAG ACC TTT GAT TCT GAG TAT GTC AAG ACT AGA ACC AAT GAT TGG GAC 5730 Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp 1865 1870 1875

TTC GTG GTT ACA ACT GAC ATT TCA GAA ATG GGT GCC AAT TTC AAG GCT 5778 Phe Val Val Thr Thr Asp He Ser Glu Met Gly Ala Asn Phe Lys Ala 1880 1885 1890

GAG AGG GTT ATA GAC CCC AGA CGC TGC ATG AAA CCA GTC ATA CTA ACA 5826

Glu Arg Val He Asp Pro Arg Arg Cys Met Lys Pro Val He Leu Thr

1895 1900 1905 1910

GAT GGT GAA GAG CGG GTG ATT CTG GCA GGA CCT ATG CCA GTG ACC CAC 5874 Asp Gly Glu Glu Arg Val He Leu Ala Gly Pro Met Pro Val Thr His 1915 1920 1925

TCT AGT GCA GCA CAA AGA AGA GGG AGA ATA GGA AGA AAT CCA AAA AAT 5922 Ser Ser Ala Ala Gin Arg Arg Gly Arg He Gly Arg Asn Pro Lys Asn 1930 1935 1940

GAG AAT GAC CAG TAC ATA TAC ATG GGG GAA CCT CTG GAA AAT GAT GAA 5970 Glu Asn Asp Gin Tyr He Tyr Met Gly Glu Pro Leu Glu Asn Asp Glu 1945 1950 1955

GAC TGT GCA CAC TGG AAA GAA GCT AAA ATG CTC CTA GAT AAC ATC AAC 6018 Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu Asp Asn He Asn 1960 1965 1970

ACG CCA GAA GGA ATC ATT CCT AGC ATG TTC GAA CCA GAG CGT GAA AAG 6066

Thr Pro Glu Gly He He Pro Ser Met Phe Glu Pro Glu Arg Glu Lys

1975 1980 1985 1990

GTG GAT GCC ATT GAT GGC GAA TAC CGC TTG AGA GGA GAA GCA AGG AAA 6114 Val Asp Ala He Asp Gly Glu Tyr Arg Leu Arg Gly Glu Ala Arg Lys 1995 2000 2005

ACC TTT GTA GAC TTA ATG AGA AGA GGA GAC CTA CCA GTC TGG TTG GCC 6162 Thr Phe Val Asp Leu Met Arg Arg Gly Asp Leu Pro Val Trp Leu Ala 2010 2015 2020

TAC AGA GTG GCA GCT GAA GGC ATC AAC TAC GCA GAC AGA AGG TGG TGT 6210 Tyr Arg Val Ala Ala Glu Gly He Asn Tyr Ala Asp Arg Arg Trp Cys 2025 2030 2035

TTT GAT GGA GTC AAG AAC AAC CAA ATC CTA GAA GAA AAC GTG GAA GTT 6258 Phe Asp Gly Val Lys Asn Asn Gin He Leu Glu Glu Asn Val Glu Val 2040 2045 2050

GAA ATC TGG ACA AAA GAA GGG GAA AGG AAG AAA TTG AAA CCC AGA TGG 6306 Glu He Trp Thr Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp 2055 2060 2065 2070

TTG GAT GCT AGG ATC TAT TCT GAC CCA CTG GCG CTA AAA GAA TTT AAG 6354 Leu Asp Ala Arg He Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys 2075 2080 2085

GAA TTT GCA GCC GGA AGA AAG TCT CTG ACC CTG AAC CTA ATC ACA GAA 6402 Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu He Thr Glu 2090 2095 2100

ATG GGT AGG CTC CCA ACC TTC ATG ACT CAG AAG GCA AGA GAC GCA CTG 6450 Met Gly Arg Leu Pro Thr Phe Met Thr Gin Lys Ala Arg Asp Ala Leu 2105 2110 2115

GAC AAC TTA GCA GTG CTG CAC ACG GCT GAG GCA GGT GGA AGG GCG TAC 6498 Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala Tyr 2120 2125 2130

AAC CAT GCT CTC AGT GAA CTG CCG GAG ACC CTG GAG ACA TTG CTT TTA 6546 Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu Leu Leu 2135 2140 2145 2150

CTG ACA CTT CTG GCT ACA GTC ACG GGA GGG ATC TTT TTA TTC TTG ATG 6594 Leu Thr Leu Leu Ala Thr Val Thr Gly Gly He Phe Leu Phe Leu Met 2155 2160 2165

AGC GGA AGG GGC ATA GGG AAG ATG ACC CTG GGA ATG TGC TGC ATA ATC 6642 Ser Gly Arg Gly He Gly Lys Met Thr Leu Gly Met Cys Cys He He 2170 2175 2180

ACG GCT AGC ATC CTC CTA TGG TAC GCA CAA ATA CAG CCA CAC TGG ATA 6690 Thr Ala Ser He Leu Leu Trp Tyr Ala Gin He Gin Pro His Trp He 2185 2190 2195

GCA GCT TCA ATA ATA CTG GAG TT.T TTT CTC ATA GTT TTG CTT ATT CCA 6738 Ala Ala Ser He He Leu Glu Phe Phe Leu He Val Leu Leu He Pro 2200 2205 2210

GAA CCT GAA AAA CAG AGA ACA CCC CAA GAC AAC CAA CTG ACC TAC GTT 6786 Glu Pro Glu Lys Gin Arg Thr Pro Gin Asp Asn Gin Leu Thr Tyr Val 2215 2220 2225 2230

GTC ATA GCC ATC CTC ACA GTG GTG GCC GCA ACC ATG GCA AAC GAG ATG 6834 Val He Ala He Leu Thr Val Val Ala Ala Thr Met Ala Asn Glu Met 2235 2240 2245

GGT TTC CTA GAA AAA ACG AAG AAA GAT CTC GGA TTG GGA AGC ATT GCA 6882 Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu Gly Leu Gly Ser He Ala 2250 2255 2260

ACC CAG CAA CCC GAG AGC AAC ATC CTG GAC ATA GAT CTA CGT CCT GCA 6930 Thr Gin Gin Pro Glu Ser Asn He Leu Asp He Asp Leu Arg Pro Ala 2265 2270 2275

TCA GCA TGG ACG CTG TAT GCC GTG GCC ACA ACA TTT GTT ACA CCA ATG 6978 Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met 2280 2285 2290

TTG AGA CAT AGC ATT GAA AAT TCC TCA GTG AAT GTG TCC CTA ACA GCT 7026

Leu Arg His Ser He Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala

2295 2300 2305 2310

ATA GCC AAC CAA GCC ACA GTG TTA ATG GGT CTC GGG AAA GGA TGG CCA 7074 He Ala Asn Gin Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro 2315 2320 2325

TTG TCA AAG ATG GAC ATC GGA GTT CCC CTT CTC GCC ATT GGA TGC TAC 7122 Leu Ser Lys Met Asp He Gly Val Pro Leu Leu Ala He Gly Cys Tyr 2330 2335 2340

TCA CAA GTC AAC CCC ATA ACT CTC ACA GCA GCT CTT TTC TTA TTG GTA 7170 Ser Gin Val Asn Pro He Thr Leu Thr Ala Ala Leu Phe Leu Leu Val 2345 2350 2355

GCA CAT TAT GCC ATC ATA GGG CCA GGA CTC CAA GCA AAA GCA ACC AGA 7218 Ala His Tyr Ala He He Gly Pro Gly Leu Gin Ala Lys Ala Thr Arg 2360 2365 2370

GAA GCT CAG AAA AGA GCA GCG GCG GGC ATC ATG AAA AAC CCA ACT GTC 7266

Glu Ala Gin Lys Arg Ala Ala Ala Gly He Met Lys Asn Pro Thr Val

2375 2380 2385 2390

GAT GGA ATA ACA GTG ATT GAC CTA GAT CCA ATA CCT TAT GAT CCA AAG 7314 Asp Gly He Thr Val He Asp Leu Asp Pro He Pro Tyr Asp Pro Lys 2395 2400 2405

TTT GAA AAG CAG TTG GGA CAA GTA ATG CTC CTA GTC CTC TGC GTG ACT 7362 Phe Glu Lys Gin Leu Gly Gin Val Met Leu Leu Val Leu Cys Val Thr 2410 2415 2420

CAA GTA TTG ATG ATG AGG ACT ACA TGG GCT CTG TGT GAG GCT TTA ACC 7410 Gin Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys Glu Ala Leu Thr 2425 2430 2435

TTA GCT ACC GGG CCC ATC TCC ACA TTG TGG GAA GGA AAT CCA GGG AGG 7458 Leu Ala Thr Gly Pro He Ser Thr Leu Trp Glu Gly Asn Pro Gly Arg 2440 2445 2450

TTT TGG AAC ACT ACC ATT GCG GTG TCA ATG GCT AAC ATT TTT AGA GGG 7506

Phe Tr Asn Thr Thr He Ala Val Ser Met Ala Asn He Phe Arg Gly

2455 2460 2465 2470

AGT TAC TTG GCC GGA GCT GGA CTT CTC TTT TCT ATT ATG AAG AAC ACA 7554 Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe Ser He Met Lys Asn Thr 2475 2480 2485

ACC AAC ACA AGA AGG GGA ACT GGC AAC ATA GGA GAG ACG CTT GGA GAG 7602 Thr Asn Thr Arg Arg Gly Thr Gly Asn He Gly Glu Thr Leu Gly Glu 2490 2495 2500

AAA TGG AAA AGC CGA TTG AAC GCA TTG GGA AAA AGT GAA TTC CAG ATC 7650 Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly Lys Ser Glu Phe Gin He 2505 2510 2515

TAC AAG AAA AGT GGA ATC CAG GAA GTG GAT AGA ACC TTA GCA AAA GAA 7698 Tyr Lys Lys Ser Gly He Gin Glu Val Asp Arg Thr Leu Ala Lys Glu 2520 2525 2530

GGC ATT AAA AGA GGA GAA ACG GAC CAT CAC GCT GTG TCG CGA GGC TCA 7746

Gly He Lys Arg Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser

2535 2540 2545 2550

GCA AAA CTG AGA TGG TTC GTT GAG AGA AAC ATG GTC ACA CCA GAA GGG 7794 Ala Lys Leu Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly 2555 2560 2565

AAA GTA GTG GAC CTC GGT TGT GGC AGA GGA GGC TGG TCA TAC TAT TGT 7842 Lys Val Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys 2570 2575 2580

GGA GGA CTA AAG AAT GTA AGA GAA GTC AAA GGC CTA ACA AAA GGA GGA 7890 Gly Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly 2585 2590 2595

CCA GGA CAC GAA GAA CCC ATC CCC ATG TCA ACA TAT GGG TGG AAT CTA 7938 Pro Gly His Glu Glu Pro He Pro Met Ser Thr Tyr Gly Trp Asn Leu 2600 2605 2610

GTG CGT CTT CAA AGT GGA GTT GAC GTT TTC TTC ATC CCG CCA GAA AAG 7986

Val Arg Leu Gin Ser Gly Val Asp Val Phe Phe He Pro Pro Glu Lys

2615 2620 2625 2630

TGT GAC ACA TTA TTG TGT GAC ATA GGG GAG TCA TCA CCA AAT CCC ACA 8034 Cys Asp Thr Leu Leu Cys Asp He Gly Glu Ser Ser Pro Asn Pro Thr 2635 2640 2645

GTG GAA GCA GGA CGA ACA CTC AGA GTC CTT AAC TTA GTA GAA AAT TGG 8082 Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu Val Glu Asn Trp 2650 2655 2660

TTG AAC AAC AAC ACT CAA TTT TGC ATA AAG GTT CTC AAC CCA TAT ATG 8130 Leu Asn Asn Asn Thr Gin Phe Cys He Lys Val Leu Asn Pro Tyr Met 2665 2670 2675

CCC TCA GTC ATA GAA AAA ATG GAA GCA CTA CAA AGG AAA TAT GGA GGA 8178 Pro Ser Val He Glu Lys Met Glu Ala Leu Gin Arg Lys Tyr Gly Gly 2680 2685 2690

GCC TTA GTG AGG AAT CCA CTC TCA CGA AAC TCC ACA CAT GAG ATG TAC 8226

Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr

2695 2700 2705 2710

TGG GTA TCC AAT GCT TCC GGG AAC ATA GTG TCA TCA GTG AAC ATG ATT 8274 Trp Val Ser Asn Ala Ser Gly Asn He Val Ser Ser Val Asn Met He 2715 2720 2725

TCA AGG ATG TTG ATC AAC AGA TTT ACA ATG AGA TAC AAG AAA GCC ACT 8322 Ser Arg Met Leu He Asn Arg Phe Thr Met Arg Tyr Lys Lys Ala Thr 2730 2735 2740

TAC GAG CCG GAT GTT GAC CTC GGA AGC GGA ACC CGT AAC ATC GGG ATT 8370 Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly Thr Arg Asn He Gly He 2745 2750 2755

GAA AGT GAG ATA CCA AAC CTA GAT ATA ATT GGG AAA AGA ATA GAA AAA 8418 Glu Ser Glu He Pro Asn Leu Asp He He Gly Lys Arg He Glu Lys 2760 2765 2770

ATA AAG CAA GAG CAT GAA ACA TCA TGG CAC TAT GAC CAA GAC CAC CCA 8466

He Lys Gin Glu His Glu Thr Ser Trp His Tyr Asp Gin Asp His Pro

2775 2780 2785 2790

TAC AAA ACG TGG GCA TAC CAT GGT AGC TAT GAA ACA AAA CAG ACT GGA 8514 Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gin Thr Gly 2795 2800 2805

TCA GCA TCA TCC ATG GTC AAC GGA GTG GTC AGG CTG CTG ACA AAA CCT 8562 Ser Ala Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro 2810 2815 2820

TGG GAC GTC GTC CCC ATG GTG ACA CAG ATG GCA ATG ACA GAC ACG ACT 8610 Trp Asp Val Val Pro Met Val Thr Gin Met Ala Met Thr Asp Thr Thr 2825 2830 2835

CCA TTT GGA CAA CAG CGC GTT TTT AAA GAG AAA GTG GAC ACG AGA ACC 8658 Pro Phe Gly Gin Gin Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr 2840 2845 2850

CAA GAA CCG AAA GAA GGC ACG AAG AAA CTA ATG AAA ATA ACA GCA GAG 8706

Gin Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys He Thr Ala Glu

2855 2860 2865 2870

TGG CTT TGG AAA GAA TTA GGG AAG AAA AAG ACA CCC AGG ATG TGC ACC 8754 Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys Thr 2875 2880 2885

AGA GAA GAA TTC ACA AGA AAG GTG AGA AGC AAT GCA GCC TTG GGG GCC 8802 Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala Ala Leu Gly Ala 2890 2895 2900

ATA TTC ACT GAT GAG AAC AAG TGG AAG TCG GCA CGT GAG GCT GTT GAA 8850 He Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu 2905 2910 2915

GAT AGT AGG TTT TGG GAG CTG GTT GAC AAG GAA AGG AAT CTC CAT CTT 8898 Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Asn Leu His Leu 2920 2925 2930

GAA GGA AAG TGT GAA ACA TGT GTG TAC AAC ATG ATG GGA AAA AGA GAG 8946

Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu

2935 2940 2945 2950

AAG AAG CTA GGG GAA TTC GGC AAG GCA AAA GGC AGC AGA GCC ATA TGG 8994 Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala He Trp 2955 2960 2965

TAC ATG TGG CTT GGA GCA CGC TTC TTA GAG TTT GAA GCC CTA GGA TTC 9042 Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe 2970 2975 2980

TTA AAT GAA GAT CAC TGG TTC TCC AGA GAG AAC TCC CTG AGT GGA GTG 9090 Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly Val 2985 2990 2995

GAA GGA GAA GGG CTG CAC AAG CTA GGT TAC ATT CTA AGA GAC GTG AGC 9138 Glu Gly Glu Gly Leu His Lys Leu Gly Tyr He Leu Arg Asp Val Ser 3000 3005 3010

AAG AAA GAG GGA GGA GCA ATG TAT GCC GAT GAC ACC GCA GGA TGG GAT 9186

Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp

3015 3020 3025 3030

ACA AGA ATC ACA CTA GAA GAC KKA AAA AAT GAA GAA ATG GTA ACA AAC 9234 Thr Arg He Thr Leu Glu Asp Xaa Lys Asn Glu Glu Met Val Thr Asn 3035 3040 3045

CAC ATG GAA GGA GAA CAC AAG AAA CTA GCC GAG GCC ATT TTC AAA CTA 9282 His Met Glu Gly Glu His Lys Lys Leu Ala Glu Ala He Phe Lys Leu 3050 3055 3060

ACG TAC CAA AAC AAG GTG GTG CGT GTG CAA AGA CCA ACA CCA AGA GGC 9330 Thr Tyr Gin Asn Lys Val Val Arg Val Gin Arg Pro Thr Pro Arg Gly 3065 3070 3075

ACA GTA ATG GAC ATC ATA TCG AGA AGA GAC CAA AGA GGT AGT GGA CAA 9378 Thr Val Met Asp He He Ser Arg Arg Asp Gin Arg Gly Ser Gly Gin 3080 3085 3090

GTT GGC ACC TAT GGA CTC AAT ACT TTC ACC AAT ATG GAA GCC CAA CTA 9426

Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gin Leu

3095 3100 3105 3110

ATC AGA CAG ATG GAG GGA GAA GGA GTC TTT AAA AGC ATT CAG CAC CTA 9474 He Arg Gin Met Glu Gly Glu Gly Val Phe Lys Ser He Gin His Leu 3115 3120 3125

ACA ATC ACA GAA GAA ATC GCT GTG CAA AAC TGG TTA GCA AGA GTG GGG 9522 Thr He Thr Glu Glu He Ala Val Gin Asn Trp Leu Ala Arg Val Gly 3130 3135 3140

CGC GAA AGG TTA TCA AGA ATG GCC ATC AGT GGA GAT GAT TGT GTT GTG 9570 Arg Glu Arg Leu Ser Arg Met Ala He Ser Gly Asp Asp Cys Val Val 3145 3150 3155

AAA CCT TTA GAT GAC AGG TTC GCA AGC GCT TTA ACA GCT CTA AAT GAC 9618 Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn Asp 3160 3165 3170

ATG GGA AAG ATT AGG AAA GAC ATA CAA CAA TGG GAA CCT TCA AGA GGA 9666

Met Gly Lys He Arg Lys Asp He Gin Gin Trp Glu Pro Ser Arg Gly

3175 3180 3185 3190

TGG AAT GAT TGG ACA CAA GTG CCC TTC TGT TCA CAC CAT TTC CAT GAG 9714 Trp Asn Asp Trp Thr Gin Val Pro Phe Cys Ser His His Phe His Glu 3195 3200 3205

TTA ATC ATG AAA GAC GGT CGC GTA CTC GTT GTT CCA TGT AGA AAC CAA 9762 Leu He Met Lys Asp Gly Arg Val Leu Val Val Pro Cys Arg Asn Gin 3210 3215 3220

GAT GAA CTG ATT GGC AGA GCC CGA ATC TCC CAA GGA GCA GGG TGG TCT 9810 Asp Glu Leu He Gly Arg Ala Arg He Ser Gin Gly Ala Gly Trp Ser 3225 3230 3235

TTG CGG GAG ACG GCC TGT TTG GGG AAG TCT TAC GCC CAA ATG TGG AGC 9858 Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser Tyr Ala Gin Met Trp Ser 3240 3245 3250

TTG ATG TAC TTC CAC AGA CGC GAC CTC AGG CTG GCG GCA AAT GCT ATT 9906 Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala He 3255 3260 3265 3270

TGC TCG GCA GTA CCA TCA CAT TGG GTT CCA ACA AGT CGA ACA ACC TGG 9954 Cys Ser Ala Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp 3275 3280 3285

TCC ATA CAT GCT AAA CAT GAA TGG ATG ACA ACG GAA GAC ATG CTG ACA 10002 Ser He His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr 3290 3295 3300

GTC TGG AAC AGG GTG TGG ATT CAA GAA AAC CCA TGG ATG GAA GAC AAA 10050 Val Trp Asn Arg Val Trp He Gin Glu Asn Pro Trp Met Glu Asp Lys 3305 3310 3315

ACT CCA GTG GAA TCA TGG GAG GAA ATC CCA TAC TTG GGG AAA AGA GAA 10098 Thr Pro Val Glu Ser Trp Glu Glu He Pro Tyr Leu Gly Lys Arg Glu 3320 3325 3330

GAC CAA TGG TGC GGC TCA TTG ATT GGG TTA ACA AGC AGG GCC ACC TGG 10146 Asp Gin Trp Cys Gly Ser Leu He Gly Leu Thr Ser Arg Ala Thr Trp 3335 3340 3345 3350

GCA AAG AAC ATC CAA GCA GCA ATA AAT CAA GTT AGA TCC CTT ATA GGC 10194 Ala Lys Asn He Gin Ala Ala He Asn Gin Val Arg Ser Leu He Gly 3355 3360 3365

AAT GAA GAA TAC ACA GAT TAC ATG CCA TCC ATG AAA AGA TTC AGA AGA 10242 Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys Arg Phe Arg Arg 3370 3375 3380

GAA GAG GAA GAA GCA GGA GTT CTG TGG TAGAAAGCAA AACTAACATG AAACAAGG 10297 Glu Glu Glu Glu Ala Gly Val Leu Trp

3385 3390

CTAGAAGTCA GGTCGGATTA AGCCATAGTA CGGAAAAAAC TATGCTACCT GTGAGCCCCG 10357

TCCAAGGACG TTAAAAGAAG TCAGGCCATC ATAAATGCCA TAGCTTGAGT AAACTATGCA 10417

GCCTGTAGCT CCACCTGAGA AGGTGTAAAA AATCCGGGAG GCCACAAACC ATGGAAGCTG 10477

TACGCATGGC GTAGTGGACT AGCGGTTAGA GAGGACCCCT CCCTTACAAA TCGCAGCAAC 10537

AATGGGGGCC CAAGGCGAGA TGAAGCTGTA GTCTCGCTGG AAGGACTAGA GGTTAGAGGA 10597

GACCCCCCCG AAACAAAAAA CAGCATATTG ACGCTGGGAA AGACCAGAGA TCCTGCTGTC 10657

TCCTCAGCAT CATTCCAGGC ACAGAACGCC AGAAAATGGA ATGGTGCTGT TGAATCAACA 10717

GGTTCT 10723

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10723 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

( ix ) FEATURE :

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 97...10269 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

AGTTGTTAGT CTACGTGGAC CGACAAAGAC AGATTCTTTG AGGGAGCTAA GCTCAATGTA 60 GTTCTAACAG TTTTTTAATT AGAGAGCAGA TCTCTG ATG AAT AAC CAA CGG AAA 114

Met Asn Asn Gin Arg Lys 1 5

AAG GCG AAA AAC ACG CCT TTC AAT ATG CTG AAA CGC GAG AGA AAC CGC 162 Lys Ala Lys Asn Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn Arg 10 15 20

GTG TCG ACT GTG CAA CAG CTG ACA AAG AGA TTC TCA CTT GGA ATG CTG 210 Val Ser Thr Val Gin Gin Leu Thr Lys Arg Phe Ser Leu Gly Met Leu 25 30 35

CAG GGA CGA GGA CCA TTA AAA CTG TTC ATG GCC CTG GTG GCG TTC CTT 258 Gin Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe Leu 40 45 50

CGT TTC CTA ACA ATC CCA CCA ACA GCA GGG ATA TTG AAG AGA TGG GGA 306 Arg Phe Leu Thr He Pro Pro Thr Ala Gly He Leu Lys Arg Trp Gly 55 60 65 70

ACA ATT AAA AAA TCA AAA GCT ATT AAT GTT TTG AGA GGG TTC AGG AAA 354 Thr He Lys Lys Ser Lys Ala He Asn Val Leu Arg Gly Phe Arg Lys 75 80 85

GAG ATT GGA AGG ATG CTG AAC ATC TTG AAT AGG AGA CGC AGA TCT GCA 402 Glu He Gly Arg Met Leu Asn He Leu Asn Arg Arg Arg Arg Ser Ala 90 95 100

GGC ATG ATC ATT ATG CTG ATT CCA ACA GTG ATG GCG TTC CAT TTA ACC 450 Gly Met He He Met Leu He Pro Thr Val Met Ala Phe His Leu Thr 105 110 115

ACA CGT AAC GGA GAA CCA CAC ATG ATC GTC AGC AGA CAA GAG AAA GGG 498 Thr Arg Asn Gly Glu Pro His Met He Val Ser Arg Gin Glu Lys Gly 120 125 130

AAA AGT CTT CTG TTT AAA ACA GAG GTT GGC GTG AAC ATG TGT ACC CTC 546 Lys Ser Leu Leu Phe Lys Thr Glu Val Gly Val Asn Met Cys Thr Leu 135 140 145 150

ATG GCC ATG GAC CTT GGT GAA TTG TGT GAA GAC ACA ATC ACG TAC AAG 594 Met Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr He Thr Tyr Lys 155 160 165

TGT CCC CTT CTC AGG CAG AAT GAG CCA GAA GAC ATA GAC TGT TGG TGC 642 Cys Pro Leu Leu Arg Gin Asn Glu Pro Glu Asp He Asp Cys Trp Cys 170 175 180

NAC TCT ACG TCC ACG TGG GTA ACT TAT GGG ACG TGT ACC ACC ATG GGA 690 Xaa Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Met Gly 185 190 195

GAA CAT AGA AGA GAA AAA AGA TCA GTG GCA CTC GTT CCA CAT GTG GGA 738 Glu His Arg Arg Glu Lys Arg Ser Val Ala Leu Val Pro His Val Gly 200 205 210

ATG GGA CTG GAG ACA CGA ACT GAA ACA TGG ATG TCA TCA GAA GGG GCC 786 Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala 215 220 225 230

TGG AAA CAT GTC CAG AGA ATT GAA ACT TGG ATC TTG AGA CAT CCA GGC 834 Trp Lys His Val Gin Arg He Glu Thr Trp He Leu Arg His Pro Gly 235 240 245

TTC ACC ATG ATG GCA GCA ATC CTG GCA TAC ACC ATA GGA ACG ACA CAT 882 Phe Thr Met Met Ala Ala He Leu Ala Tyr Thr He Gly Thr Thr His 250 255 260

TTC CAA AGA GCC CTG ATT TTC ATC TTA CTG ACA GCT GTC ACT CCT TCA 930 Phe Gin Arg Ala Leu He Phe He Leu Leu Thr Ala Val Thr Pro Ser 265 270 275

ATG ACA ATG CGT TGC ATA GGA ATG TCA AAT AGA GAC TTT GTG GAA GGG 978 Met Thr Met Arg Cys He Gly Met Ser Asn Arg Asp Phe Val Glu Gly 280 285 290

GTT TCA GGA GGA AGC TGG GTT GAC ATA GTC TTA GAA CAT GGA AGC TGT 1026 Val Ser Gly Gly Ser Trp Val Asp He Val Leu Glu His Gly Ser Cys 295 300 305 310

GTG ACG ACG ATG GCA AAA AAC AAA CCA ACA TTG GAT TTT GAA CTG ATA 1074 Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Glu Leu He 315 320 325

AAA ACA GAA GCC AAA CAG CCT GCC ACC CTA AGG AAG TAC TGT ATA GAG 1122 Lys Thr Glu Ala Lys Gin Pro Ala Thr Leu Arg Lys Tyr Cys He Glu 330 335 340

GCA AAG CTA ACC NAC ACA ACA ACA GAA TCT CGC TGC CCA ACA CAA GGG 1170 Ala Lys Leu Thr Xaa Thr Thr Thr Glu Ser Arg Cys Pro Thr Gin Gly 345 350 355

GAA CCC AGC CTA AAT GAA GAG CAG GAC AAA AGG TTC GTC TGC AAA CAC 1218 Glu Pro Ser Leu Asn Glu Glu Gin Asp Lys Arg Phe Val Cys Lys His 360 365 370

TCC ATG GTA GAC AGA GGA TGG GGA AAT GGA TGT GGA CTA TTT GGA AAG 1266 Ser Met Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 375 380 385 390

GGA GGC ATT GTG ACC TGT GCT ATG TTC AGA TGC AAA AAG AAC ATG GAA 1314 Gly Gly He Val Thr Cys Ala Met Phe Arg Cys Lys Lys Asn Met Glu 395 400 405

GGA AAA GTT GTG CAA CCA GAA AAC TTG GAA TAC ACC ATT GTG ATA ACA 1362 Gly Lys Val Val Gin Pro Glu Asn Leu Glu Tyr Thr He Val He Thr 410 415 420

CCT CAC TCA GGG GAA GAG CAT GCA GTC GGA NAT GAC ACA GGA AAA CAT 1410 Pro His Ser Gly Glu Glu His Ala Val Gly Xaa Asp Thr Gly Lys His 425 430 435

GGC AAG GAA ATC AAA ATA ACA CCA CAG AGT TCC ATC ACA GAA GCA GAA 1458 Gly Lys Glu He Lys He Thr Pro Gin Ser Ser He Thr Glu Ala Glu 440 445 450

TTG ACA GGT TAT GGC ACT GTC ACA ATG GAG TGC TCT CCA AGA ACG GGC 1506 Leu Thr Gly Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Arg Thr Gly 455 460 465 470

CTC GAC TTC AAT GAG ATG GTG TTG CTG CAG ATG GAA AAT AAA GCT TGG 1554 Leu Asp Phe Asn Glu Met Val Leu Leu Gin Met Glu Asn Lys Ala Trp 475 480 485

CTG GTG CAC AGG CAA TGG TTC CTA GAC CTG CCG TTA CCA TGG TTG CCC 1602 Leu Val His Arg Gin Trp Phe Leu Asp Leu Pro Leu Pro Trp Leu Pro 490 495 500

GGA GCG GAC ACA CAA GGG TCA AAT TGG ATA CAG AAA GAG ACA TTG GTC 1650 Gly Ala Asp Thr Gin Gly Ser Asn Trp He Gin Lys Glu Thr Leu Val 505 510 515

ACT TTC AAA AAT CCC CAT GCG AAG AAA CAG GAT GTT GTT GTT TTA GGA 1698 Thr Phe Lys Asn Pro His Ala Lys Lys Gin Asp Val Val Val Leu Gly 520 525 530

TCC CAA GAA GGG GCC ATG CAC ACA GCA CTT ACA GGG GCC ACA GAA ATC 1746 Ser Gin Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu He 535 540 545 550

CAA ATG TCA TCA GGA AAC TTA CTC TTC ACA GGA CAT CTC AAG TGC AGG 1794 Gin Met Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys Arg 555 560 565

CTG AGA ATG GAC AAG CTA CAG CTC AAA GGA ATG TCA TAC TCT ATG TGC 1842 Leu Arg Met Asp Lys Leu Gin Leu Lys Gly Met Ser Tyr Ser Met Cys 570 575 580

ACA GGA AAG TTT AAA GTT GTG AAG GAA ATA GCA GAA ACA CAA CAT GGA 1890 Thr Gly Lys Phe Lys Val Val Lys Glu He Ala Glu Thr Gin His Gly 585 590 595

ACA ATA GTT ATC AGA GTG CAA TAT GAA GGG GAC GGC TCT CCA TGC AAG 1938 Thr He Val He Arg Val Gin Tyr Glu Gly Asp Gly Ser Pro Cys Lys 600 605 610

ATC CCT TTT GAG ATA ATG GAT TTG GAA AAA AGA CAT GTC TTA GGT CGC 1986 He Pro Phe Glu He Met Asp Leu Glu Lys Arg His Val Leu Gly Arg 615 620 625 630

CTG ATT ACA GTC AAC CCA ATT GTG ACA GAA AAA GAT AGC CCA GTC AAC 2034 Leu He Thr Val Asn Pro He Val Thr Glu Lys Asp Ser Pro Val Asn 635 640 645

ATA GAA GCA GAA CCT CCA TTT GGA GAC AGC TAC ATC ATC ATA GGA GTA 2082 He Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr He He He Gly Val 650 655 660

GAG CCG GGA CAA CTG AAG CTC AAC TGG TTT AAG AAA GGA AGT TCT ATC 2130 Glu Pro Gly Gin Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser He 665 670 675

GGC CAA ATG TTT GAG ACA ACA ATG AGG GGG GCG AAG AGA ATG GCC ATT 2178 Gly Gin Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala He 680 685 690

TTA GGT GAC ACA GCC TGG GAT TTT GGA TCC TTG GGA GGA GTG TTT ACA 2226 Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly Val Phe Thr 695 700 705 710

TCT ATA GGA AAG GCT CTC CAC CAA GTC TTT GGA GCA ATC TAT GGA GCT 2274 Ser He Gly Lys Ala Leu His Gin Val Phe Gly Ala He Tyr Gly Ala 715 720 725

GCC TTC AGT GGG GTT TCA TGG ACT ATG AAA ATC CTC ATA GGA GTC ATT 2322 Ala Phe Ser Gly Val Ser Trp Thr Met Lys He Leu He Gly Val He 730 735 740

ATC ACA TGG ATA GGA ATG AAT TCA CGC AGC ACC TCA CTG TCT GTG ACA 2370 He Thr Trp He Gly Met Asn Ser Arg Ser Thr Ser Leu Ser Val Thr 745 750 755

CTA GTA TTG GTG GGA ATT GTG ACA CTG TAT TTG GGA GTC ATG GTG CAG 2418 Leu Val Leu Val Gly He Val Thr Leu Tyr Leu Gly Val Met Val Gin 760 765 770

GCC GAT AGT GGT TGC GTT GTG AGC TGG AAA AAC AAA GAA CTG AAA TGT 2466 Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys 775 780 785 790

GGC AGT GGG ATT TTC ATC ACA GAC AAC GTG CAC ACA TGG ACA GAA CAA 2514 Gly Ser Gly He Phe He Thr Asp Asn Val His Thr Trp Thr Glu Gin 795 800 805

TAC AAG TTC CAA CCA GAA TCC CCT TCA AAA CTA GCT TCA GCT ATC CAG 2562 Tyr Lys Phe Gin Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala He Gin 810 815 820

AAA GCC CAT GAA GAG GAC ATT TGT GGA ATC CGC TCA GTA ACA AGA CTG 2610 Lys Ala His Glu Glu Asp He Cys Gly He Arg Ser Val Thr Arg Leu 825 830 835

GAG AAT CTG ATG TGG AAA CAA ATA ACA CCA GAA TTG AAT CAC ATT CTA 2658 Glu Asn Leu Met Trp Lys Gin He Thr Pro Glu Leu Asn His He Leu 840 845 850

TCA GAA AAT GAG GTG AAG TTA ACT ATT ATG ACA GGA GAC ATC AAA GGA 2706 Ser Glu Asn Glu Val Lys Leu Thr He Met Thr Gly Asp He Lys Gly 855 860 865 870

ATC ATG CAG GCA GGA AAA CGA TCT CTG CGG CCT CAG CCC ACT GAG CTG 2754 He Met Gin Ala Gly Lys Arg Ser Leu Arg Pro Gin Pro Thr Glu Leu 875 880 885

AAG TAT TCA TGG AAA ACA TGG GGC AAA GCA AAA ATG CTC TCT ACA GAG 2802 Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu 890 895 900

TCT CAT NAC CAG ACC TTT CTC ATT GAT GGC CCC GAA ACA GCA GAA TGC 2850 Ser His Xaa Gin Thr Phe Leu He Asp Gly Pro Glu Thr Ala Glu Cys

905 910 915

CCC AAC ACA AAT AGA GCT TGG AAT TCG TTG GAA GTT GAA GAC TAT GGC 2898 Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly 920 925 930

TTT GGA GTA TTC ACC ACC AAT ATA TGG CTA AAA TTG AAA GAA AAA CAG 2946 Phe Gly Val Phe Thr Thr Asn He Trp Leu Lys Leu Lys Glu Lys Gin 935 940 945 950

GAT GTA TTC TGC GAC TCA AAA CTC ATG TCA GCG GCC ATA AAA GAC AAC 2994 Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala He Lys Asp Asn 955 960 965

AGA GCC GTC CAT GCC GAT ATG GGT TAT TGG ATA GAA AGT GCA CTC NAT 3042 Arg Ala Val His Ala Asp Met Gly Tyr Trp He Glu Ser Ala Leu Xaa 970 975 980

GAC ACA TGG AAG ATA GAG AAA GCC TCT TTC ATT GAA GTT AAA AAC TGC 3090 Asp Thr Trp Lys He Glu Lys Ala Ser Phe He Glu Val Lys Asn Cys 985 990 995

CAC TGG CCA AAA TCA CAC ACC CTC TGG AGC AAT GGA GTG CTA GAA AGT 3138 His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser 1000 1005 1010

GAG ATG ATA ATT CCA AAG AAT CTC GCT GGA CCA GTG TCT CAA CAC AAC 3186 Glu Met He He Pro Lys Asn Leu Ala Gly Pro Val Ser Gin His Asn 1015 1020 1025 1030

TAT AGA CCA GGC TAC CAT ACA CAA ATA ACA GGA CCA TGG CAT CTA GGT 3234 Tyr Arg Pro Gly Tyr His Thr Gin He Thr Gly Pro Trp His Leu Gly 1035 1040 1045

AAG CTT GAG ATG GAC TTT GAT TTC TGT GAT GGA ACA ACA GTG GTA GTG 3282 Lys Leu Glu Met Asp Phe Asp Phe Cys Asp Gly Thr Thr Val Val Val 1050 1055 1060

ACT GAG GAC TGC GGA AAT AGA GGA CCC TCT TTG AGA ACA ACC ACT GCC 3330 Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala 1065 1070 1075

TCT GGA AAA CTC ATA ACA GAA TGG TGC TGC CGA TCT TGC ACA TTA CCA 3378 Ser Gly Lys Leu He Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro 1080 1085 1090

CCG CTA AGA TAC AGA GGT GAG GAT GGG TGC TGG TAC GGG ATG GAA ATC 3426 Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu He 1095 1100 1105 1110

AGA CCA TTG AAG GAG AAA GAA GAG AAT TTG GTC AAC TCC TTG GTC ACA 3474 Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr 1115 1120 1125

GCT GGA CAT GGG CAG GTC GAC AAC TTT TCA CTA GGA GTC TTG GGA ATG 3522 Ala Gly His Gly Gin Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met 1130 ^' 1135 1140

GCA TTG TTC CTG GAG GAA ATG CTT AGG ACC CGA GTA GGA ACG AAA CAT 3570 Ala Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His 1145 1150 1155

GCA ATA CTA CTA GTT GCA GTT TCT TTT GTG ACA TTG ATC ACA GGG AAC 3618

Ala He Leu Leu Val Ala Val Ser Phe Val Thr Leu He Thr Gly Asn 1160 1165 1170

ATG TCC TTT AGA GAC CTG GGA AGA GTG ATG GTT ATG GTA GGC GCC ACT 3666

Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly Ala Thr 1175 1180 1185 1190

ATG ACG GAT GAC ATA GGT ATG GGC GTG ACT TAT CTT GCC CTA CTA GCA 3714

Met Thr Asp Asp He Gly Met Gly Val Thr Tyr Leu Ala Leu Leu Ala 1195 1200 1205

GCC TTC AAA GTC AGA CCA ACT TTT GCA GCT GGA CTA CTC TTG AGA AAG 3762

Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu Leu Leu Arg Lys 1210 1215 1220

CTG ACC TCC AAG GAA TTG ATG ATG ACT ACT ATA GGA ATT GTA CTC CTC 3810

Leu Thr Ser Lys Glu Leu Met Met Thr Thr He Gly He Val Leu Leu 1225 1230 1235

TCC CAG AGC ACC ATA CCA GAG ACC ATT CTT GAG TTG ACT GAT GCG TTA 3858

Ser Gin Ser Thr He Pro Glu Thr He Leu Glu Leu Thr Asp Ala Leu 1240 1245 1250

GCC TTA GGC ATG ATG GTC CTC AAA ATG GTG AGA AAT ATG GAA AAG TAT 3906

Ala Leu Gly Met Met Val Leu Lys Met Val Arg Asn Met Glu Lys Tyr 1255 1260 1265 1270

CAA TTG GCA GTG ACT ATC ATG GCT ATC TTG TGC GTC CCA AAC GCA GTG 3954

Gin Leu Ala Val Thr He Met Ala He Leu Cys Val Pro Asn Ala Val 1275 1280 1285

ATA TTA CAA AAC GCA TGG AAA GTG AGT TGC ACA ATA TTG GCA GTG GTG 4002

He Leu Gin Asn Ala Trp Lys Val Ser Cys Thr He Leu Ala Val Val 1290 1295 1300

TCC GTT TCC CCA CTG TTC TTA ACA TCC TCA CAG CAA AAA ACA GAT TGG 4050

Ser Val Ser Pro Leu Phe Leu Thr Ser Ser Gin Gin Lys Thr Asp Trp 1305 1310 1315

ATA CCA TTA GCA TTG ACG ATC AAA GGT CTC AAT CCA ACA GCT ATT TTT 4098

He Pro Leu Ala Leu Thr He Lys Gly Leu Asn Pro Thr Ala He Phe 1320 1325 1330

CTA ACA ACC CTC TCA AGA ACC AGC AAG AAA AGG AGC TGG CCA TTA AAT 4146

Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn 1335 1340 1345 1350

GAG GCT ATC ATG GCA GTC GGG ATG GTG AGC ATT TTA GCC AGT TCT CTC 4194

Glu Ala He Met Ala Val Gly Met Val Ser He Leu Ala Ser Ser Leu 1355 1360 1365

CTA AAA AAT GAT ATT CCC ATG ACA GGA CCA TTA GTG GCT GGA GGG CTC 4242

Leu Lys Asn Asp He Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu 1370 1375 1380

CTC ACT GTG TGC TAC GTG CTC ACT GGA CGA TCG GCC GAT TTG GAA CTG 4290

Leu Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu 1385 1390 1395

GAG AGA GCA GCC GAT GTC AAA TGG GAA GAC CAG GCA GAG ATA TCA GGA 4338 Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gin Ala Glu He Ser Gly 1400 1405 1410

AGC AGT CCA ATC CTG TCA ATA ACA ATA TCA GAA GAT GGT AGC ATG TCG 4386 Ser Ser Pro He Leu Ser He Thr He Ser Glu Asp Gly Ser Met Ser 1415 1420 1425 1430

ATA AAA AAT GAA GAG GAA GAA CAA ACA CTG ACC ATA CTC ATT AGA ACA 4434 He Lys Asn Glu Glu Glu Glu Gin Thr Leu Thr He Leu He Arg Thr 1435 1440 1445

GGA TTG CTG GTG ATC TCA GGA CTT TTT CCT GTA TCA ATA CCA ATC ACG 4482 Gly Leu Leu Val He Ser Gly Leu Phe Pro Val Ser He Pro He Thr 1450 1455 1460

GCA GCA GCA TGG TAC CTG TGG GAA GTG AAG AAA CAA CGG GCC GGA GTA 4530 Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gin Arg Ala Gly Val 1465 1470 1475

TTG TGG GAT GTT CCT TCA CCC CCA CCC ATG GGA AAG GCT GAA CTG GAA 4578 Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys Ala Glu Leu Glu 1480 1485 1490

GAT GGA GCC TAT AGA ATT AAG CAA AAA GGG ATT CTT GGA TAT TCC CAG 4626 Asp Gly Ala Tyr Arg He Lys Gin Lys Gly He Leu Gly Tyr Ser Gin 1495 1500 1505 1510

ATC GGA GCC GGA GTT TAC AAA GAA GGA ACA TTC CAT ACA ATG TGG CAT 4674 He Gly Ala Gly Val Tyr Lys Glu Gly Thr Phe His Thr Met Trp His 1515 1520 1525

GTC ACA CGT GGC GCT GTT CTA ATG CAT AAA GGA AAG AGG ATT GAA CCA 4722 Val Thr Arg Gly Ala Val Leu Met His Lys Gly Lys Arg He Glu Pro 1530 1535 1540

TCA TGG GCG GAC GTC AAG AAA GAC CTA ATA TCA TAT GGA GGA GGC TGG 4770 Ser Trp Ala Asp Val Lys Lys Asp Leu He Ser Tyr Gly Gly Gly Trp 1545 1550 1555

AAG TTA GAA GGA GAA TGG AAG GAA GGA GAA GAA GTC CAG GTA TTG GCA 4818 Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu Glu Val Gin Val Leu Ala 1560 1565 1570

CTG GAG CCT GGA AAA AAT CCA AGA GCC GTC CAA ACG AAA CCT GGT CTT 4866 Leu Glu Pro Gly Lys Asn Pro Arg Ala Val Gin Thr Lys Pro Gly Leu 1575 1580 1585 1590

TTC AAA ACC AAC GCC GGA ACA ATA GGT GCT GTA TCT CTG GAC TTT TCT 4914 Phe Lys Thr Asn Ala Gly Thr He Gly Ala Val Ser Leu Asp Phe Ser 1595 1600 1605

CCT GGA ACG TCA GGA TCT CCA ATT ATC GAC AAA AAA GGA AAA GTT GTG 4962 Pro Gly Thr Ser Gly Ser Pro He He Asp Lys Lys Gly Lys Val Val 1610 1615 1620

GGT CTT TAT GGT AAT GGT GTT GTT ACA AGG AGT GGA GCA TAT GTG AGT 5010 Gly Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser 1625 1630 1635

GCT ATA GCC CAG ACT GAA AAA AGC ATT GAA GAC AAC CCA GAG ATC GAA 5058 Ala He Ala Gin Thr Glu Lys Ser He Glu Asp Asn Pro Glu He Glu 1640 1645 1650

GAT GAC ATT TTC CGA AAG AGA AGA CTG ACC ATC ATG GAC CTC CAC CCA 5106 Asp Asp He Phe Arg Lys Arg Arg Leu Thr He Met Asp Leu His Pro 1655 1660 1665 1670

GGA GCG GGA AAG ACG AAG AGA TAC CTT CCG GCC ATA GTC AGA GAA GCT 5154 Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala He Val Arg Glu Ala 1675 1680 1685

ATA AAA CGG GGT TTG AGA ACA TTA ATC TTG GCC CCC ACT AGA GTT GTG 5202 He Lys Arg Gly Leu Arg Thr Leu He Leu Ala Pro Thr Arg Val Val 1690 1695 1700

GCA GCT GAA ATG GAG GAA GCC CTT AGA GGA CTT CCA ATA AGA TAC CAG 5250 Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro He Arg Tyr Gin 1705 1710 1715

ACC CCA GCC ATC AGA GCT GAG CAC ACC GGG CGG GAG ATT GTG GAC CTA 5298 Thr Pro Ala He Arg Ala Glu His Thr Gly Arg Glu He Val Asp Leu 1720 1725 1730

ATG TGT CAT GCC ACA TTT ACC ATG AGG CTG CTA TCA CCA GTT AGA GTG 5346 Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser Pro Val Arg Val 1735 1740 1745 1750

CCA AAC TAC AAC CTG ATT ATC ATG GAC GAA GCC CAT TTC ACA GAC CCA 5394 Pro Asn Tyr Asn Leu He He Met Asp Glu Ala His Phe Thr Asp Pro 1755 1760 1765

GCA AGT ATA GCA GCT AGA GGA TAC ATC TCA ACT CGA GTG GAG ATG GGT 5442 Ala Ser He Ala Ala Arg Gly Tyr He Ser Thr Arg Val Glu Met Gly 1770 1775 1780

GAG GCA GCT GGG ATT TTT ATG ACA GCC ACT CCC CCG GGA AGC AGA GAC 5490 Glu Ala Ala Gly He Phe Met Thr Ala Thr Pro Pro Gly Ser Arg Asp 1785 1790 1795

CCA TTT CCT CAG AGC AAT GCA CCA ATC ATA GAT GAA GAA AGA GAA ATC 5538 Pro Phe Pro Gin Ser Asn Ala Pro He He Asp Glu Glu Arg Glu He 1800 1805 1810

CCT GAA CGT TCG TGG AAT TCC GGA CAT GAA TGG GTC ACG GAT TTT AAA 5586 Pro Glu Arg Ser Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys 1815 1820 1825 1830

GGG AAG ACT GTT TGG TTC GTT CCA AGT ATA AAA GCA GGA AAT GAT ATA 5634 Gly Lys Thr Val Trp Phe Val Pro Ser He Lys Ala Gly Asn Asp He 1835 1840 1845

GCA GCT TGC CTG AGG AAA AAT GGA AAG AAA GTG ATA CAA CTC AGT AGG 5682 Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys Val He Gin Leu Ser Arg 1850 1855 1860

AAG ACC TTT GAT TCT GAG TAT GTC AAG ACT AGA ACC AAT GAT TGG GAC 5730 Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp 1865 1870 1875

TTC GTG GTT ACA ACT GAC ATT TCA GAA ATG GGT GCC AAT TTC AAG GCT 5778 Phe Val Val Thr Thr Asp He Ser Glu Met Gly Ala Asn Phe Lys Ala 1880 1885 1890

GAG AGG GTT ATA GAC CCC AGA CGC TGC ATG AAA CCA GTC ATA CTA ACA 5826 Glu Arg Val He Asp Pro Arg Arg Cys Met Lys Pro Val He Leu Thr 1895 1900 1905 1910

GAT GGT GAA GAG CGG GTG ATT CTG GCA GGA CCT ATG CCA GTG ACC CAC 5874 Asp Gly Glu Glu Arg Val He Leu Ala Gly Pro Met Pro Val Thr His 1915 1920 1925

TCT AGT GCA GCA CAA AGA AGA GGG AGA ATA GGA AGA AAT CCA AAA AAT 5922 Ser Ser Ala Ala Gin Arg Arg Gly Arg He Gly Arg Asn Pro Lys Asn 1930 1935 1940

GAG AAT GAC CAG TAC ATA TAC ATG GGG GAA CCT CTG GAA AAT GAT GAA 5970 Glu Asn Asp Gin Tyr He Tyr Met Gly Glu Pro Leu Glu Asn Asp Glu 1945 1950 1955

GAC TGT GCA CAC TGG AAA GAA GCT AAA ATG CTC CTA GAT AAC ATC AAC 6018 Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu Asp Asn He Asn 1960 1965 1970

ACG CCA GAA GGA ATC ATT CCT AGC ATG TTC GAA CCA GAG CGT GAA AAG 6066 Thr Pro Glu Gly He He Pro Ser Met Phe Glu Pro Glu Arg Glu Lys 1975 1980 1985 1990

GTG GAT GCC ATT GAT GGC GAA TAC CGC TTG AGA GGA GAA GCA AGG AAA 6114 Val Asp Ala He Asp Gly Glu Tyr Arg Leu Arg Gly Glu Ala Arg Lys 1995 2000 2005

ACC TTT GTA GAC TTA ATG AGA AGA GGA GAC CTA CCA GTC TGG TTG GCC 6162 Thr Phe Val Asp Leu Met Arg Arg Gly Asp Leu Pro Val Trp Leu Ala 2010 2015 2020

TAC AGA GTG GCA GCT GAA GGC ATC AAC TAC GCA GAC AGA AGG TGG TGT 6210 Tyr Arg Val Ala Ala Glu Gly He Asn Tyr Ala Asp Arg Arg Trp Cys 2025 2030 2035

TTT GAT GGA GTC AAG AAC AAC CAA ATC CTA GAA GAA AAC GTG GAA GTT 6258 Phe Asp Gly Val Lys Asn Asn Gin He Leu Glu Glu Asn Val Glu Val 2040 2045 2050

GAA ATC TGG ACA AAA GAA GGG GAA AGG AAG AAA TTG AAA CCC AGA TGG 6306 Glu He Trp Thr Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp 2055 2060 2065 2070

TTG GAT GCT AGG ATC TAT TCT GAC CCA CTG GCG CTA AAA GAA TTT AAG 6354 Leu Asp Ala Arg He Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys 2075 2080 2085

GAA TTT GCA GCC GGA AGA AAG TCT CTG ACC CTG AAC CTA ATC ACA GAA 6402 Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu He Thr Glu 2090 2095 2100

ATG GGT AGG CTC CCA ACC TTC ATG ACT CAG AAG GCA AGA GAC GCA CTG 6450 Met Gly Arg Leu Pro Thr Phe Met Thr Gin Lys Ala Arg Asp Ala Leu 2105 2110 2115

GAC AAC TTA GCA GTG CTG CAC ACG GCT GAG GCA GGT GGA AGG GCG TAC 6498 Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala Tyr 2120 2125 2130

AAC CAT GCT CTC AGT GAA CTG CCG GAG ACC CTG GAG ACA TTG CTT TTA 6546

Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu Leu Leu

2135 2140 2145 2150

CTG ACA CTT CTG GCT ACA GTC ACG GGA GGG ATC TTT TTA TTC TTG ATG 6594 Leu Thr Leu Leu Ala Thr Val Thr Gly Gly He Phe Leu Phe Leu Met 2155 2160 2165

AGC GCA AGG GGC ATA GGG AAG ATG ACC CTG GGA ATG TGC TGC ATA ATC 6642 Ser Ala Arg Gly He Gly Lys Met Thr Leu Gly Met Cys Cys He He 2170 2175 2180

ACG GCT AGC ATC CTC CTA TGG TAC GCA CAA ATA CAG CCA CAC TGG ATA 6690 Thr Ala Ser He Leu Leu Trp Tyr Ala Gin He Gin Pro His Trp He 2185 2190 2195

GCA GCT TCA ATA ATA CTG GAG TTT TTT CTC ATA GTT TTG CTT ATT CCA 6738 Ala Ala Ser He He Leu Glu Phe Phe Leu He Val Leu Leu He Pro 2200 2205 2210

GAA CCT GAA AAA CAG AGA ACA CCC CAA GAC AAC CAA CTG ACC TAC GTT 6786

Glu Pro Glu Lys Gin Arg Thr Pro Gin Asp Asn Gin Leu Thr Tyr Val

2215 2220 2225 2230

GTC ATA GCC ATC CTC ACA GTG GTG GCC GCA ACC ATG GCA AAC GAG ATG 6834 Val He Ala He Leu Thr Val Val Ala Ala Thr Met Ala Asn Glu Met 2235 2240 2245

GGT TTC CTA GAA AAA ACG AAG AAA GAT CTC GGA TTG GGA AGC ATT GCA 6882 Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu Gly Leu Gly Ser He Ala 2250 2255 2260

ACC CAG CAA CCC GAG AGC AAC ATC CTG GAC ATA GAT CTA CGT CCT GCA 6930 Thr Gin Gin Pro Glu Ser Asn He Leu Asp He Asp Leu Arg Pro Ala 2265 2270 2275

TCA GCA TGG ACG CTG TAT GCC GTG GCC ACA ACA TTT GTT ACA CCA ATG 6978 Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met 2280 2285 2290

TTG AGA CAT AGC ATT GAA AAT TCC TCA GTG AAT GTG TCC CTA ACA GCT 7026

Leu Arg His Ser He Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala

2295 2300 2305 2310

ATA GCC AAC CAA GCC ACA GTG TTA ATG GGT CTC GGG AAA GGA TGG CCA 7074 He Ala Asn Gin Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro 2315 2320 2325

TTG TCA AAG ATG GAC ATC GGA GTT CCC CTT CTC GCC ATT GGA TGC TAC 7122 Leu Ser Lys Met Asp He Gly Val Pro Leu Leu Ala He Gly Cys Tyr 2330 2335 2340

TCA CAA GTC AAC CCC ATA ACT CTC ACA GCA GCT CTT TTC TTA TTG GTA 7170 Ser Gin Val Asn Pro He Thr Leu Thr Ala Ala Leu Phe Leu Leu Val 2345 2350 2355

GCA CAT TAT GCC ATC ATA GGG CCA GGA CTC CAA GCA AAA GCA ACC AGA 7218 Ala His Tyr Ala He He Gly Pro Gly Leu Gin Ala Lys Ala Thr Arg 2360 2365 2370

GAA GCT CAG AAA AGA GCA GCG GCG GGC ATC ATG AAA AAC CCA ACT GTC 7266

Glu Ala Gin Lys Arg Ala Ala Ala Gly He Met Lys Asn Pro Thr Val

2375 2380 2385 2390

GAT GGA ATA ACA GTG ATT GAC CTA GAT CCA ATA CCT TAT GAT CCA AAG 7314 Asp Gly He Thr Val He Asp Leu Asp Pro He Pro Tyr Asp Pro Lys 2395 2400 2405

TTT GAA AAG CAG TTG GGA CAA GTA ATG CTC CTA GTC CTC TGC GTG ACT 7362 Phe Glu Lys Gin Leu Gly Gin Val Met Leu Leu Val Leu Cys Val Thr 2410 2415 2420

CAA GTA TTG ATG ATG AGG ACT ACA TGG GCT CTG TGT GAG GCT TTA ACC 7410 Gin Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys Glu Ala Leu Thr 2425 2430 2435

TTA GCT ACC GGG CCC ATC TCC ACA TTG TGG GAA GGA AAT CCA GGG AGG 7458 Leu Ala Thr Gly Pro He Ser Thr Leu Trp Glu Gly Asn Pro Gly Arg 2440 2445 2450

TTT TGG AAC ACT ACC ATT GCG GTG TCA ATG GCT AAC ATT TTT AGA GGG 7506

Phe Trp Asn Thr Thr He Ala Val Ser Met Ala Asn He Phe Arg Gly

2455 2460 2465 2470

AGT TAC TTG GCC GGA GCT GGA CTT CTC TTT TCT ATT ATG AAG AAC ACA 7554 Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe Ser He Met Lys Asn Thr 2475 2480 2485

ACC AAC ACA AGA AGG GGA ACT GGC AAC ATA GGA GAG ACG CTT GGA GAG 7602 Thr Asn Thr Arg Arg Gly Thr Gly Asn He Gly Glu Thr Leu Gly Glu 2490 2495 2500

AAA TGG AAA AGC CGA TTG AAC GCA TTG GGA AAA AGT GAA TTC CAG ATC 7650 Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly Lys Ser Glu Phe Gin He 2505 2510 2515

TAC AAG AAA AGT GGA ATC CAG GAA GTG GAT AGA ACC TTA GCA AAA GAA 7698 Tyr Lys Lys Ser Gly He Gin Glu Val Asp Arg Thr Leu Ala Lys Glu 2520 2525 2530

GGC ATT AAA AGA GGA GAA ACG GAC CAT CAC GCT GTG TCG CGA GGC TCA 7746

Gly He Lys Arg Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser

2535 2540 2545 2550

GCA AAA CTG AGA TGG TTC GTT GAG AGA AAC ATG GTC ACA CCA GAA GGG 7794 Ala Lys Leu Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly 2555 2560 2565

AAA GTA GTG GAC CTC GGT TGT GGC AGA GGA GGC TGG TCA TAC TAT TGT 7842 Lys Val Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys 2570 2575 2580

GGA GGA CTA AAG AAT GTA AGA GAA GTC AAA GGC CTA ACA AAA GGA GGA 7890 Gly Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly 2585 2590 2595

CCA GGA CAC GAA GAA CCC ATC CCC ATG TCA ACA TAT GGG TGG AAT CTA 7938 Pro Gly His Glu Glu Pro He Pro Met Ser Thr Tyr Gly Trp Asn Leu 2600 2605 2610

GTG CGT CTT CAA AGT GGA GTT GAC GTT TTC TTC ATC CCG CCA GAA AAG 7986

Val Arg Leu Gin Ser Gly Val Asp Val Phe Phe He Pro Pro Glu Lys

2615 2620 2625 2630

TGT GAC ACA TTA TTG TGT GAC ATA GGG GAG TCA TCA CCA AAT CCC ACA 8034 Cys Asp Thr Leu Leu Cys Asp He Gly Glu Ser Ser Pro Asn Pro Thr 2635 2640 2645

GTG GAA GCA GGA CGA ACA CTC AGA GTC CTT AAC TTA GTA GAA AAT TGG 8082 Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu Val Glu Asn Trp 2650 2655 2660

TTG AAC AAC AAC ACT CAA TTT TGC ATA AAG GTT CTC AAC CCA TAT ATG 8130 Leu Asn Asn Asn Thr Gin Phe Cys He Lys Val Leu Asn Pro Tyr Met 2665 2670 2675

CCC TCA GTC ATA GAA AAA ATG GAA GCA CTA CAA AGG AAA TAT GGA GGA 8178 Pro Ser Val He Glu Lys Met Glu Ala Leu Gin Arg Lys Tyr Gly Gly 2680 2685 2690

GCC TTA GTG AGG AAT CCA CTC TCA CGA AAC TCC ACA CAT GAG ATG TAC 8226

Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr

2695 2700 2705 2710

TGG GTA TCC AAT GCT TCC GGG AAC ATA GTG TCA TCA GTG AAC ATG ATT 8274 Trp Val Ser Asn Ala Ser Gly Asn He Val Ser Ser Val Asn Met He 2715 2720 2725

TCA AGG ATG TTG ATC AAC AGA TTT ACA ATG AGA TAC AAG AAA GCC ACT 8322 Ser Arg Met Leu He Asn Arg Phe Thr Met Arg Tyr Lys Lys Ala Thr 2730 2735 2740

TAC GAG CCG GAT GTT GAC CTC GGA AGC GGA ACC CGT AAC ATC GGG ATT 8370 Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly Thr Arg Asn He Gly He 2745 2750 2755

GAA AGT GAG ATA CCA AAC CTA GAT ATA ATT GGG AAA AGA ATA GAA AAA 8418 Glu Ser Glu He Pro Asn Leu Asp He He Gly Lys Arg He Glu Lys 2760 2765 2770

ATA AAG CAA GAG CAT GAA ACA TCA TGG CAC TAT GAC CAA GAC CAC CCA 8466

He Lys Gin Glu His Glu Thr Ser Trp His Tyr Asp Gin Asp His Pro

2775 2780 2785 2790

TAC AAA ACG TGG GCA TAC CAT GGT AGC TAT GAA ACA AAA CAG ACT GGA 8514 Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gin Thr Gly 2795 2800 2805

TCA GCA TCA TCC ATG GTC AAC GGA GTG GTC AGG CTG CTG ACA AAA CCT 8562 Ser Ala Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro 2810 2815 2820

TGG GAC GTT GTC CCC ATG GTG ACA CAG ATG GCA ATG ACA GAC ACG ACT 8610 Trp Asp Val Val Pro Met Val Thr Gin Met Ala Met Thr Asp Thr Thr 2825 2830 2835

CCA TTT GGA CAA CAG CGC GTT TTT AAA GAG AAA GTG GAC ACG AGA ACC 8658 Pro Phe Gly Gin Gin Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr 2840 2845 2850

CAA GAA CCG AAA GAA GGC ACG AAG AAA CTA ATG AAA ATA ACA GCA GAG 8706 Gin Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys He Thr Ala Glu 2855 2860 2865 2870

TGG CTT TGG AAA GAA TTA GGG AAG AAA AAG ACA CCC AGG ATG TGC ACC 8754 Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys Thr 2875 2880 2885

AGA GAA GAA TTC ACA AGA AAG GTG AGA AGC AAT GCA GCC TTG GGG GCC 8802 Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala Ala Leu Gly Ala 2890 2895 2900

ATA TTC ACT GAT GAG AAC AAG TGG AAG TCG GCA CGT GAG GCT GTT GAA 8850 He Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu 2905 2910 2915

GAT AGT AGG TTT TGG GAG CTG GTT GAC AAG GAA AGG AAT CTC CAT CTT 8898 Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Asn Leu His Leu 2920 2925 2930

GAA GGA AAG TGT GAA ACA TGT GTG TAC AAC ATG ATG GGA AAA AGA GAG 8946 Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu 2935 2940 2945 2950

AAG AAG CTA GGG GAA TTC GGC AAG GCA AAA GGC AGC AGA GCC ATA TGG 8994 Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala He Trp 2955 2960 2965

TAC ATG TGG CTT GGA GCA CGC TTC TTA GAG TTT GAA GCC CTA GGA TTC 9042 Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe 2970 2975 2980

TTA AAT GAA GAT CAC TGG TTC TCC AGA GAG AAC TCC CTG AGT GGA GTG 9090 Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly Val 2985 2990 2995

GAA GGA GAA GGG CTG CAC AAG CTA GGT TAC ATT CTA AGA GAC GTG AGC 9138 Glu Gly Glu Gly Leu His Lys Leu Gly Tyr He Leu Arg Asp Val Ser 3000 3005 3010

AAG AAA GAG GGA GGA GCA ATG TAT GCC GAT GAC ACC GCA GGA TGG GAT 9186 Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp 3015 3020 3025 3030

ACA AGA ATC ACA CTA GAA GAC KKA AAA AAT GAA GAA ATG GTA ACA AAC 9234 Thr Arg He Thr Leu Glu Asp Xaa Lys Asn Glu Glu Met Val Thr Asn 3035 3040 3045

CAC ATG GAA GGA GAA CAC AAG AAA CTA GCC GAG GCC ATT TTC AAA CTA 9282 His Met Glu Gly Glu His Lys Lys Leu Ala Glu Ala He Phe Lys Leu 3050 3055 3060

ACG TAC CAA AAC AAG GTG GTG CGT GTG CAA AGA CCA ACA CCA AGA GGC 9330 Thr Tyr Gin Asn Lys Val Val Arg Val Gin Arg Pro Thr Pro Arg Gly 3065 3070 3075

ACA GTA ATG GAC ATC ATA TCG AGA AGA GAC CAA AGA GGT AGT GGA CAA 9378 Thr Val Met Asp He He Ser Arg Arg Asp Gin Arg Gly Ser Gly Gin 3080 3085 3090

GTT GGC ACC TAT GGA CTC AAT ACT TTC ACC AAT ATG GAA GCC CAA CTA 9426 Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gin Leu 3095 3100 3105 3110

ATC AGA CAG ATG GAG GGA GAA GGA GTC TTT AAA AGC ATT CAG CAC CTA 9474 He Arg Gin Met Glu Gly Glu Gly Val Phe Lys Ser He Gin His Leu 3115 3120 3125

ACA ATC ACA GAA GAA ATC GCT GTG CAA AAC TGG TTA GCA AGA GTG GGG 9522 Thr He Thr Glu Glu He Ala Val Gin Asn Trp Leu Ala Arg Val Gly 3130 3135 3140

CGC GAA AGG TTA TCA AGA ATG GCC ATC AGT GGA GAT GAT TGT GTT GTG 9570 Arg Glu Arg Leu Ser Arg Met Ala He Ser Gly Asp Asp Cys Val Val 3145 3150 3155

AAA CCT TTA GAT GAC AGG TTC GCA AGC GCT TTA ACA GCT CTA AAT GAC 9618 Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn Asp 3160 3165 3170

ATG GGA AAG ATT AGG AAA GAC ATA CAA CAA TGG GAA CCT TCA AGA GGA 9666 Met Gly Lys He Arg Lys Asp He Gin Gin Trp Glu Pro Ser Arg Gly 3175 3180 3185 3190

TGG AAT GAT TGG ACA CAA GTG CCC TTC TGT TCA CAC CAT TTC CAT GAG 9714 Trp Asn Asp Trp Thr Gin Val Pro Phe Cys Ser His His Phe His Glu 3195 3200 3205

TTA ATC ATG AAA GAC GGT CGC GTA CTC GTT GTT CCA TGT AGA AAC CAA 9762 Leu He Met Lys Asp Gly Arg Val Leu Val Val Pro Cys Arg Asn Gin 3210 3215 3220

GAT GAA CTG ATT GGC AGA GCC CGA ATC TCC CAA GGA GCA GGG TGG TCT 9810 Asp Glu Leu He Gly Arg Ala Arg He Ser Gin Gly Ala Gly Trp Ser 3225 3230 3235

TTG CGG GAG ACG GCC TGT TTG GGG AAG TCT TAC GCC CAA ATG TGG AGC 9858 Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser Tyr Ala Gin Met Trp Ser 3240 3245 3250

TTG ATG TAC TTC CAC AGA CGC GAC CTC AGG CTG GCG GCA AAT GCT ATT 9906 Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala He 3255 3260 3265 3270

TGC TCG GCA GTA CCA TCA CAT TGG GTT CCA ACA AGT CGA ACA ACC TGG 9954 Cys Ser Ala Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp 3275 3280 3285

TCC ATA CAT GCT AAA CAT GAA TGG ATG ACA ACG GAA GAC ATG CTG ACA 10002 Ser He His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr 3290 3295 3300

GTC TGG AAC AGG GTG TGG ATT CAA GAA AAC CCA TGG ATG GAA GAC AAA 10050 Val Trp Asn Arg Val Trp He Gin Glu Asn Pro Trp Met Glu Asp Lys 3305 3310 3315

ACT CCA GTG GAA TCA TGG GAG GAA ATC CCA TAC TTG GGG AAA AGA GAA 10098

Thr Pro Val Glu Ser Trp Glu Glu He Pro Tyr Leu Gly Lys Arg Glu 3320 3325 3330

GAC CAA TGG TGC GGC TCA TTG ATT GGG TTA ACA AGC AGG GCC ACC TGG 10146

Asp Gin Trp Cys Gly Ser Leu He Gly Leu Thr Ser Arg Ala Thr Trp 3335 3340 3345 3350

GCA AAG AAC ATC CAA GCA GCA ATA AAT CAA GTT AGA TCC CTT ATA GGC 10194

Ala Lys Asn He Gin Ala Ala He Asn Gin Val Arg Ser Leu He Gly 3355 3360 3365

AAT GAA GAA TAC ACA GAT TAC ATG CCA TCC ATG AAA AGA TTC AGA AGA 10242

Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys Arg Phe Arg Arg 3370 3375 3380

GAA GAG GAA GAA GCA GGA GTT CTG TGG TAGAAAGCAA AACTAACATG AAACAAGG 10297 Glu Glu Glu Glu Ala Gly Val Leu Trp

3385 3390

CTAGAAGTCA GGTCGGATTA AGCCATAGTA CGGAAAAAAC TATGCTACCT GTGAGCCCCG 10357 TCCAAGGACG TTAAAAGAAG TCAGGCCATC ATAAATGCCA TAGCTTGAGT AAACTATGCA 10417 GCCTGTAGCT CCACCTGAGA AGGTGTAAAA AATCCGGGAG GCCACAAACC ATGGAAGCTG 10477 TACGCATGGC GTAGTGGACT AGCGGTTAGA GAGGACCCCT CCCTTACAAA TCGCAGCAAC 10537 AATGGGGGCC CAAGGCGAGA TGAAGCTGTA GTCTCGCTGG AAGGACTAGA GGTTAGAGGA 10597 GACCCCCCCG AAACAAAAAA CAGCATATTG ACGCTGGGAA AGACCAGAGA TCCTGCTGTC 10657 TCCTCAGCAT CATTCCAGGC ACAGAACGCC AGAAAATGGA ATGGTGCTGT TGAATCAACA 10717 GGTTCT 10723

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CCCAGTCACG ACGTTGTAAA ACGAC 25

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GGATGTGCTG CAAGGCGATT AAGTTGG 27

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

TGAGCGGATA ACAATTTCAC ACAGG 25

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GGCTTTACAC TTTATGCTTC CGGCTCG 27

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GCGGATATTG GAATTCTCTA GAAATTTAAT ACGACTCACT ATAAGTTGTT AGTCTACGTG 60 GACCGACAAA GACAG 75

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

CCAGTGAATT CGAGCTCACG CGTAAATTTA ATACGACTCA CTATAAGTTG TTAGTCTACG 60 TGGACCGACA AAGACAG 77

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

AGTTGTTAGT CTACGTGGAC CGAC 24

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GACAGATTCT TTGAGGGAGC TGAGCTCAAC GTAG 34

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

TCAATATGCT GAAACGCGAG AGAAACCG 28

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GGGATTGTTA GGAAACGAAG GAACGC 26

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CCACCAACAG CAGGGATACT GAAAAGATGG GG 32

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TGCAGATCTG CGTCTCCTAT TCAAG 25

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

CGTGAACATG TGTACCCTCA TGGCC 25

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

TTGCACCAAC AGTCAATGTC TTCAGG 26

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

ACCAGAAGAC ATAGATTGTT GGTGC 25

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

GCACCAACAG TCTATGTCTT CTGGC 25

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

ATGTTTCCAG GCCCCTTCTG ATGAC 25

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GCAGCAATCC TGGCATACAC CATAG 25

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21

GGTTGACATA GTCTTAGAAC ATGGAAG 27

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

CTTCCATGTT CTAAGACTAT GTCAACC 27

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GTCTTAGAAC ATGGAAGTTG TGTGACGACG ATGGC 35

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ACAACAGAAT CTCGCTGCCC AACAC 25

1 43

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

GCAAACACTC CATGGTAGAC AGAGG 25

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

CCTCTGTCTA CCATGGAGTG TTTGC 25

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

CCACATCCAT TTCCCCATCC TCTGTCT 27

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

GGAAAGGGAG GCATTGTGAC CTGTGCTATG 30

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

GGAAATCAAA ATAACACCAC AGAGTTCC 28

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

CTGCAGCAAC ACCATCTCAT TGAAGTCGAG GCCC 34

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GACTTCAATG AGATGGTGCT GCTGC 25

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

GCAGCAGCAC CATCTCATTG AAGTC 25

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

AAGCTTGGCT GGTGCACAGG CAATGGTT 28

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

TGGTAACGGC AGGTCTAGGA ACCATTG 27

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

GGACATCTCA AGTGCAGGCT GAG 23

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

CTCAGCCTGC ACTTGAGATG TCC 23

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

GAAGGAAATA GCAGAAACAC AACATGG 27

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

CCCTTCATAT TGTACTCTGA TAACTATTGT TCC 33

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

CCTCCATTCG GAGACAGCTA CATCATCATA GG 32

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

CCTATGATGA TGTAGCTGTC TCCGAATGGA GG 32

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: ATGGCCATTT TAGGTGACAC AGCCTGGGA 29

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

TGTAAACACT CCTCCCAGGG ATCCAAA 27

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

CTCATAGGAG TCATTATCAC ATGGATAGG 29

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

GGGGATTCTG GTTGGAACTT ATATTGTTCT GTCC 34

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

TGATTCAATT CTGGTGTTAT TTGTTTCCAC 30

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

AAGGAATCAT GCAGGCAGGA AAACG 25

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

ACTTCCAGCG AGTTCCAAGC TC 22

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

AACAGAGCCG TCCATGCCGA TATGG 25

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

TCCATTGCTC CAAAGGGTGT GT 22

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

AGCTTGAGAT GGACTTTGAT TTCTG 25

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: GGTCTGATTT CCATCCCGTA CC 22

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

GTCCTTTAGA GACCTGGGAA GAG 23

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

GTTTTCTCAA GAGTAGTCCA GCTGC 25

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

ATCAATTGGC AGTGACTATC ATGGC 25

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

TGTTAAGAGC AGTGGAGAAA CGGAC 25

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA . (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

GATTGAGACC TTTGATCGTC AACGC 25

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

TGACAGGACC ATTAGTGGCT GGAGG 25

(2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

CGTGCTCACT GGACGATCGG CCGATTTGGA ACTG 34

(2) INFORMATION FOR SEQ ID NO:59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

GGGCTGCTTC CTGATATTTC TGCC 24

(2) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:

CCTGTGGGAA GTGAAGAAAC AACGG 25

(2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: GCTCCATCTT CCAGTTCAGC CTTTCCCATG 30

(2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

CTCCGGCTCC AATCTGAGAG TATCC 25

(2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

CCTAATATCA TATGGAGGAG GCTGG 25

(2) INFORMATION FOR SEQ ID NO:64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

GAAGGAGAAG AAGTCCAGGT ATTGG 25

(2) INFORMATION FOR SEQ ID NO:65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

CTGTCGACAA TTGGAGATCC TGACG 25

(2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

GTGGAGCATA TGTGAGTGCT ATAGC 25

(2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:

TCTGACTATG GCCGGAAGGT ATCTC 25

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:

ACATTAATCT TGGCCCCCAC TAGAG 25

(2) INFORMATION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

CGATCTCCCG CCCGGTGTG 19

(2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:

CTAACTGGTG ATAGCAGCCT CATGG 25

(2) INFORMATION FOR SEQ ID NO:71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: CCTACTGAGT TGTATCACTT TCTTTCC 27

(2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

TGGATTTCTT CCTATTCTCC CTCTTC 26

(2) INFORMATION FOR SEQ ID NO:73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:

TTCAAGGCTG AGAGGGTTAT AGACC 25

(2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

TCTGGTTGGC CTACAGAGTG GCAGC 25

(2) INFORMATION FOR SEQ ID NO:75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

CCTTCTTTTG TCCAGATTTC CACTTCC 27

(2) INFORMATION FOR SEQ ID NO:76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:

GCGTACAACC ATGCTCTCAG TGAACTGCCG GAGAC 35

(2) INFORMATION FOR SEQ ID NO:77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:

TTCCCAGGGT CATCTTCCCT ATAC 24

(2) INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:

GATGCTAGCC GTGATTATGC AGCACATTCC C 31

(2) INFORMATION FOR SEQ ID NO:79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:

AAACAGAGAA CACCCCAAGA CAACC 25

(2) INFORMATION FOR SEQ ID NO:80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:

CGGCATACAG CGTCCATGCT G 21

(2) INFORMATION FOR SEQ ID NO:81:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81 GTCTCGGGAA AGGATGGCCA TTGTC 25

(2) INFORMATION FOR SEQ ID NO:82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:

CTCTGGTTGC TTTTGCTTGA AGTCC 25

(2) INFORMATION FOR SEQ ID NO:83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:

CCGCCGCTGC TCTTTTCTGA GCTTCTC 27

(2) INFORMATION FOR SEQ ID NO:84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:

AGGACTACAT GGGCTCTGTG TGAGG 25

(2) INFORMATION FOR SEQ ID NO:85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:

GAGAAGTCCA GCTCCGGCC 19

(2) INFORMATION FOR SEQ ID NO:86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:

AGAGAAACAT GGTCACACCA GAAGG 25

(2) INFORMATION FOR SEQ ID NO:87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:

GTTCTTCGTG TCCTGGTCCT CC 22

(2) INFORMATION FOR SEQ ID NO:88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:

GGAAATATGG AGGAGCCTAG TGAGG 25

(2) INFORMATION FOR SEQ ID NO:89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:

ACCCAGTACA TCTCATGTGT GG 22

(2) INFORMATION FOR SEQ ID NO:90:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:

GAGCATGAAA CATCATGGCA CTATGACC 28

(2) INFORMATION FOR SEQ ID NO:91 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:91: TCATGGCACT ATGACCAAGA CCACC 25

(2) INFORMATION FOR SEQ ID NO:92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:

CAGTCTGACC ACTCCGTTCA CC 22

(2) INFORMATION FOR SEQ ID NO:93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:

AAGGTGAGAA GCAATGCAGC CTTGG 25

(2) INFORMATION FOR SEQ ID NO:94:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:

GGGCCATATT CACTGATGAG AACAAGTGG 29

(2) INFORMATION FOR SEQ ID NO:95:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:

TCTTTCCCTG TCAACCAGCT CC 22

(2) INFORMATION FOR SEQ ID NO:96:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:

AATGAAGATC ACTGGTTCTC CAGAG 25

(2) INFORMATION FOR SEQ ID NO:97:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:

ACGTGAGCAA GAAAGAGGGA GGAGC 25

(2) INFORMATION FOR SEQ ID NO:98:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:

TGTCCCATCC TGCTGTGTCA TC 22

(2) INFORMATION FOR SEQ ID NO:99:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:

GCTAGTTTCT TGTGTTCTCC TTCCATGTGG 30

(2) INFORMATION FOR SEQ ID NO:100:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:

TCATATCGAG AAGAGACCAA AGAGG 25

(2) INFORMATION FOR SEQ ID NO:101:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:

ACTCCTTCTC CCTCCATCTG TCTG 24

(2) INFORMATION FOR SEQ ID NO:102:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:

ATGCTTTTGA AGATTCCTTC TCCCTCC 27

(2) INFORMATION FOR SEQ ID NO:103:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:

GCACAGCGAT TTCTTCTGTG ATTGTTAGGT GC 32

(2) INFORMATION FOR SEQ ID NO:104:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:

ACAATGGGAA CCTTCAAGAG GATGG 25

(2) INFORMATION FOR SEQ ID NO:105:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:

TTATCACATT GGATCCTTCA AGAGGATGGA ATGATTGGAC ACAAG 45

(2) INFORMATION FOR SEQ ID NO:106:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:

CAGAAGGGCA CTTGTGTCCA ATCATTCC 28

(2) INFORMATION FOR SEQ ID NO:107:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:

CTCCCTGGGA AATTCGGGCT C 21

(2) INFORMATION FOR SEQ ID NO:108:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: CCGTCTCCCG CAAAGACCAC CCTGCTCC 28

(2) INFORMATION FOR SEQ ID NO:109:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:

TTATCACCTA TCTAGACCGT CTCCCGCAAA GACCACCCTG CTCC 44

(2) INFORMATION FOR SEQ ID NO:110:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:

GTTGGAACCC AATGTGATGG TACTGC 26

(2) INFORMATION FOR SEQ ID NO:111:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:

ACAAGTCGAA CAACCTGGTC CATAC 25

(2) INFORMATION FOR SEQ ID NO:112:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:

GCATGTCTTC CGTCGTCATC C 21

(2) INFORMATION FOR SEQ ID NO:113:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:

CTTGAATCCA CACCCTGTTC CAGAC 25

(2) INFORMATION FOR SEQ ID NO:114:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:

ATACACAGAT TACATGCCAT CCATG 25

(2) INFORMATION FOR SEQ ID NO: 115:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (Vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:

TTTTGCCTTC TACCACAGGA C 21

(2) INFORMATION FOR SEQ ID NO:116:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:

GAAACAAGGC TAGAAGTCAG GTCGG 25

(2) INFORMATION FOR SEQ ID NO:117:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (Vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:

GACGGGGCTC ACAGGTAGCA TAG 23

(2) INFORMATION FOR SEQ ID NO:118:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: GCCTGTAGCT CCACCTGAGA AGGTG 25

(2) INFORMATION FOR SEQ ID NO:119:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:

GGAAGCTGTA CGCATGGCGT AGTGG 25

(2) INFORMATION FOR SEQ ID NO:120:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:

GGGCCCCCGT TGTTGCTGC 19

(2) INFORMATION FOR SEQ ID NO:121:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:

AGAACCTGTT GATTCAACAG CACCATTCCA TTTTCTG 37

(2) INFORMATION FOR SEQ ID NO:122:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:

TTATCACCTA GCATGCTCTA GAAGAACCTG TTGATTCAAC AGCACCATTC CATTTTCTG 59

(2) INFORMATION FOR SEQ ID NO:123:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:

TTATCACCTA TCTAGAGAAC CTGTTGATTC AACAGCACCA TTCCATTTTC TG 52

(2) INFORMATION FOR SEQ ID NO:124:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2394 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 1...2394 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:

AGA TTC TCA AAA GGA TTG CTC TCA GGC CAA GGA CCC ATG AAA TTG GTG 48 Arg Phe Ser Lys Gly Leu Leu Ser Gly Gin Gly Pro Met Lys Leu Val 1 5 10 15

ATG GCT TTC ATA GCA TTC TTA AGA TTT CTA GCC ATA CCC CCA ACA GCA 96 Met Ala Phe He Ala Phe Leu Arg Phe Leu Ala He Pro Pro Thr Ala 20 25 30

GGA ATT TTG GCT AGA TGG GGC TCA TTC AAG AAG AAT GGA GCG ATT AAA 144 Gly He Leu Ala Arg Trp Gly Ser Phe Lys Lys Asn Gly Ala He Lys 35 40 45

GTG TTA CGG GGT TTC AAG AGA GAA ATC TCA AAC ATG CTA AAC ATA ATG 192 Val Leu Arg Gly Phe Lys Arg Glu He Ser Asn Met Leu Asn He Met 50 55 60

AAC AGG AGG AAA AGA TCC GTG ACC ATG CTC CTT ATG CTG CTG CCC ACA 240 Asn Arg Arg Lys Arg Ser Val Thr Met Leu Leu Met Leu Leu Pro Thr 65 70 75 80

GCC CTG GCG TTC CAT CTG ACG ACA CGA GGG GGA GAG CCG CAT ATG ATA 288 Ala Leu Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met He 85 90 95

GTT AGC AAG CAG GAA AGA GGA AAG TCA CTT TTG TTC AAG ACC TCT GCA 336 Val Ser Lys Gin Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala 100 105 110

GGT GTC AAC ATG TGC ACC CTC ATT GCG ATG GAT TTG GGA GAG TTG TGT 384 Gly Val Asn Met Cys Thr Leu He Ala Met Asp Leu Gly Glu Leu Cys 115 120 125

GAG GAC ACG ATG ACC TAC AAA TGC CCC CGG ATC ACT GAG GCG GAA CCA 432 Glu Asp Thr Met Thr Tyr Lys Cys Pro Arg He Thr Glu Ala Glu Pro 130 135 140

GAT GAC GTT GAC TGT TGG TGC AAT GCC ACG GAC ACA TGG GTG ACC TAT 480 Asp Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr 145 150 155 160

GGA ACG TGC TCT CAA ACT GGC GAA CAC CGA CGA GAC AAA CGT TCC GTC 528 Gly Thr Cys Ser Gin Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val 165 170 175

GCA TTG GCC CCA CAC GTG GGG CTT GGC CTA GAA ACA AGA GCC GAA ACG 576 Ala Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Ala Glu Thr 180 185 190

TGG ATG TCC TCT GAA GGT GCT TGG AAA CAG ATA CAA AAA GTA GAG ACT 624 Trp Met Ser Ser Glu Gly Ala Trp Lys Gin He Gin Lys Val Glu Thr 195 200 205

TGG GCT CTG AGA CAT CCA GGA TTC ACG GTG ATA GCC CTT TTT CTA GCA 672 Trp Ala Leu Arg His Pro Gly Phe Thr Val He Ala Leu Phe Leu Ala 210 215 220

CAT GCC ATA GGA ACA TCC ATC ACC CAG AAA GGG ATC ATT TTC ATT TTG 720 His Ala He Gly Thr Ser He Thr Gin Lys Gly He He Phe He Leu 225 230 235 240

CTG ATG CTG GTA ACA CCA TCT ATG GCC ATG CGA TGC GTG GGA ATA GGC 768 Leu Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly He Gly 245 250 255

AAC AGA GAC TTC GTG GAA GGA CTG TCA GGA GCA ACA TGG GTG GAT GTG 816 Asn Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val 260 265 270

GTA CTG GAG CAT GGA AGT TGC GTC ACC ACC ATG GCA AAA AAC AAA CCA 864 Val Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro 275 280 285

ACA CTG GAC ATT GAA CTC TTG AAG ACG GAG GTC ACA AAC CCT GCA GTT 912 Thr Leu Asp He Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val 290 295 300

CTG CGT AAA TTG TGC ATT GAA GCT AAA ATA TCA AAC ACC ACC ACC GAT 960 Leu Arg Lys Leu Cys He Glu Ala Lys He Ser Asn Thr Thr Thr Asp 305 310 315 320

TCG AGA TGT CCA ACA CAA GGA GAA GCC ACA CTG GTG GAA GAA CAA GAC 1008 Ser Arg Cys Pro Thr Gin Gly Glu Ala Thr Leu Val Glu Glu Gin Asp 325 330 335

GCG AAC TTT GTG TGC CGA CGA ACG TTC GTG GAC AGA GGC TGG GGC AAT 1056 Ala Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn 340 345 350

GGC TGT GGG CTA TTC GGA AAA GGT AGT CTA ATA ACG TGT GCC AAG TTT 1104 Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu He Thr Cys Ala Lys Phe 355 360 365

AAG TGT GTG ACA AAA CTA GAA GGA AAG ATA GCT CAA TAT GAA AAC CTA 1152 Lys Cys Val Thr Lys Leu Glu Gly Lys He Ala Gin Tyr Glu Asn Leu 370 375 380

AAA TAT TCA GTG ATA GTC ACC GTC CAC ACT GGA GAT CAG CAC CAG GTG 1200 Lys Tyr Ser Val He Val Thr Val His Thr Gly Asp Gin His Gin Val 385 390 395 400

GGA AAT GAG ACT ACA GAA CAT GGA ACA ACT GCA ACC ATA ACA CCT CAA 1248 Gly Asn Glu Thr Thr Glu His Gly Thr Thr Ala Thr He Thr Pro Gin 405 410 415

GCT CCT ACG TCG GAA ATA CAG CTG ACC GAC TAC GGA ACC CTT ACA TTA 1296 Ala Pro Thr Ser Glu He Gin Leu Thr Asp Tyr Gly Thr Leu Thr Leu 420 425 430

GAT TGT TCA CCT AGG ACA GGG CTA GAT TTT AAC GAG ATG GTG TTG CTG 1344 Asp Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu 435 440 445

ACA ATG AAA AAG AAA TCA TGG CTT GTC CAC AAA CAG TGG TTT CTA GAC 1392 Thr Met Lys Lys Lys Ser Trp Leu Val His Lys Gin Trp Phe Leu Asp 450 455 460

TTA CCA CTG CCT TGG ACC TCT GGG GCT TTA ACA TCC CAA GAG ACT TGG 1440 Leu Pro Leu Pro Trp Thr Ser Gly Ala Leu Thr Ser Gin Glu Thr Trp 465 470 475 480

AAC AGA CAA GAT TTA CTG GTC ACA TTT AAG ACA GCT CAT GCA AAG AAG 1488 Asn Arg Gin Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys 485 490 495

CAG GAA GTA GTC GTA CTA GGA TCA CAA GAA GGA GCA ATG CAC ACT GCG 1536 Gin Glu Val Val Val Leu Gly Ser Gin Glu Gly Ala Met His Thr Ala 500 505 510

CTG ACT GGA GCG ACA GAA ATC CAA ACG TCA GGA ACG ACA ACA ATT TTC 1584 Leu Thr Gly Ala Thr Glu He Gin Thr Ser Gly Thr Thr Thr He Phe 515 520 525

GCA GGA CAC CTA AAA TGC AGA CTA AAA ATG GAC AAA CTA ACT TTA AAA 1632 Ala Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys 530 535 540

GGG ATG TCA TAT GTG ATG TGC ACA GGC TCA TTC AAG TTA GAG AAA GAA 1680 Gly Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu 545 550 555 560

GTG GCT GAG ACC CAG CAT GGA ACT GTT CTG GTG CAG GTT AAA TAT GAA 1728 Val Ala Glu Thr Gin His Gly Thr Val Leu Val Gin Val Lys Tyr Glu 565 570 575

GGA ACA GAC GCA CCA TGC AAG ATT CCC TTT TCG ACC CAA GAT GAG AAA 1776 Gly Thr Asp Ala Pro Cys Lys He Pro Phe Ser Thr Gin Asp Glu Lys 580 585 590

GGA GCA ACC CAG AAT GGG AGA TTA ATA ACA GCC AAC CCC ATA GTC ACT 1824 Gly Ala Thr Gin Asn Gly Arg Leu He Thr Ala Asn Pro He Val Thr 595 600 605

GAC AAA GAA AAA CCA GTC AAT ATT GAG GCA GAA CCA CCC TTT GGT GAG 1872 Asp Lys Glu Lys Pro Val Asn He Glu Ala Glu Pro Pro Phe Gly Glu 610 615 620

AGC TAC ATC GTG GTA GGA GCA GGT GAA AAA GCT TTG AAA CTA AGC TGG 1920 Ser Tyr He Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp 625 630 635 640

TTC AAG AAA GGA AGC AGC ATA GGG AAA ATG TTT GAA GCA ACT GCC CGA 1968 Phe Lys Lys Gly Ser Ser He Gly Lys Met Phe Glu Ala Thr Ala Arg 645 650 655

GGA GCA CGA AGG ATG GCC ATT CTG GGA GAC ACC GCA TGG GAC TTC GGT 2016 Gly Ala Arg Arg Met Ala He Leu Gly Asp Thr Ala Trp Asp Phe Gly 660 665 670

TCT ATA GGA GGA GTG TTC ACG TCT ATG GGA AAA CTG GTA CAC CAG GTT 2064 Ser He Gly Gly Val Phe Thr Ser Met Gly Lys Leu Val His Gin Val 675 680 685

TTT GGA ACT GCA TAT GGA GTT TTG TTT AGC GGA GTT TCT TGG ACC ATG 2112 Phe Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met 690 695 700

AAA ATA GGA ATA GGG ATT CTG CTG ACA TGG CTA GGA TTA AAT TCA AGG 2160 Lys He Gly He Gly He Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg 705 710 715 720

AAC ACG TCC CTT TCG GTG ATG TGC ATC GCA GTT GGC ATG GTC ACA CTG 2208 Asn Thr Ser Leu Ser Val Met Cys He Ala Val Gly Met Val Thr Leu 725 730 735

TAC CTA GGA GTC ATG GTT CAG GCA GAT TCG GGA TGT GTA ATC AAC TGG 2256 Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys Val He Asn Trp 740 745 750

AAA GGC AGA GAA CTT AAA TGT GGA AGC GGC ATT TTT GTC ACT AAT GAA 2304 Lys Gly Arg Glu Leu Lys Cys Gly Ser Gly He Phe Val Thr Asn Glu 755 760 765

GTT CAC ACT TGG ACA GAG CAA TAC AAA TTC CAG GCT GAC TCC CCC AAG 2352 Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Ala Asp Ser Pro Lys 770 775 780

AGA CTA TCA GCA GCC ATT GGG AAG GCA TGG GAG GAG GGT GTG 2394

Arg Leu Ser Ala Ala He Gly Lys Ala Trp Glu Glu Gly Val 785 790 795

(2) INFORMATION FOR SEQ ID NO:125:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2145 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 1...2145 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:

AAG GTC TTA AAA GGC TTC AAG AAG GAG ATC TCA AAC ATG CTG AGC ATT 48 Lys Val Leu Lys Gly Phe Lys Lys Glu He Ser Asn Met Leu Ser He 1 5 10 15

ATC AAC AAA CGG AAA AAG ACA TCG CTC TGT CTC ATG ATG ATG TTA CCA 96 He Asn Lys Arg Lys Lys Thr Ser Leu Cys Leu Met Met Met Leu Pro 20 25 30

GCA ACA CTT GCT TTC CAC TTA ACT TCA CGA GAT GGA GAG CCG CGC ATG 144 Ala Thr Leu Ala Phe His Leu Thr Ser Arg Asp Gly Glu Pro Arg Met 35 40 45

ATT GTG GGG AAG AAT GAA AGA GGA AAA TCC CTA CTT TTC AAG ACA GCC 192 He Val Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala 50 55 60

TCT GGA ATC AAC ATG TGC ACA CTC ATA GCT ATG GAT CTG GGA GAG ATG 240 Ser Gly He Asn Met Cys Thr Leu He Ala Met Asp Leu Gly Glu Met 65 70 75 80

TGT GAT GAC ACG GTC ACT TAC AAA TGC CCC CAC ATT ACC GAA GTG GAG 288 Cys Asp Asp Thr Val Thr Tyr Lys Cys Pro His He Thr Glu Val Glu 85 90 95

CCT GAA GAC ATT GAC TGC TGG TGC AAC CTT ACA TCG ACA TGG GTG ACT 336 Pro Glu Asp He Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr 100 105 110

TAT GGA ACA TGC AAT CAA GCT GGA GAG CAT AGA CGC GAT AAG AGA TCA 384 Tyr Gly Thr Cys Asn Gin Ala Gly Glu His Arg Arg Asp Lys Arg Ser 115 120 125

GTG GCG TTA GCT CCC CAT GTT GGC ATG GGA CTG GAC ACA CGC ACT CAA 432 Val Ala Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gin 130 135 140

ACC TGG ATG TCG GCT GAA GGA GCT TGG AGA CAA GTC GAG AAG GTA GAG 480 Thr Trp Met Ser Ala Glu Gly Ala Trp Arg Gin Val Glu Lys Val Glu 145 150 155 160

ACA TGG GCC CTT AGG CAC CCA GGG TTT ACC ATA CTA GCC CTA TTT CTT 528 Thr Trp Ala Leu Arg His Pro Gly Phe Thr He Leu Ala Leu Phe Leu 165 170 175

GCC CAT TAC ATA GGC ACT TCC TTG ACC CAG AAA GTG GTT ATT TTT ATA 576 Ala His Tyr He Gly Thr Ser Leu Thr Gin Lys Val Val He Phe He 180 185 190

CTA TTA ATG CTG GTT ACC CCA TCC ATG ACA ATG AGA TGT GTA GGA GTA 624 Leu Leu Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val 195 200 205

GGA AAC AGA GAT TTT GTG GAA GGC CTA TCG GGA GCT ACG TGG GTT GAC 672 Gly Asn Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp 210 215 220

GTG GTG CTC GAG CAC GGT GGG TGT GTG ACT ACC ATG GCT AAG AAC AAG 720 Val Val Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys 225 230 235 240

CCC ACG CTG GAC ATA GAG CTT CAG AAG ACC GAG GCC ACC CAA CTG GCG 768 Pro Thr Leu Asp He Glu Leu Gin Lys Thr Glu Ala Thr Gin Leu Ala 245 250 255

ACC CTA AGG AAG CTA TGC ATT GAG GGA AAA ATT ACC AAC ATA ACA ACC 816 Thr Leu Arg Lys Leu Cys He Glu Gly Lys He Thr Asn He Thr Thr 260 265 270

GAC TCA AGA TGT CCC ACC CAA GGG GAA GCG ATT TTA CCT GAG GAG CAG 864 Asp Ser Arg Cys Pro Thr Gin Gly Glu Ala He Leu Pro Glu Glu Gin 275 280 285

GAC CAG AAC TAC GTG TGT AAG CAT ACA TAC GTG GAC AGA GGC TGG GGA 912 Asp Gin Asn Tyr Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly 290 295 300

AAC GGT TGT GGT TTG TTT GGC AAG GGA AGC TTG GTG ACA TGC GCG AAA 960 Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys 305 310 315 320

TTT CAA TGT TTA GAA TCA ATA GAG GGA AAA GTG GTG CAA CAT GAG AAC 1008 Phe Gin Cys Leu Glu Ser He Glu Gly Lys Val Val Gin His Glu Asn 325 330 335

CTC AAA TAC ACC GTC ATC ATC ACA GTG CAC ACA GGA GAC CAA CAC CAG 1056 Leu Lys Tyr Thr Val He He Thr Val His Thr Gly Asp Gin His Gin 340 345 350

GTG GGA AAT GAA ACG CAG GGA GTC ACG GCT GAG ATA ACA CCC CAG GCA 1104 Val Gly Asn Glu Thr Gin Gly Val Thr Ala Glu He Thr Pro Gin Ala 355 360 365

TCA ACC GCT GAA GCC ATT TTA CCT GAA TAT GGA ACC CTC GGG CTA GAA 1152 Ser Thr Ala Glu Ala He Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu 370 375 380

TGC TCA CCA CGG ACA GGT TTG GAT TTC AAT GAA ATG ATC TCA TTG ACA 1200 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met He Ser Leu Thr 385 390 395 400

ATG AAG AAC AAA GCA TGG ATG GTA CAT AGA CAA TGG TTC TTT GAC TTA 1248 Met Lys Asn Lys Ala Trp Met Val His Arg Gin Trp Phe Phe Asp Leu 405 410 415

CCC CTA CCA TGG ACA TCA GGA GCT ACA GCA GAA ACA CCA ACT TGG AAC 1296 Pro Leu Pro Trp Thr Ser Gly Ala Thr Ala Glu Thr Pro Thr Trp Asn 420 425 430

AGG AAA GAG CTT CTT GTG ACA TTT AAA AAT GCA CAT GCA AAA AAG CAA 1344 Arg Lys Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gin 435 440 445

GAA GTA GTT GTT CTT GGA TCA CAA GAG GGA GCA ATG CAT ACA GCA CTG 1392 Glu Val Val Val Leu Gly Ser Gin Glu Gly Ala Met His Thr Ala Leu 450 455 460

ACA GGA GCT ACA GAG ATC CAA ACC TCA GGA GGC ACA AGT ATC TTT GCG 1440 Thr Gly Ala Thr Glu He Gin Thr Ser Gly Gly Thr Ser He Phe Ala 465 470 475 480

GGG CAC TTA AAA TGT AGA CTC AAG ATG GAC AAA TTG GAA CTC AAA GGG 1488 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly 485 490 495

ATG AGC TAT GCA ATG TGC TTG GGT AGC TTT GTG TTG AAG AAA GAA GTC 1536 Met Ser Tyr Ala Met Cys Leu Gly Ser Phe Val Leu Lys Lys Glu Val 500 505 510

TCC GAA ACG CAG CAT GGG ACA ATA CTC ATT AAG GTT GAG TAC AAA GGG 1584 Ser Glu Thr Gin His Gly Thr He Leu He Lys Val Glu Tyr Lys Gly 515 520 525

AAA GAT GCA CCC TGC AAG ATT CCT TTC TCC ACG GAG GAT GGA CAA GGA 1632 Lys Asp Ala Pro Cys Lys He Pro Phe Ser Thr Glu Asp Gly Gin Gly 530 535 540

AAA GCT CAC AAT GGC AGA CTG ATC ACA GCC AAT CCA GTG GTG ACC AAG 1680 Lys Ala His Asn Gly Arg Leu He Thr Ala Asn Pro Val Val Thr Lys 545 550 555 560

AAG GAG GAG CCT GTC AAC ATT GAG GCT GAA CCT CCT TTT GGA GAA AGT 1728 Lys Glu Glu Pro Val Asn He Glu Ala Glu Pro Pro Phe Gly Glu Ser 565 570 575

AAC ATA GTA ATT GGA ATT GGA GAC AAA GCC CTG AAA ATC AAC TGG TAC 1776 Asn He Val He Gly He Gly Asp Lys Ala Leu Lys He Asn Trp Tyr 580 585 590

AAG AAG GGA AGC TCG ATT GGG AAG ATG TTC GAG GCT ACT GCC AGA GGT 1824 Lys Lys Gly Ser Ser He Gly Lys Met Phe Glu Ala Thr Ala Arg Gly 595 600 605

GCA AGG CGC ATG GCC ATC TTG GGA GAC ACA GCC TGG GAC TTT GGA TCA 1872 Ala Arg Arg Met Ala He Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 610 615 620

GTG GGT GGT GTT TTG AAT TCA TTA GGG AAA ATG GTC CAC CAA ATA TTT 1920 Val Gly Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gin He Phe 625 630 635 640

GGG AGT GCT TAC ACA GCC CTA TTT GGT GGA GTC TCC TGG ATG ATG AAA 1968 Gly Ser Ala Tyr Thr Ala Leu Phe Gly Gly Val Ser Trp Met Met Lys 645 650 655

ATT GGA ATA GGT GTC CTC TTA ACC TGG ATA GGG TTG AAC TCA AAA AAT 2016 He Gly He Gly Val Leu Leu Thr Trp He Gly Leu Asn Ser Lys Asn 660 665 670

ACT TCT ATG TCA TTT TCA TGC ATC GCG ATA GGA ATC ATT ACA CTC TAT 2064 Thr Ser Met Ser Phe Ser Cys He Ala He Gly He He Thr Leu Tyr 675 680 685

CTG GGA GCC GTG GTG CAA GCT GAC ATG GGG TGT GTC ATA AAC TGG AAA 2112 Leu Gly Ala Val Val Gin Ala Asp Met Gly Cys Val He Asn Trp Lys 690 695 700

GGC AAA GAA CTC AAA TGT GGA AGT GGA ATT TTC 2145

Gly Lys Glu Leu Lys Cys Gly Ser Gly He Phe 705 710 715

(2) INFORMATION FOR SEQ ID NO:126:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2175 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 1...2175 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:

ATT CTG AAG AGA TGG GGA CAG TTG AAG AAA AAT AAG GCC ATC AGG ATA 48 He Leu Lys Arg Trp Gly Gin Leu Lys Lys Asn Lys Ala He Arg He 1 5 10 15

CTG ATT GGA TTC AGG AAG GAG ATA GGC CGC ATG CTG AAC ATC TTG AAC 96 Leu He Gly Phe Arg Lys Glu He Gly Arg Met Leu Asn He Leu Asn 20 25 30

GGG AGA AAA AGG TCA ACG ATA ACA TTG CTG TGC TTG ATT CCC ACC GTA 144 Gly Arg Lys Arg Ser Thr He Thr Leu Leu Cys Leu He Pro Thr Val 35 40 45

ATG GCG TTT CAC TTG TCA ACA AGA GAT GGC GAA CCC CTC ATG ATA GTG 192 Met Ala Phe His Leu Ser Thr Arg Asp Gly Glu Pro Leu Met He Val 50 55 60

GCA AAA CAT GAA AGG GGG AGA CCT CTC TTG TTT AAG ACA ACA GAG GGG 240 Ala Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly 65 70 75 80

ATC AAC AAA TGC ACT CTC ATT GCC ATG GAC TTG GGT GAA ATG TGT GAG 288 He Asn Lys Cys Thr Leu He Ala Met Asp Leu Gly Glu Met Cys Glu 85 90 95

GAC ACT GTC ACG TAT AAA TGC CCC TTA CTG GTC AAT ACC GAA CCT GAA 336 Asp Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr Glu Pro Glu 100 105 110

GAC ATT GAT TGC TGG TGC AAT CTC ACG TCT ACC TGG GTC ACA TAT GGG 384 Asp He Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly 115 120 125

ACA TAC ACC CAG AGC GGA GAA CGG AGA CGA GAG AAG CGC TCA GTA GCT 432 Thr Tyr Thr Gin Ser Gly Glu Arg Arg Arg Glu Lys Arg Ser Val Ala 130 135 140

TTA ACA CCA CAT TCA GGA ATG GGA TTG GAA ACA AGA GCT GAG ACA TGG 480 Leu Thr Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp 145 150 155 160

ATG TCA TCG GAA GGG GCT TGG AAG CAT GCT CAG AGA GTA GAG AGC TGG 528 Met Ser Ser Glu Gly Ala Trp Lys His Ala Gin Arg Val Glu Ser Trp 165 170 175

ATA CTC AGA AAC CCA GGA TTC GCG CTC TTG GCA GGA TTT ATG GCT TAT 576 He Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr 180 185 190

ATG ATT GGG CAA ACA GGA ATC CAG CGA ACT GTC TTC TTT GTC CTA ATG 624 Met He Gly Gin Thr Gly He Gin Arg Thr Val Phe Phe Val Leu Met 195 200 205

ATG CTG GTC GCC CCA TCC TAC GGA ATG CGA TGC GTA GGA GTA GGA AAC 672 Met Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val Gly Asn 210 215 220

AGA GAC TTT GTG GAA GGA GTC TCA GGT GGA GCA TGG GTC GAT CTG GTG 720 Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val Asp Leu Val 225 230 235 240

CTA GAA CAT GGA GGA TGC GTC ACA ACC ATG GCC CAG GGA AAA CCA ACC 768 Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Gin Gly Lys Pro Thr 245 250 255

TTG GAT TTT GAA CTG ACT AAG ACA ACA GCC AAG GAA GTG GCT CTG TTA 816 Leu Asp Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu 260 265 270

AGA ACC TAT TGC ATT GAA GCC TCA ATA TCA AAC ATA ACC ACG GCA ACA 864 Arg Thr Tyr Cys He Glu Ala Ser He Ser Asn He Thr Thr Ala Thr 275 280 285

AGA TGT CCA ACG CAA GGA GAG CCT TAT CTA AAA GAG GAA CAA GAC CAA 912 Arg Cys Pro Thr Gin Gly Glu Pro Tyr Leu Lys Glu Glu Gin Asp Gin 290 295 300

CAG TAC ATT TGC CGG AGA GAT GTG GTA GAC AGA GGG TGG GGC AAT GGC 960 Gin Tyr He Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly 305 310 315 320

TGT GGC TTG TTT GGA AAA GGA GGA GTT GTG ACA TGT GCG AAG TTT TCA 1008 Cys Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser 325 330 335

TGT TCG GGG AAG ATA ACA GGC AAT TTG GTC CAA ATT GAG AAC CTT GAA 1056 Cys Ser Gly Lys He Thr Gly Asn Leu Val Gin He Glu Asn Leu Glu 340 345 350

TAC ACA GTG GTT GTA ACA GTC CAC AAT GGA GAC ACC CAT GCA GTA GGA 1104 Tyr Thr Val Val Val Thr Val His Asn Gly Asp Thr His Ala Val Gly 355 360 365

AAT GAC ACA TCC AAT CAT GGA GTT ACA GCC ACG ATA ACT CCC AGG TCA 1152 Asn Asp Thr Ser Asn His Gly Val Thr Ala Thr He Thr Pro Arg Ser 370 375 380

CCA TCG GTG GAA GTC AAA TTG CCG GAC TAT GGA GAA CTA ACA CTC GAT 1200 Pro Ser Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp 385 390 395 400

TGT GAA CCC AGG TCT GGA ATT GAC TTT AAT GAG ATG ATT CTG ATG AAA 1248 Cys Glu Pro Arg Ser Gly He Asp Phe Asn Glu Met He Leu Met Lys 405 410 415

ATG AAA AAG AAA ACA TGG CTT GTG CAT AAG CAA TGG TTT TTG GAT CTA 1296 Met Lys Lys Lys Thr Trp Leu Val His Lys Gin Trp Phe Leu Asp Leu 420 425 430

CCT CTA CCA TGG ACA GCA GGA GCA GAC ACA TCA GAG GTT CAC TGG AAT 1344 Pro Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn 435 440 445

TAC AAA GAG AGA ATG GTG ACA TTT AAG GTT CCT CAT GCC AAG AGA CAG 1392 Tyr Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gin 450 455 460

GAT GTG ACA GTG CTG GGA TCT CAG GAA GGA GCC ATG CAT TCT GCC CTC 1440 Asp Val Thr Val Leu Gly Ser Gin Glu Gly Ala Met His Ser Ala Leu 465 470 475 480

GCT GGA GCC ACA GAA GTG GAC TCC GGT GAT GGA AAT CAC ATG TTT GCA 1488 Ala Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala 485 490 495

GGA CAT CTC AAG TGC AAA GTC CGT ATG GAG AAA TTG AGA ATC AAG GGA 1536 Gly His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg He Lys Gly 500 505 510

ATG TCA TAC ACG ATG TGT TCA GGA AAG TTC TCA ATT GAC AAA GAG ATG 1584 Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser He Asp Lys Glu Met 515 520 525

GCA GAA ACA CAG CAT GGG ACA ACA GTG GTG AAA GTC AAG TAT GAA GGT 1632 Ala Glu Thr Gin His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly 530 535 540

GCT GGA GCT CCG TGT AAA GTC CCC ATA GAG ATA AGA GAT GTG AAC AAG 1680 Ala Gly Ala Pro Cys Lys Val Pro He Glu He Arg Asp Val Asn Lys 545 550 555 560

AAA AAA GTG GTT GGG CGT ATC ATC TCA TCC ACC CCT TTG GCT GAG AAT 1728 Lys Lys Val Val Gly Arg He He Ser Ser Thr Pro Leu Ala Glu Asn 565 570 575

ACC AAC AGT GCA ACC AAC ATA GAG TTA GAA CCC CCC TTT GGG GAC AGC 1776 Thr Asn Ser Ala Thr Asn He Glu Leu Glu Pro Pro Phe Gly Asp Ser 580 585 590

TAC ATA GTG ATA GGT GTT GGA AAC AGT GCA TTA ACA CTC CAT TGG TTC 1824 Tyr He Val He Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe 595 600 605

AGG AAA GGG AGT TCC ATT GGC AAG ATG TTT GAG TCC ACA TAC AGA GGT 1872 Arg Lys Gly Ser Ser He Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly 610 615 620

GCA AAA CGA ATG GCC ATT CTA GGT GAA ACA GCT TGG GAT TTT GGT TCC 1920 Ala Lys Arg Met Ala He Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser 625 630 635 640

GTT GGT GGA CTG TTC ACA TCA TTG GGA AAG GCT GTG CAC CAG GTT TTT 1968 Val Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala Val His Gin Val Phe 645 650 655

GGA AGT GTG TAT ACA ACC ATG TTT GGA GGA GTC TCA TGG ATG ATT AGA 2016 Gly Ser Val Tyr Thr Thr Met Phe Gly Gly Val Ser Trp Met He Arg 660 665 670

ATC CTA ATT GGG TTC CTA GTG TTG TGG ATT GGC ACG AAC TCA AGG AAC 2064 He Leu He Gly Phe Leu Val Leu Trp He Gly Thr Asn Ser Arg Asn 675 680 685

ACT TCA ATG GCT ATG ACG TGC ATA GCT GTT GGA GGA ATC ACT CTG TTT 2112 Thr Ser Met Ala Met Thr Cys He Ala Val Gly Gly He Thr Leu Phe 690 695 700

CTG GGC TTC ACA GTT CAA GCA GAG ATG GGT TGT GTG GTG TCA TGG AGT 2160 Leu Gly Phe Thr Val Gin Ala Glu Met Gly Cys Val Val Ser Trp. Ser 705 710 715 720

GGG AAA GAA TTG AGG 2175

Gly Lys Glu Leu Arg 725

(2) INFORMATION FOR SEQ ID NO:127:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:

CACTACCGCA AGGTAGAGAG CTCGGCATT1 CCTCTTGGTG 40

(2) INFORMATION FOR SEQ ID NO:128:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:

GTGATGGCGT TCCATCTCTC GAGCCGTAAC GGAGAACCAC 40

(2) INFORMATION FOR SEQ ID NO:129:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: GCCCTGGCGT TCCATCTCTC GAGCCGAGGG GGAGAGCCGC 40 (2) INFORMATION FOR SEQ ID NO:130:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:

ACACTTGCTT TCCACCTCTC GAGCCGAGAT GGAGAGCCGC 40

(2) INFORMATION FOR SEQ ID NO:131:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: GTAATGGCGT TTCACCTCTC GAGCAGAGAT GGCGAACCCC 40 (2) INFORMATION FOR SEQ ID NO:132:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:

CCTATCCTTA CTTAAGATCT TCGTGGAGTG ACAGAC 36

(2) INFORMATION FOR SEQ ID NO:133:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:

GGATAGGAAT GAATTCTAGA AGCACCTCAC TGTCTG 36

(2) INFORMATION FOR SEQ ID NO:134:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:

CCGCAGAGAT CGTTTTCCTG CCTGCATGAT TCC 33

(2) INFORMATION FOR SEQ ID NO:135:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:

CCGATCCTAA TTTAAGATCT TTGTGCAGGG AAAGCC 36

(2) INFORMATION FOR SEQ ID NO:136:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:

CCTATCCCAA CTTGAGATCT TTATGAAGAT ACAGTA 36

(2) INFORMATION FOR SEQ ID NO:137:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:

CCTAACCGTG CTTGAGATCT TTGTGAAGTT ACCGAC 36