Plants and cells transformed with a nucleic acid from Bacillus thuringiensis strain KB59A4-6 encoding a novel SUP toxin

Culturing of Bacillus Isolates Useful According to the Invention

The cellular host containing the Bacillus insecticidal gene may be grown in any convenient nutrient medium. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

The Bacillus cells of the invention can be cultured using standard art media and fermentation techniques. During the fermentation cycle, the bacteria can be harvested by first separating the Bacillus vegetative cells, spores, crystals, and lysed cellular debris from the fermentation broth by means well known in the art. Any Bacillus spores or crystal δ-endotoxins formed can be recovered employing well-known techniques and used as a conventional δ-endotoxin B.t . preparation. The supernatant from the fermentation process contains toxins of the present invention. The toxins are isolated and purified employing well-known techniques.

A subculture of Bacillus isolates, or mutants thereof, can be used to inoculate the following medium, known as TB broth:

The potassium phosphate was added to the autoclaved broth after cooling. Flasks were incubated at 30° C. on a rotary shaker at 250 rpm for 24-36 hours.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The Bacillus obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation. In a specific embodiment, Bacillus proteins useful according the present invention can be obtained from the supernatant. The culture supernatant containing the active protein(s) can be used in bioassays.

Alternatively, a subculture of Bacillus isolates, or mutants thereof, can be used to inoculate the following peptone, glucose, salts medium:

The salts solution and CaCl 2 solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The Bacillus spores and/or crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.

Isolation and Preparation of Cellular DNA for PCR

DNA can be prepared from cells grown on Spizizen's agar, or other minimal or enriched agar known to those skilled in the art, for approximately 16 hours. Spizizen's casamino acid agar comprises 23.2 g/l Spizizen's minimal salts [(NH 4 ) 2 SO 4 , 120 g; K 2 HPO 4 , 840 g; KH 2 PO 4 , 360 g; sodium citrate, 60 g; MgSO 4 . 7H 2 O, 12 g. Total: 1392 g]; 1.0 g/l vitamin-free casamino acids; 15.0 g/l Difco agar. In preparing the agar, the mixture was autoclaved for 30 minutes, then a sterile, 50% glucose solution can be added to a final concentration of 0.5% (1/100 vol). Once the cells are grown for about 16 hours, an approximately 1 cm 2 patch of cells can be scraped from the agar into 300 μl of 10 mM Tris-HCl (pH 8.0)−1 mM EDTA. Proteinase K was added to 50 μg/ml and incubated at 55° C. for 15 minutes. Other suitable proteases lacking nuclease activity can be used. The samples were then placed in a boiling water bath for 15 minutes to inactivate the proteinase and denature the DNA. This also precipitates unwanted components. The samples are then centrifuged at 14,000×g in an Eppendorf microfuge at room temperature for 5 minutes to remove cellular debris. The supernatants containing crude DNA were transferred to fresh tubes and frozen at −20° C. until used in PCR reactions.

Alternatively, total cellular DNA may be prepared from plate-grown cells using the QIAamp Tissue Kit from Qiagen (Santa Clarita, Calif.) following instructions from the manufacturer.

Primers Useful for Characterizing and/or Identifying Toxin Genes

The following set of PCR primers can be used to identify and/or characterize genes of the subject invention, which encode pesticidal toxins:

GGRTTAMTTGGRTAYTATTT (SEQ ID NO. 3)

ATATCKWAYATTKGCATTTA (SEQ ID NO. 4)

Redundant nucleotide codes used throughout the subject disclosure are in accordance with the IUPAC convention and include:

R=A or G

M=A or C

Y=C or T

K=G or T

W=A or T

Identification and Sequencing of Genes Encoding Novel Soluble Protein Toxins From Bacillus Strains

PCR using primers SEQ ID NO. 3 and SEQ ID NO. 4 was performed on total cellular genomic DNA isolated from a broad range of B.t . strains. Those samples yielding an approximately 1 kb band were selected for characterization by DNA sequencing. Amplified DNA fragments were first cloned into the PCR DNA TA-cloning plasmid vector, pCR2.1, as described by the supplier (Invitrogen, San Diego, Calif.). Plasmids were isolated from recombinant clones and tested for the presence of an approximately 1 kbp insert by PCR using the plasmid vector primers, T3 and T7.

The following strains yielded the expected band of approximately 1000 bp, thus indicating the presence of a MIS-type toxin gene: PS66D3, PS177C8, PS177I8, PS33F1, PS157C1 (157C1-A), PS201Z, PS31F2, and PS185Y2.

Plasmids were then isolated for use as sequencing templates using QIAGEN (Santa Clarita, Calif.) miniprep kits as described by the supplier. Sequencing reactions were performed using the Dye Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems. Sequencing reactions were run on a ABI PRISM 377 Automated Sequencer. Sequence data was collected, edited, and assembled using the ABI PRISM 377 Collection, Factura, and AutoAssembler software from PE ABI. DNA sequences were determined for portions of novel toxin genes from the following isolates: PS66D3, PS177C8, PS177I8, PS33F1, PS157C1 (157C1-A), PS201Z, PS31F2, and PS185Y2. These nucleotide sequences are shown in SEQ ID NOS. 5, 7, 9, 38, 33, 35, 36, and 37, respectively. Polypeptide sequences were deduced for portions of the encoded, novel soluble toxins from the following isolates: PS66D3, PS177C8, PS177I8, and PS157C1 (toxin 157C1-A). These nucleotide sequences are shown in SEQ ID NOS. 6, 8, 10, and 34, respectively.

Restriction Fragment Length Polymorphism (RFLP) of Toxins From Bacillus thuringiensis Strains

Total cellular DNA was prepared from various Bacillus thuriengensis ( B.t .) strains grown to an optical density of 0.5-0.8 at 600 nm visible light. DNA was extracted using the Qiagen Genomic-tip 500/G kit and Genomic DNA Buffer Set according to protocol for Gram positive bacteria (Qiagen Inc.; Valencia, Calif.).

Standard Southern hybridizations using 32 P-lableled probes were used to identifiy and characterize novel toxin genes within the total genomic DNA preparations. Prepared total genomic DNA was digested with various restriction enzymes, electrophoresed on a 1% agarose gel, and immobilized on a supported nylon membrane using standard methods (Maniatis et al.).

PCR-amplified DNA fragments 1.0-1.1 kb in length were gel purified for use as probes. Approximately 25 ng of each DNA fragment was used as a template for priming nascent DNA synthesis using DNA polymerase I Klenow fragment (New England Biolabs), random hexanucleotide primers (Boehringer Mannheim) and 32 PdCTP.

Each 32 P-lableled fragment served as a specific probe to its corresponding genomic DNA blot. Hybridizations of immobilized DNA with randomly labeled 32 P probes were performed in standard aqueous buffer consisting of 5×SSPE, 5×Denhardt's solution, 0.5% SDS, 0.1 mg/ml at 65° C. overnight. Blots were washed under moderate stringency in 0.2×SSC, 0.1% SDS at 65° C. and exposed to film. RFLP data showing specific hybridization bands containing all or part of the novel gene of interest was obtained for each strain.

In separate experiments, alternative probes for MIS and WAR genes were used to detect novel toxin genes on Southern blots of genomic DNA by 32 P autoradiography or by non-radioactive methods using the DIG nucleic acid labeling and detection system (Boehringer Mannheim; Indianapolis, Ind.). DNA fragments approximately 2.6 kbp (PS177C8 MIS toxin gene; SEQ ID NO. 7) and 1.3 kbp (PS177C8 WAR toxin gene; SEQ ID NO. 11) in length were PCR amplified from plasmid pMYC2450 using primers homologous to the 5′ and 3′ ends of each respective gene. pMYC2450 is a recombinant plasmid containing the PS177C8 MIS and WAR genes on an approximately 14 kbp ClaI fragment in pHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised of pBluescript S/K [Stratagene, La Jolla, Calif.] and the replication origin from a resident B.t . plasmed [D. Lereclus et al. 1989; FEMS Microbiology Letters 60:211-218]). These DNA fragments were used as probes for MIS RFLP classes A through N and WAR RFLP classes A through L. RFLP data in Table 4 for class O was generated using MIS fragments approximately 1636 bp amplified with primers S1-633F (CACTCAAAAAATGAAAAGGGAAA; SEQ ID NO. 39) and S1-2269R (CCGGTTTTATTGATGCTAC; SEQ ID NO. 40). RFLP data in Table 5 for class M was generated using WAR fragments approximately 495 bp amplified with primers S2-501F (AGAACAATTTTTAGATAGGG; SEQ ID NO. 41) and S2-995R (TCCCTAAAGCATCAGAAATA; SEQ ID NO 42).

Fragments were gel purified and approximately 25 ng of each DNA fragment was randomly labeled with 32 P for radioactive detection or approximately 300 ng of each DNA fragment was randomly labeled with the DIG High Prime kit for nonradioactive detection. Hybridization of immobilized DNA with randomly labeled 32 P probes were performed in standard formamide conditions: 50% formamide, 5×SSPE, 5×Denhardt's solution, 2% SDS, 0.1 mg/ml sonicated sperm DNA at 42° C. overnight. Blots were washed under low stringency in 2×SSC, 0.1% SDS at 42° C. and exposed to film. RFLP data showing DNA bands containing all or part of the novel gene of interest was obtained for each strain.

RFLP data using MIS probes as discussed above were as follows:

RFLP data using WAR probes as discussed above were as follows:

Characterization and/or Identification of WAR Toxins

In a further embodiment of the subject invention, pesticidal toxins can be characterized and/or identified by their level of reactivity with antibodies to pesticidal toxins exemplified herein. In a specific embodiment, antibodies can be raised to WAR toxins such as the toxin obtainable from PS177C8a. Other WAR toxins can then be identified and/or characterized by their reactivity with the antibodies. In a preferred embodiment, the antibodies are polyclonal antibodies. In this example, toxins with the greatest similarity to the 177C8a-WAR toxin would have the greatest reactivity with the polyclonal antibodies. WAR toxins with greater diversity react with the 177C8a polyclonal antibodies, but to a lesser extent. Toxins which immunoreact with polyclonal antibodies raised to the 177C8a WAR toxin can be obtained from, for example, the isolates designated PS177C8a, PS177I8, PS66D3, KB68B55-2, PS185Y2, KB53A49-4, KB68B51-2, PS31F2, PS74H3, PS28M, PS71G6, PS71G7, PS71I1, PS71N1, PS201JJ7, KB73, KB68B46-2, KB71A35-4, KB71A116-1, PS70B2, PS71C2, PS86D1, HD573B, PS33F1, PS67B3, PS205C, PS40C1, PS130A3, PS143A2, PS157C1, PS201Z, PS71G4, KB42A33-8, KB71A72-1, KB71A133-11, KB71A134-2, KB69A125-3, KB69A127-7, KB69A136-2, and KB71A20-4. Isolates PS31F2 and KB68B46-2 show very weak antibody reactivity, suggesting advantageous diversity.

Molecular Cloning and DNA Sequence Analysis of Soluble Insecticidal Protein (MIS and WAR) Genes From Bacillus thuringiensis Strain PS205C

Total cellular DNA was prepared from Bacillus thuringensis strain PS205C grown to an optical density of 0.5-0.8 at 600 nm visible light in Luria Bertani (LB) broth. DNA was extracted using the Qiagen Genomic-tip 500/G kit and Genomic DNA Buffer Set according to the protocol for Gram positive bacteria (Qiagen Inc.; Valencia, Calif.). A PS205C cosmid library was constructed in the SuperCos vector (Stratragene) using inserts of PS205C total cellular DNA partially digested with Nde II. XL1-Blue cells (Stratagene) were transfected with packaged cosmids to obtain clones resistant to carbenicillin and kanamycin. 576 cosmid colonies were grown in 96-well blocks in 1 ml LB+carbenicillin (100 μg/ml)+kanamycin (50 μg/ml) at 37° C. for 18 hours and replica plated onto nylon filters for screening by hybridization.

A PCR amplicon containing approximately 1000 bp of the PS205C MIS gene was amplified from PS205 genomic DNA using primers SEQ ID NO. 3 and SEQ ID NO. 4 as described in Example 4. The DNA fragment was gel purified using QiaexII extraction (Qiagen). The probe was radiolabeled with 32 P-dCTP using the Prime-It II kit (Stratgene) and used in aqueous hybridization solution (6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA) with the colony lift filters at 65° C. for 16 hours. The colony lift filters were briefly washed 1× in 2×SSC/0. 1% SDS at room temperature followed by two additional washes for 10 minutes in 0.5×SSC/0.1% SDS. The filters were then exposed to X-ray film for 5.5 hours. One cosmid clone that hybridized strongly to the probe was selected for further analysis. This cosmid clone was confirmed to contain the MIS gene by PCR amplification with primers SEQ ID NO. 3 and SEQ ID NO. 4. This cosmid clone was designated as pMYC3105; recombinant E. coli XL-1Blue MR cells containing pMYC3105 are designated MR992.

A subculture of MR992 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Ill. 61604 USA on May 4, 1999. The accession number is NRRL B-30124. A truncated plasmid clone for PS205C was also deposited on May 4, 1999. The accession number is NRRL B-30122.

To sequence the PS205C MIS and WAR genes, random transposon insertions into pMYC3105 were generated using the GPS-1 Genome Priming System and protocols (New England Biolabs). The GPS2 trasposition vector encoding chloramphenicol resistance was chosen for selection of cosmids containing insertions. pMYC3105 cosmids that acquired transposons were identified by transformation and selection of E. coli XL1-Blue MR on media containing ampicillin, kanamycin and chloramphenicol. Cosmid templates were prepared from individual colonies for use as sequencing templates using the Multiscreen 96-well plasmid prep (Millipore). The MIS and WAR toxin genes encoded by pMYC3105 were sequenced with GPS2 primers using the ABI377 automated sequencing system and associated software. The MIS and WAR genes were found to be located next to one another in an apparent transcriptional operon. The nucleotide and deduced polypeptide sequences are designated as new SEQ ID NOS. 43-46.

Molecular Cloning and DNA Sequence Analysis of Soluble Insecticidal Protein (MIS and WAR) Genes From Bacillus Thuringensis Strain PS 31F2

a. Preparation and Cloning of Genomic DNA

Total cellular DNA was prepared from the Bacillus thuringensis strain PS31F2 grown to an optical density of 0.5-0.8 at 600 nm visible light in Luria Bertani (LB) broth. DNA was extracted using the Qiagen Genomic-tip 500/G kit or Genomic-Tip 20/G and Genomic DNA Buffer Set (Qiagen Inc.; Valencia, Calif.) according to the protocol for Gram positive bacteria.

Lambda libraries containing total genomic DNA from Bacillus thuringensis strain PS31F2 were prepared from DNA partially digested with NdeII. Partial NdeII restriction digests were electrophoresed on a 0.7% agarose gel and the region of the gel containing DNA fragments within the size range of 9-20 kbp was excised from the gel. DNA was electroeluted from the gel fragment in 0.1×TAE buffer at approximately 30 V for one hour and purified using Elutip-d columns (Schleicher and Schuell; Keene, N.H.).

Purified, fractionated DNA was ligated into BamHI-digested Lambda-GEM-11 arms (Promega Corp., Madison, Wis.). Ligated DNA was then packaged into lambda phage using Gigapack III Gold packaging extract (Stratagene Corp., La Jolla, Calif.). E. coli strain KW251 was infected with recombinant phage and plated onto LB plates in LB top agarose. Plaques were lifted onto nitrocellulose filters and prepared for hybridization using standard methods (Maniatis, et al.). DNA fragments approximately 1.1 kb (PS177C8 MIS) or 700 bp (PS177C8 WAR) in length were PCR amplified from plasmid pMYC2450 and used as the probes. Fragments were gel purified and approximately 25 ng of each DNA fragment was randomly labeled with 32 P-dCTP. Hybridization of immobilized DNA with randomly 32 P-labeled PS177C8 probes was performed in standard formamide conditions: 50% formamide, 5×SSPE, 5×Denhardt's solution, 2% SDS, 0.1 mg/ml at 42° C. overnight. Blots were washed under low stringency in 2×SSC, 0.1% SDS at 42° C. and exposed to film. Hybridizing plaques were isolated from the plates and suspended in SM buffer. Phage DNA was prepared using LambdaSorb phage adsorbent (Promega, Madison, Wis.). PCR using the oligonucleotide primers SEQ ID NO. 3 and SEQ ID NO. 4 was performed using phage DNA templates to verify the presence of the target gene. The PCR reactions yielded the expected 1 kb band in both DNA samples confirming that those phage clones contain the gene of interest. For subcloning, phage DNA was digested with various enzymes, fractionated on a 1% agarose gel and blotted for Southern analysis. Southern analysis was performed as decribed above. A HindIII fragment approximately 8 kb in size was identified that contained the PS31F2 toxin genes. This fragment was gel purified and cloned into the HindIII site of pBluescriptII (SK+); this plasmid clone is designated pMYC2610. The recombinant E. coli XL10Gold [pMYC2610] strain was designated MR983.

A subculture of MR983 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Ill. 61604 USA on May 4, 1999. The accession number is NRRL B-30123.

b. DNA Sequencing

The pMYC2610 HindIII fragment containing the PS31F2 toxin genes was isolated by restriction digestion, fractionation on a 0.7% agarose gel and purification from the gel matrix using the QiaexII kit (Qiagen Inc.; Valencia, Calif.). Gel purified insert DNA was then digested separately with restriction enzymes AluI, MseI, or RsaI and fractionated on a 1% agarose gel. DNA fragments between 0.5 and 1.5 kb were excised from the gel and purified using the QiaexII kit. Recovered fragments were ligated into EcoRV digested pBluescriptII and transformed into E. coli XL10 Gold cells. Plasmid DNA was prepared from randomly chosen transformants, digested with NotI and ApaI to verify insert size and used as sequencing templates with primers homologous to plasmid vector sequences. Primer walking was used to complete the sequence. Sequencing reactions were performed using dRhodamine or BigDye Sequencing kit (ABI Prism/Perkin Elmer Applied Biosystems) and run on ABI 373 or 377 automated sequencers. Data was analyzed using Factura, Autoassembler (ABI Prism) and Gentics Computer Group (Madison, Wis.) programs. The MIS and WAR genes were found to be located next to one another in an apparent transcriptional operon. The WAR gene is 5′ to the MIS gene, and the two genes are separated by 4 nucleotide bases.

The nucleotide sequences and deduced peptide sequences for the novel MIS and WAR genes from PS31F2 are reported as new SEQ ID NOS. 47-50.

c. Subcloning and Transformation of B. thuringiensis

The PS31F2 toxin genes were subcloned on the 8 kbp HinDIII fragment from pMYC2610 into the E. coli/B.t . shuttle vector, pHT370 (O. Arantes and D. Lereclus. 1991. Gene 108: 115-119), for expression from the native Bacillus promoter. The resulting plasmid construct was designated pMYC2615. pMYC2415 plasmid DNA was prepared from recombinant E.coli XL10Gold for transformation into the acrystallierous (Cry-) B.t . host, CryB (A. Aronson, Purdue University, West Lafayette, Ind.), by electroporation. The recombinant CryB [pMYC2615] strain was designated MR558.

Molecular Cloning and DNA Sequence Analysis of a Novel SUP Toxin Gene From Bacillus Thuringiensis strain KB59A4-6

Total cellular DNA was prepared from the Bacillus thuringensis strain KB59A4-6 grown to an optical density of 0.5-0.8 at 600 nm visible light in Luria Bertani (LB) broth. DNA was extracted using the Qiagen Genomic-tip 500/G kit and Genomic DNA Buffer Set according to the protocol for Gram positive bacteria (Qiagen Inc.; Valencia, Calif.). DNA was digested with HinDIII and run on 0.7% agarose gels for Southern blot analysis by standard methods (Maniatis et al.). A PCR amplicon containing the SUP-like gene (SEQ ID NO. 1) from Javelin-90 genomic DNA was obtained by using the oligos “3A-atg (GCTCTAGAAGGAGGTAACTTATGAACAAGAATAATACTAAATTAAGC) (SEQ ID NO. 51) and “3A-taa” (GGGGTACCTTACTTAATAGAGACATCG) (SEQ ID NO. 52). This DNA fragment was gel purified and labeled with radioactive 32 P-dCTP using Prime-It II Random Primer Labeling Kit (Stratagene) for use as a probe. Hybridization of Southern blot filters was carried out in a solution of 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA at 42° C. overnight in a shaking water bath. The filters were subsequently washed in 1×SSPE and 0.1% SDS once at 25° C. followed by two additional washes at 37° C. Hybridized filters were then exposed to X-ray film at −80° C. An approximately 1 kbp HinDIII fragment of KB59A4-6 genomic DNA was identified that hybridized to the Javelin 90 SUP probe.

A lambda library of KB59A4-6 genomic DNA was constructed as follows. DNA was partially digested with Sau3A and size-fractionated on agarose gels. The region of the gel containing fragments between 9.0 and 23 kbp was excised and DNA was isolated by electroelution in 0.1×TAE buffer followed by purification over Elutip-d columns (Schleicher and Schuell, Keene, N.H.). Size-fractionated DNA inserts were ligated into BamHI-digested Lambda-Gem 11 (Promega) and recombinant phage were packaged using GigapackIII XL Packing Extract (Stratagene). Phage were plated on E. coli VCS257 cells for screening by hybridization. Plaques were transferred to nylon filters and dried under vacuum at 80° C. Hybridization was then performed with the Javelin 90 Sup gene probe as described above. One plaque that gave a positive signal was selected using a Pasteur pipette to obtain a plug. The plug was soaked over-night at room temperature in 1 mL SM buffer+10 uL CHCl 3 . Large-scale phage DNA preparations (Maniatis et al.) were obtained from liquid lysates of E. coli KW251 infected with this phage.

The KB59A4-6 toxin gene was subcloned into the E. coli/B. thuringiensis shuttle vector, pHT370 (O. Arantes and D. Lereclus. 1991. Gene 108: 115-119), on an approximately 5.5 kbp SacI/XbaI fragment identified by Southern hybridization. This plasmid subclone was designated pMYC2473. Recombinant E. coli XL10-Gold cells (Stratagene) containing this construct are designated MR993. The insecticidal toxin gene was sequenced by primer walking using pMYC2473 plasmid and PCR amplicons as DNA templates. Sequencing reactions were performed using the Dye Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems and run on a ABI PRISM 377 Automated Sequencer. Sequence data was analyzed using the PE ABI PRISM 377 Collection, Factura, and AutoAssembler software. The DNA sequence and deduced peptide sequence of the KB59A4-6 toxin are reported as new SEQ ID NOS. 53 and 54, respectively.

A subculture of MR993 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Ill. 61604 USA on May 4, 1999. The accession number is NRRL B-30125.

Bioassays for Activity Against Lepidopterans and Coleopterans

Biological activity of the toxins and isolates of the subject invention can be confirmed using standard bioassay procedures. One such assay is the budworm-bollworm ( Heliothis virescens [Fabricius] and Helicoverpa zea [Boddie]) assay. Lepidoptera bioassays were conducted with either surface application to artificial insect diet or diet incorporation of samples. All Lepidopteran insects were tested from the neonate stage to the second instar. All assays were conducted with either toasted soy flour artificial diet or black cutworm artificial diet (BioServ, Frenchtown, N.J.).

Diet incorporation can be conducted by mixing the samples with artificial diet at a rate of 6 mL suspension plus 54 mL diet. After vortexing, this mixture is poured into plastic trays with compartmentalized 3-ml wells (Nutrend Container Corporation, Jacksonville, Fla.). A water blank containing no B.t . serves as the control. First instar larvae (USDA-ARS, Stoneville, Miss.) are placed onto the diet mixture. Wells are then sealed with Mylar sheeting (ClearLam Packaging, Ill.) using a tacking iron, and several pinholes are made in each well to provide gas exchange. Larvae were held at 25° C. for 6 days in a 14:10 (light:dark) holding room. Mortality and stunting are recorded after six days.

Bioassay by the top load method utilizes the same sample and diet preparations as listed above. The samples are applied to the surface of the insect diet. In a specific embodiment, surface area ranged from 0.3 to approximately 0.8 cm 2 depending on the tray size, 96 well tissue culture plates were used in addition to the format listed above. Following application, samples are allowed to air dry before insect infestation. A water blank containing no B.t . can serve as the control. Eggs are applied to each treated well and were then sealed with Mylar sheeting (ClearLam Packaging, Ill.) using a tacking iron, and pinholes are made in each well to provide gas exchange. Bioassays are held at 25° C. for 7 days in a 14:10 (light:dark) or 28° C. for 4 days in a 14:10 (light:dark) holding room. Mortality and insect stunting are recorded at the end of each bioassay.

Another assay useful according to the subject invention is the Western corn rootworm assay. Samples can be bioassayed against neonate western corn rootworm larvae ( Diabrotica virgifera virgifera ) via top-loading of sample onto an agar-based artificial diet at a rate of 160 ml/cm 2 . Artificial diet can be dispensed into 0.78 cm 2 wells in 48-well tissue culture or similar plates and allowed to harden. After the diet solidifies, samples are dispensed by pipette onto the diet surface. Excess liquid is then evaporated from the surface prior to transferring approximately three neonate larvae per well onto the diet surface by camel's hair brush. To prevent insect escape while allowing gas exchange, wells are heat-sealed with 2-mil punched polyester film with 27HT adhesive (Oliver Products Company, Grand Rapids, Mich.). Bioassays are held in darkness at 25° C., and mortality scored after four days.

Analogous bioassays can be performed by those skilled in the art to assess activity against other pests, such as the black cutworm ( Agrotis ipsilon ).

Results are shown in Table 6.

Results of Western Corn Rootworm Bioassays and Further Characterization of the Toxins

Concentrated liquid supernatant solutions, obtained according to the subject invention, were tested for activity against Western corn rootworm (WCRW). Supernatants from the following isolates were found to cause mortality against WCRW: PS31F2, PS66D3, PS177I8, KB53A49-4, KB68B46-2, KB68B51-2, KB68B55-2, and PS177C8.

Supernatants from the following isolates were also found to cause mortality against WCRW: PS205A3, PS185V2, PS234E1, PS71G4, PS248N10, PS191A21, KB63B19-13, KB63B19-7, KB68B62-7, KB68B63-2, KB69A125-1, KB69A125-3, KB69A125-5, KB69A127-7, KB69A132-1, KB69B2-1, KB70B5-3, KB71A125-15, and KB71A35-6; it was confirmed that this activity was heat labile. Furthermore, it was determined that the supernatants of the following isolates did not react (yielded negative test results) with the WAR antibody (see Example 12), and did not react with the MIS (SEQ ID NO. 31) and WAR (SEQ ID NO. 51) probes: PS205A3, PS185V2, PS234E1, PS71G4, PS248N10, PS191A21, KB63B19-13, KB63B19-7, KB68B62-7, KB68B63-2, KB69A125-1, KB69A125-5, KB69A132-1, KB69B2-1, KB70B5-3, KB71A125-15, and KB71A35-6; the supernatants of isolates KB69A125-3 and KB69A127-7 yielded positive test results.

Culturing of 31F2 Clones and Bioassay of 31F2 Toxins on Western Corn Rootworm (wCRW)

E. coli MR983 and the negative control strain MR948 ( E. coli XL1-Blue [pSupercos]; vector control) were grown in 250 ml bottom baffled flasks containing 50 ml of DIFCO Terrific Broth medium. Cultures were incubated in New Brunswick shaker agitating at 250 RPM, 30° C. for ˜23 hours. After 23 hours of incubation samples were aseptically taken to examine the cultures under the microscope to check for presence of contaminants. 30 ml of culture were dispensed into a 50 ml centrifuge tube and centrifuged in a Sorvall centrifuge at 15,000 rpm for 20 minutes. The 1× supernatant was saved and submitted for bioassay against wCRW. The pellet was resuspended 5× with 10 mM TRIS buffer, and was sonicated prior to submission for bioassay against wCRW.

B.t . strain MR558 and the negative control MR539 ( B.t . cry B[pHT Blue II]; vector control) were grown in the same manner except for the omission of glycerol from the Terrific Broth medium. B.t . cell pellets were resuspended in water rather than buffer prior to sonication.

Assays for the E. coli clone MR983 and B. thuringiensis clone MR558 containing the 31F2 toxin genes were conducted using the same experimental design as in Example 10 for western corn rootworm with the following exceptions: Supernatant samples were top-loaded onto diet at a dose of ˜160 ul/cm 2 . B.t . cellular pellet samples at a 5× concentration were top-loaded onto the diet at a dose of˜150 ul/ cm 2 for both clones, and at ˜75, and at doses of ˜35 ul/ cm2 for the MR558 B. thuringiensis clone (quantity of active toxin unknown for either clone). Approximately 6-8 larvae were transferred onto the diet immediately after the sample had evaporated. The bioassay plate was sealed with mylar sheeting using a tacking iron and pinholes were made above each well to provide gas exchange. Both the MR983 and MR558 clones demonstrated degrees of bioactivity (greater mortality) against western corn rootworm as compared to the toxin-negative clones MR948 and MR539.

Table 7 presents the results showing the bioactivity of cloned PS31F2 toxins against western corn rootworm.

Target Pests

Toxins of the subject invention can be used, alone or in combination with other toxins, to control one or more non-mammalian pests. These pests may be, for example, those listed in Table 8. Activity can readily be confirmed using the bioassays provided herein, adaptations of these bioassays, and/or other bioassays well known to those skilled in the art.

Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants with genes encoding the insecticidal toxin of the present invention. The transformed plants are resistant to attack by the target pest.

Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the Bacillus toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli . The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System , Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci . 4:1-46; and An et al. (1985) EMBO J 4:277-287.

Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978 ] Mol. Gen. Genet . 163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives. In biolistic transformation, plasmid DNA or linear DNA can be employed.

The transformed cells are regenerated into morphologically normal plants in the usual manner. If a transformation event involves a germ line cell, then the inserted DNA and corresponding phenotypic trait(s) will be transmitted to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about 80% of the full length toxin. Methods for creating synthetic Bacillus genes for use in plants are known in the art.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and of the appended claims.

54

2375 base pairs

nucleic acid

single

linear

DNA (genomic)

Jav90

1

ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT 60

AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAACATGAT TTTTAAAACG 120

GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGAT 180

ATTTCTGGTA AATTGGATGG GGTGAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAAC 240

TTAAATACAG AATTATCTAA GGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTTA 300

AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCGGGTATA TCTACCTAAA 360

ATTACCTCTA TGTTGAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGAA 420

TACTTAAGTA AACAATTGCA AGAGATTTCT GATAAGTTGG ATATTATTAA TGTAAATGTA 480

CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AAAGGATTAA ATATGTGAAC 540

GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAGTT CAAAAGTAAA AAAGGATGGC 600

TCTCCTGCAG ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAACA 660

AAAAATGATG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGGA 720

AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAAT 780

GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGCT 840

CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGAT 900

ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGTA 960

AACATCCTCC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAGGA 1020

AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GATTGGGTTT 1080

GAAATTAGTA ATGATTCAAT TACAGTATTA AAAGTATATG AGGCTAAGCT AAAACAAAAT 1140

TATCAAGTCG ATAAGGATTC CTTATCGGAA GTTATTTATG GTGATATGGA TAAATTATTG 1200

TGCCCAGATC AATCTGAACA AATCTATTAT ACAAATAACA TAGTATTTCC AAATGAATAT 1260

GTAATTACTA AAATTGATTT CACTAAAAAA ATGAAAACTT TAAGATATGA GGTAACAGCG 1320

AATTTTTATG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAAAAAGT AGAATCAAGT 1380

GAAGCGGAGT ATAGAACGTT AAGTGCTAAT GATGATGGGG TGTATATGCC GTTAGGTGTC 1440

ATCAGTGAAA CATTTTTGAC TCCGATTAAT GGGTTTGGCC TCCAAGCTGA TGAAAATTCA 1500

AGATTAATTA CTTTAACATG TAAATCATAT TTAAGAGAAC TACTGCTAGC AACAGACTTA 1560

AGCAATAAAG AAACTAAATT GATYGTCCCG CCAAGTGGTT TTATTAGCAA TATTGTAGAG 1620

AACGGGTCCA TAGAAGAGGA CAATTTAGAG CCGTGGAAAG CAAATAATAA GAATGCGTAT 1680

GTAGATCATA CAGGCGGAGT GAATGGAACT AAAGCTTTAT ATGTTCATAA GGACGGAGGA 1740

ATTTCACAAT TTATTGGAGA TAAGTTAAAA CCGAAAACTG AGTATGTAAT CCAATATACT 1800

GTTAAAGGAA AACCTTCTAT TCATTTAAAA GATGAAAATA CTGGATATAT TCATTATGAA 1860

GATACAAATA ATAATTTAGA AGATTATCAA ACTATTAATA AACGTTTTAC TACAGGAACT 1920

GATTTAAAGG GAGTGTATTT AATTTTAAAA AGTCAAAATG GAGATGAAGC TTGGGGAGAT 1980

AACTTTATTA TTTTGGAAAT TAGTCCTTCT GAAAAGTTAT TAAGTCCAGA ATTAATTAAT 2040

ACAAATAATT GGACGAGTAC GGGATCAACT AATATTAGCG GTAATACACT CACTCTTTAT 2100

CAGGGAGGAC GAGGGATTCT AAAACAAAAC CTTCAATTAG ATAGTTTTTC AACTTATAGA 2160

GTGTATTTTT CTGTGTCCGG AGATGCTAAT GTAAGGATTA GAAATTCTAG GGAAGTGTTA 2220

TTTGAAAAAA GATATATGAG CGGTGCTAAA GATGTTTCTG AAATGTTCAC TACAAAATTT 2280

GAGAAAGATA ACTTTTATAT AGAGCTTTCT CAAGGGAATA ATTTATATGG TGGTCCTATT 2340

GTACATTTTT ACGATGTCTC TATTAAGTAA CCCAA 2375

790 amino acids

amino acid

single

linear

protein

Jav90

2

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu

35 40 45

Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys

50 55 60

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn

65 70 75 80

Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln

85 90 95

Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr

100 105 110

Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val

115 120 125

Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys

130 135 140

Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val

145 150 155 160

Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile

165 170 175

Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr

180 185 190

Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu

195 200 205

Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val

210 215 220

Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly

225 230 235 240

Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile

245 250 255

Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr

260 265 270

Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Thr

275 280 285

Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr

290 295 300

Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val

305 310 315 320

Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala

325 330 335

Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys

340 345 350

Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr

355 360 365

Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp

370 375 380

Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu

385 390 395 400

Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe

405 410 415

Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys

420 425 430

Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly

435 440 445

Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr

450 455 460

Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val

465 470 475 480

Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala

485 490 495

Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg

500 505 510

Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile

515 520 525

Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile

530 535 540

Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr

545 550 555 560

Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His

565 570 575

Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys

580 585 590

Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His

595 600 605

Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn

610 615 620

Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr

625 630 635 640

Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu

645 650 655

Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys

660 665 670

Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly

675 680 685

Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg

690 695 700

Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg

705 710 715 720

Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser

725 730 735

Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val

740 745 750

Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu

755 760 765

Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr

770 775 780

Asp Val Ser Ile Lys Pro

785 790

20 base pairs

nucleic acid

single

linear

DNA (genomic)

3

GGRTTAMTTG GRTAYTATTT 20

20 base pairs

nucleic acid

single

linear

DNA (genomic)

4

ATATCKWAYA TTKGCATTTA 20

1042 base pairs

nucleic acid

single

linear

DNA (genomic)

66D3

5

TTAATTGGGT ACTATTTTAA AGGAAAAGAT TTTAATAATC TTACTATATT TGCTCCAACA 60

CGTGAGAATA CTCTTATTTA TGATTTAGAA ACAGCGAATT CTTTATTAGA TAAGCAACAA 120

CAAACCTATC AATCTATTCG TTGGATCGGT TTAATAAAAA GCAAAAAAGC TGGAGATTTT 180

ACCTTTCAAT TATCGGATGA TGAGCATGCT ATTATAGAAA TCGATGGGAA AGTTATTTCG 240

CAAAAAGGCC AAAAGAAACA AGTTGTTCAT TTAGAAAAAG ATAAATTAGT TCCCATCAAA 300

ATTGAATATC AATCTGATAA AGCGTTAAAC CCAGATAGTC AAATGTTTAA AGAATTGAAA 360

TTATTTAAAA TAAATAGTCA AAAACAATCT CAGCAAGTGC AACAAGACGA ATTGAGAAAT 420

CCTGAATTTG GTAAAGAAAA AACTCAAACA TATTTAAAGA AAGCATCGAA AAGCAGCCTG 480

TTTAGCAATA AAAGTAAACG AGATATAGAT GAAGATATAG ATGAGGATAC AGATACAGAT 540

GGAGATGCCA TTCCTGATGT ATGGGAAGAA AATGGGTATA CCATCAAAGG AAGAGTAGCT 600

GTTAAATGGG ACGAAGGATT AGCTGATAAG GGATATAAAA AGTTTGTTTC CAATCCTTTT 660

AGACAGCACA CTGCTGGTGA CCCCTATAGT GACTATGAAA AGGCATCAAA AGATTTGGAT 720

TTATCTAATG CAAAAGAAAC ATTTAATCCA TTGGTGGCTG CTTTTCCAAG TGTCAATGTT 780

AGCTTGGAAA ATGTCACCAT ATCAAAAGAT GAAAATAAAA CTGCTGAAAT TGCGTCTACT 840

TCATCGAATA ATTGGTCCTA TACAAATACA GAGGGGGCAT CTATTGAAGC TGGAATTGGA 900

CCAGAAGGTT TGTTGTCTTT TGGAGTAAGT GCCAATTATC AACATTCTGA AACAGTGGCC 960

AAAGAGTGGG GTACAACTAA GGGAGACGCA ACACAATATA ATACAGCTTC AGCAGGATAT 1020

CTAAATGCCA ATGTACGATA TA 1042

347 amino acids

amino acid

single

linear

peptide

66D3

6

Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr Ile

1 5 10 15

Phe Ala Pro Thr Arg Glu Asn Thr Leu Ile Tyr Asp Leu Glu Thr Ala

20 25 30

Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr Gln Ser Ile Arg Trp

35 40 45

Ile Gly Leu Ile Lys Ser Lys Lys Ala Gly Asp Phe Thr Phe Gln Leu

50 55 60

Ser Asp Asp Glu His Ala Ile Ile Glu Ile Asp Gly Lys Val Ile Ser

65 70 75 80

Gln Lys Gly Gln Lys Lys Gln Val Val His Leu Glu Lys Asp Lys Leu

85 90 95

Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Lys Ala Leu Asn Pro Asp

100 105 110

Ser Gln Met Phe Lys Glu Leu Lys Leu Phe Lys Ile Asn Ser Gln Lys

115 120 125

Gln Ser Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu Phe Gly

130 135 140

Lys Glu Lys Thr Gln Thr Tyr Leu Lys Lys Ala Ser Lys Ser Ser Leu

145 150 155 160

Phe Ser Asn Lys Ser Lys Arg Asp Ile Asp Glu Asp Ile Asp Glu Asp

165 170 175

Thr Asp Thr Asp Gly Asp Ala Ile Pro Asp Val Trp Glu Glu Asn Gly

180 185 190

Tyr Thr Ile Lys Gly Arg Val Ala Val Lys Trp Asp Glu Gly Leu Ala

195 200 205

Asp Lys Gly Tyr Lys Lys Phe Val Ser Asn Pro Phe Arg Gln His Thr

210 215 220

Ala Gly Asp Pro Tyr Ser Asp Tyr Glu Lys Ala Ser Lys Asp Leu Asp

225 230 235 240

Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro

245 250 255

Ser Val Asn Val Ser Leu Glu Asn Val Thr Ile Ser Lys Asp Glu Asn

260 265 270

Lys Thr Ala Glu Ile Ala Ser Thr Ser Ser Asn Asn Trp Ser Tyr Thr

275 280 285

Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Ile Gly Pro Glu Gly Leu

290 295 300

Leu Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr Val Ala

305 310 315 320

Lys Glu Trp Gly Thr Thr Lys Gly Asp Ala Thr Gln Tyr Asn Thr Ala

325 330 335

Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr

340 345

2645 base pairs

nucleic acid

single

linear

DNA (genomic)

PS177C8a

7

ATGAAGAAGA AGTTAGCAAG TGTTGTAACG TGTACGTTAT TAGCTCCTAT GTTTTTGAAT 60

GGAAATGTGA ATGCTGTTTA CGCAGACAGC AAAACAAATC AAATTTCTAC AACACAGAAA 120

AATCAACAGA AAGAGATGGA CCGAAAAGGA TTACTTGGGT ATTATTTCAA AGGAAAAGAT 180

TTTAGTAATC TTACTATGTT TGCACCGACA CGTGATAGTA CTCTTATTTA TGATCAACAA 240

ACAGCAAATA AACTATTAGA TAAAAAACAA CAAGAATATC AGTCTATTCG TTGGATTGGT 300

TTGATTCAGA GTAAAGAAAC GGGAGATTTC ACATTTAACT TATCTGAGGA TGAACAGGCA 360

ATTATAGAAA TCAATGGGAA AATTATTTCT AATAAAGGGA AAGAAAAGCA AGTTGTCCAT 420

TTAGAAAAAG GAAAATTAGT TCCAATCAAA ATAGAGTATC AATCAGATAC AAAATTTAAT 480

ATTGACAGTA AAACATTTAA AGAACTTAAA TTATTTAAAA TAGATAGTCA AAACCAACCC 540

CAGCAAGTCC AGCAAGATGA ACTGAGAAAT CCTGAATTTA ACAAGAAAGA ATCACAGGAA 600

TTCTTAGCGA AACCATCGAA AATAAATCTT TTCACTCAAA AAATGAAAAG GGAAATTGAT 660

GAAGACACGG ATACGGATGG GGACTCTATT CCTGACCTTT GGGAAGAAAA TGGGTATACG 720

ATTCAAAATA GAATCGCTGT AAAGTGGGAC GATTCTYTAG CAAGTAAAGG GTATACGAAA 780

TTTGTTTCAA ATCCGCTAGA AAGTCACACA GTTGGTGATC CTTATACAGA TTATGAAAAG 840

GCAGCAAGAG ACCTAGATTT GTCAAATGCA AAGGAAACGT TTAACCCATT GGTAGCTGCT 900

TTTCCAAGTG TGAATGTTAG TATGGAAAAG GTGATATTAT CACCAAATGA AAATTTATCC 960

AATAGTGTAG AGTCTCATTC ATCCACGAAT TGGTCTTATA CAAATACAGA AGGTGCTTCT 1020

GTTGAAGCGG GGATTGGACC AAAAGGTATT TCGTTCGGAG TTAGCGTAAA CTATCAACAC 1080

TCTGAAACAG TTGCACAAGA ATGGGGAACA TCTACAGGAA ATACTTCGCA ATTCAATACG 1140

GCTTCAGCGG GATATTTAAA TGCAAATGTT CGATATAACA ATGTAGGAAC TGGTGCCATC 1200

TACGATGTAA AACCTACAAC AAGTTTTGTA TTAAATAACG ATACTATCGC AACTATTACG 1260

GCGAAATCTA ATTCTACAGC CTTAAATATA TCTCCTGGAG AAAGTTACCC GAAAAAAGGA 1320

CAAAATGGAA TCGCAATAAC ATCAATGGAT GATTTTAATT CCCATCCGAT TACATTAAAT 1380

AAAAAACAAG TAGATAATCT GCTAAATAAT AAACCTATGA TGTTGGAAAC AAACCAAACA 1440

GATGGTGTTT ATAAGATAAA AGATACACAT GGAAATATAG TAACTGGCGG AGAATGGAAT 1500

GGTGTCATAC AACAAATCAA GGCTAAAACA GCGTCTATTA TTGTGGATGA TGGGGAACGT 1560

GTAGCAGAAA AACGTGTAGC GGCAAAAGAT TATGAAAATC CAGAAGATAA AACACCGTCT 1620

TTAACTTTAA AAGATGCCCT GAAGCTTTCA TATCCAGATG AAATAAAAGA AATAGAGGGA 1680

TTATTATATT ATAAAAACAA ACCGATATAC GAATCGAGCG TTATGACTTA CTTAGATGAA 1740

AATACAGCAA AAGAAGTGAC CAAACAATTA AATGATACCA CTGGGAAATT TAAAGATGTA 1800

AGTCATTTAT ATGATGTAAA ACTGACTCCA AAAATGAATG TTACAATCAA ATTGTCTATA 1860

CTTTATGATA ATGCTGAGTC TAATGATAAC TCAATTGGTA AATGGACAAA CACAAATATT 1920

GTTTCAGGTG GAAATAACGG AAAAAAACAA TATTCTTCTA ATAATCCGGA TGCTAATTTG 1980

ACATTAAATA CAGATGCTCA AGAAAAATTA AATAAAAATC GTACTATTAT ATAAGTTTAT 2040

ATATGAAGTC AGAAAAAAAC ACACAATGTG AGATTACTAT AGATGGGGAG ATTTATCCGA 2100

TCACTACAAA AACAGTGAAT GTGAATAAAG ACAATTACAA AAGATTAGAT ATTATAGCTC 2160

ATAATATAAA AAGTAATCCA ATTTCTTCAA TTCATATTAA AACGAATGAT GAAATAACTT 2220

TATTTTGGGA TGATATTTCT ATAACAGATG TAGCATCAAT AAAACCGGAA AATTTAACAG 2280

ATTCAGAAAT TAAACAGATT TATAGTAGGT ATGGTATTAA GTTAGAAGAT GGAATCCTTA 2340

TTGATAAAAA AGGTGGGATT CATTATGGTG AATTTATTAA TGAAGCTAGT TTTAATATTG 2400

AACCATTGCA AAATTATGTG ACAAAATATA AAGTTACTTA TAGTAGTGAG TTAGGACAAA 2460

ACGTGAGTGA CACACTTGAA AGTGATAAAA TTTACAAGGA TGGGACAATT AAATTTGATT 2520

TTACAAAATA TAGTRAAAAT GAACAAGGAT TATTTTATGA CAGTGGATTA AATTGGGACT 2580

TTAAAATTAA TGCTATTACT TATGATGGTA AAGAGATGAA TGTTTTTCAT AGATATAATA 2640

AATAG 2645

881 amino acids

amino acid

single

linear

peptide

PS177C8a

8

Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pro

1 5 10 15

Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser Lys Thr

20 25 30

Asn Gln Ile Ser Thr Thr Gln Lys Asn Gln Gln Lys Glu Met Asp Arg

35 40 45

Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn Leu

50 55 60

Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu Ile Tyr Asp Gln Gln

65 70 75 80

Thr Ala Asn Lys Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Ile

85 90 95

Arg Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe

100 105 110

Asn Leu Ser Glu Asp Glu Gln Ala Ile Ile Glu Ile Asn Gly Lys Ile

115 120 125

Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gly

130 135 140

Lys Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe Asn

145 150 155 160

Ile Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Ser

165 170 175

Gln Asn Gln Pro Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu

180 185 190

Phe Asn Lys Lys Glu Ser Gln Glu Phe Leu Ala Lys Pro Ser Lys Ile

195 200 205

Asn Leu Phe Thr Gln Lys Met Lys Arg Glu Ile Asp Glu Asp Thr Asp

210 215 220

Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr

225 230 235 240

Ile Gln Asn Arg Ile Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys

245 250 255

Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His Thr Val Gly

260 265 270

Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser

275 280 285

Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val

290 295 300

Asn Val Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser

305 310 315 320

Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr

325 330 335

Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly Ile Ser Phe

340 345 350

Gly Val Ser Val Asn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Trp

355 360 365

Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly

370 375 380

Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala Ile

385 390 395 400

Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asp Thr Ile

405 410 415

Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn Ile Ser Pro

420 425 430

Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly Ile Ala Ile Thr Ser

435 440 445

Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys Lys Gln Val

450 455 460

Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr Asn Gln Thr

465 470 475 480

Asp Gly Val Tyr Lys Ile Lys Asp Thr His Gly Asn Ile Val Thr Gly

485 490 495

Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Lys Ala Lys Thr Ala Ser

500 505 510

Ile Ile Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg Val Ala Ala

515 520 525

Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys

530 535 540

Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gly

545 550 555 560

Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser Val Met Thr

565 570 575

Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gln Leu Asn Asp

580 585 590

Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val Lys Leu

595 600 605

Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu Tyr Asp Asn

610 615 620

Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr Asn Thr Asn Ile

625 630 635 640

Val Ser Gly Gly Asn Asn Gly Lys Lys Gln Tyr Ser Ser Asn Asn Pro

645 650 655

Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gln Glu Lys Leu Asn Lys

660 665 670

Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr

675 680 685

Gln Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile Thr Thr Lys

690 695 700

Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp Ile Ile Ala

705 710 715 720

His Asn Ile Lys Ser Asn Pro Ile Ser Ser Ile His Ile Lys Thr Asn

725 730 735

Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr Asp Val Ala

740 745 750

Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys Gln Ile Tyr

755 760 765

Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile Asp Lys Lys

770 775 780

Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser Phe Asn Ile

785 790 795 800

Glu Pro Leu Gln Asn Tyr Val Thr Lys Tyr Lys Val Thr Tyr Ser Ser

805 810 815

Glu Leu Gly Gln Asn Val Ser Asp Thr Leu Glu Ser Asp Lys Ile Tyr

820 825 830

Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser Xaa Asn Glu

835 840 845

Gln Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe Lys Ile Asn

850 855 860

Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His Arg Tyr Asn

865 870 875 880

Lys

1022 base pairs

nucleic acid

single

linear

DNA (genomic)

177I8

9

TGGATTAATT GGGTATTATT TCAAAGGAAA AGATTTTAAT AATCTTACTA TGTTTGCACC 60

GACACGTGAT AATACCCTTA TGTATGACCA ACAAACAGCG AATGCATTAT TAGATAAAAA 120

ACAACAAGAA TATCAGTCCA TTCGTTGGAT TGGTTTGATT CAGAGTAAAG AAACGGGCGA 180

TTTCACATTT AACTTATCAA AGGATGAACA GGCAATTATA GAAATCGATG GGAAAATCAT 240

TTCTAATAAA GGGAAAGAAA AGCAAGTTGT CCATTTAGAA AAAGAAAAAT TAGTTCCAAT 300

CAAAATAGAG TATCAATCAG ATACGAAATT TAATATTGAT AGTAAAACAT TTAAAGAACT 360

TAAATTATTT AAAATAGATA GTCAAAACCA ATCTCAACAA GTTCAACTGA GAAACCCTGA 420

ATTTAACAAA AAAGAATCAC AGGAATTTTT AGCAAAAGCA TCAAAAACAA ACCTTTTTAA 480

GCAAAAAATG AAAAGAGATA TTGATGAAGA TACGGATACA GATGGAGACT CCATTCCTGA 540

TCTTTGGGAA GAAAATGGGT ACACGATTCA AAATAAAGTT GCTGTCAAAT GGGATGATTC 600

GCTAGCAAGT AAGGGATATA CAAAATTTGT TTCGAATCCA TTAGACAGCC ACACAGTTGG 660

CGATCCCTAT ACTGATTATG AAAAGGCCGC AAGGGATTTA GATTTATCAA ATGCAAAGGA 720

AACGTTCAAC CCATTGGTAG CTGCTTTYCC AAGTGTGAAT GTTAGTATGG AAAAGGTGAT 780

ATTATCACCA AATGAAAATT TATCCAATAG TGTAGAGTCT CATTCATCCA CGAATTGGTC 840

TTATACGAAT ACAGAAGGAG CTTCCATTGA AGCTGGTGGC GGTCCATTAG GCCTTTCTTT 900

TGGAGTGAGT GTTAATTATC AACACTCTGA AACAGTTGCA CAAGAATGGG GAACATCTAC 960

AGGAAATACT TCACAATTCA ATACGGCTTC AGCGGGATAT TTAAATGCCA ATATACGATA 1020

TA 1022

340 amino acids

amino acid

single

linear

peptide

177I8

10

Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr

1 5 10 15

Met Phe Ala Pro Thr Arg Asp Asn Thr Leu Met Tyr Asp Gln Gln Thr

20 25 30

Ala Asn Ala Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Ile Arg

35 40 45

Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe Asn

50 55 60

Leu Ser Lys Asp Glu Gln Ala Ile Ile Glu Ile Asp Gly Lys Ile Ile

65 70 75 80

Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Glu Lys

85 90 95

Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe Asn Ile

100 105 110

Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Ser Gln

115 120 125

Asn Gln Ser Gln Gln Val Gln Leu Arg Asn Pro Glu Phe Asn Lys Lys

130 135 140

Glu Ser Gln Glu Phe Leu Ala Lys Ala Ser Lys Thr Asn Leu Phe Lys

145 150 155 160

Gln Lys Met Lys Arg Asp Ile Asp Glu Asp Thr Asp Thr Asp Gly Asp

165 170 175

Ser Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr Ile Gln Asn Lys

180 185 190

Val Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys

195 200 205

Phe Val Ser Asn Pro Leu Asp Ser His Thr Val Gly Asp Pro Tyr Thr

210 215 220

Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu

225 230 235 240

Thr Phe Asn Pro Leu Val Ala Ala Xaa Pro Ser Val Asn Val Ser Met

245 250 255

Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu

260 265 270

Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser

275 280 285

Ile Glu Ala Gly Gly Gly Pro Leu Gly Leu Ser Phe Gly Val Ser Val

290 295 300

Asn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Trp Gly Thr Ser Thr

305 310 315 320

Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala

325 330 335

Asn Ile Arg Tyr

340

1341 base pairs

nucleic acid

single

linear

DNA (genomic)

PS177C8a

11

ATGTTTATGG TTTCTAAAAA ATTACAAGTA GTTACTAAAA CTGTATTGCT TAGTACAGTT 60

TTCTCTATAT CTTTATTAAA TAATGAAGTG ATAAAAGCTG AACAATTAAA TATAAATTCT 120

CAAAGTAAAT ATACTAACTT GCAAAATCTA AAAATCACTG ACAAGGTAGA GGATTTTAAA 180

GAAGATAAGG AAAAAGCGAA AGAATGGGGG AAAGAAAAAG AAAAAGAGTG GAAACTAACT 240

GCTACTGAAA AAGGAAAAAT GAATAATTTT TTAGATAATA AAAATGATAT AAAGACAAAT 300

TATAAAGAAA TTACTTTTTC TATGGCAGGC TCATTTGAAG ATGAAATAAA AGATTTAAAA 360

GAAATTGATA AGATGTTTGA TAAAACCAAT CTATCAAATT CTATTATCAC CTATAAAAAT 420

GTGGAACCGA CAACAATTGG ATTTAATAAA TCTTTAACAG AAGGTAATAC GATTAATTCT 480

GATGCAATGG CACAGTTTAA AGAACAATTT TTAGATAGGG ATATTAAGTT TGATAGTTAT 540

CTAGATACGC ATTTAACTGC TCAACAAGTT TCCAGTAAAG AAAGAGTTAT TTTGAAGGTT 600

ACGGTTCCGA GTGGGAAAGG TTCTACTACT CCAACAAAAG CAGGTGTCAT TTTAAATAAT 660

AGTGAATACA AAATGCTCAT TGATAATGGG TATATGGTCC ATGTAGATAA GGTATCAAAA 720

GTGGTGAAAA AAGGGGTGGA GTGCTTACAA ATTGAAGGGA CTTTAAAAAA GAGTCTTGAC 780

TTTAAAAATG ATATAAATGC TGAAGCGCAT AGCTGGGGTA TGAAGAATTA TGAAGAGTGG 840

GCTAAAGATT TAACCGATTC GCAAAGGGAA GCTTTAGATG GGTATGCTAG GCAAGATTAT 900

AAAGAAATCA ATAATTATTT AAGAAATCAA GGCGGAAGTG GAAATGAAAA ACTAGATGCT 960

CAAATAAAAA ATATTTCTGA TGCTTTAGGG AAGAAACCAA TACCGGAAAA TATTACTGTG 1020

TATAGATGGT GTGGCATGCC GGAATTTGGT TATCAAATTA GTGATCCGTT ACCTTCTTTA 1080

AAAGATTTTG AAGAACAATT TTTAAATACA ATCAAAGAAG ACAAAGGATA TATGAGTACA 1140

AGCTTATCGA GTGAACGTCT TGCAGCTTTT GGATCTAGAA AAATTATATT ACGATTACAA 1200

GTTCCGAAAG GAAGTACGGG TGCGTATTTA AGTGCCATTG GTGGATTTGC AAGTGAAAAA 1260

GAGATCCTAC TTGATAAAGA TAGTAAATAT CATATTGATA AAGTAACAGA GGTAATTATT 1320

AAGGTGTTAA GCGATATGTA G 1341

446 amino acids

amino acid

single

linear

peptide

PS177C8a

12

Met Phe Met Val Ser Lys Lys Leu Gln Val Val Thr Lys Thr Val Leu

1 5 10 15

Leu Ser Thr Val Phe Ser Ile Ser Leu Leu Asn Asn Glu Val Ile Lys

20 25 30

Ala Glu Gln Leu Asn Ile Asn Ser Gln Ser Lys Tyr Thr Asn Leu Gln

35 40 45

Asn Leu Lys Ile Thr Asp Lys Val Glu Asp Phe Lys Glu Asp Lys Glu

50 55 60

Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr

65 70 75 80

Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu Asp Asn Lys Asn Asp

85 90 95

Ile Lys Thr Asn Tyr Lys Glu Ile Thr Phe Ser Met Ala Gly Ser Phe

100 105 110

Glu Asp Glu Ile Lys Asp Leu Lys Glu Ile Asp Lys Met Phe Asp Lys

115 120 125

Thr Asn Leu Ser Asn Ser Ile Ile Thr Tyr Lys Asn Val Glu Pro Thr

130 135 140

Thr Ile Gly Phe Asn Lys Ser Leu Thr Glu Gly Asn Thr Ile Asn Ser

145 150 155 160

Asp Ala Met Ala Gln Phe Lys Glu Gln Phe Leu Asp Arg Asp Ile Lys

165 170 175

Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala Gln Gln Val Ser Ser

180 185 190

Lys Glu Arg Val Ile Leu Lys Val Thr Val Pro Ser Gly Lys Gly Ser

195 200 205

Thr Thr Pro Thr Lys Ala Gly Val Ile Leu Asn Asn Ser Glu Tyr Lys

210 215 220

Met Leu Ile Asp Asn Gly Tyr Met Val His Val Asp Lys Val Ser Lys

225 230 235 240

Val Val Lys Lys Gly Val Glu Cys Leu Gln Ile Glu Gly Thr Leu Lys

245 250 255

Lys Ser Leu Asp Phe Lys Asn Asp Ile Asn Ala Glu Ala His Ser Trp

260 265 270

Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp Leu Thr Asp Ser Gln

275 280 285

Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gln Asp Tyr Lys Glu Ile Asn

290 295 300

Asn Tyr Leu Arg Asn Gln Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala

305 310 315 320

Gln Ile Lys Asn Ile Ser Asp Ala Leu Gly Lys Lys Pro Ile Pro Glu

325 330 335

Asn Ile Thr Val Tyr Arg Trp Cys Gly Met Pro Glu Phe Gly Tyr Gln

340 345 350

Ile Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe Glu Glu Gln Phe Leu

355 360 365

Asn Thr Ile Lys Glu Asp Lys Gly Tyr Met Ser Thr Ser Leu Ser Ser

370 375 380

Glu Arg Leu Ala Ala Phe Gly Ser Arg Lys Ile Ile Leu Arg Leu Gln

385 390 395 400

Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser Ala Ile Gly Gly Phe

405 410 415

Ala Ser Glu Lys Glu Ile Leu Leu Asp Lys Asp Ser Lys Tyr His Ile

420 425 430

Asp Lys Val Thr Glu Val Ile Ile Lys Val Leu Ser Asp Met

435 440 445

21 base pairs

nucleic acid

single

linear

DNA (genomic)

13

GCTGATGAAC CATTTAATGC C 21

22 base pairs

nucleic acid

single

linear

DNA (genomic)

14

CTCTTTAAAG TAGATACTAA GC 22

24 base pairs

nucleic acid

single

linear

DNA (genomic)

15

GATGAGAACT TATCAAATAG TATC 24

33 base pairs

nucleic acid

single

linear

DNA (genomic)

16

CGAATTCTTT ATTAGATAAG CAACAACAAA CCT 33

24 base pairs

nucleic acid

single

linear

DNA (genomic)

17

GTTATTTCGC AAAAAGGCCA AAAG 24

31 base pairs

nucleic acid

single

linear

DNA (genomic)

18

GAATATCAAT CTGATAAAGC GTTAAACCCA G 31

23 base pairs

nucleic acid

single

linear

DNA (genomic)

19

GCAGCYTGTT TAGCAATAAA AGT 23

20 base pairs

nucleic acid

single

linear

DNA (genomic)

20

CAAAGGAAGA GTAGCTGTTA 20

25 base pairs

nucleic acid

single

linear

DNA (genomic)

21

CAATGTTAGC TTGGAAAATG TCACC 25

22 base pairs

nucleic acid

single

linear

DNA (genomic)

22

GCTTAGTATC TACTTTAAAG AG 22

24 base pairs

nucleic acid

single

linear

DNA (genomic)

23

GATACTATTT GATAAGTTCT CATC 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

24

CTTTTGGCCT TTTTGCGAAA TAAC 24

31 base pairs

nucleic acid

single

linear

DNA (genomic)

25

CTGGGTTTAA CGCTTTATCA GATTGATATT C 31

23 base pairs

nucleic acid

single

linear

DNA (genomic)

26

ACTTTTATTG CTAAACARGC TGC 23

20 base pairs

nucleic acid

single

linear

DNA (genomic)

27

TAACAGCTAC TCTTCCTTTG 20

25 base pairs

nucleic acid

single

linear

DNA (genomic)

28

GGTGACATTT TCCAAGCTAA CATTG 25

21 base pairs

nucleic acid

single

linear

DNA (genomic)

29

CCAGTCCAAT GAACCTCTTA C 21

21 base pairs

nucleic acid

single

linear

DNA (genomic)

30

AGGGAACAAA CCTTCCCAAC C 21

20 base pairs

nucleic acid

single

linear

DNA (genomic)

31

CARMTAKTAA MTAGGGATAG 20

22 base pairs

nucleic acid

single

linear

DNA (genomic)

32

AGYTTCTATC GAAGCTGGGR ST 22

1035 base pairs

nucleic acid

single

linear

DNA (genomic)

33

GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA 60

ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCAG 120

CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGAC 180

TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGTT 240

TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAATC 300

AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCTT 360

AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAAA 420

ATCCTGAATT TAATAAAAAG GAAACGAAAG AGCTTTTATC AAAAACAGCA AAAAGAACCT 480

TTTCTCTTCA AAACGGGGTT GAGAAGCGAT GAGGATGATG ATCTAGGATA CAGATGGTGA 540

TAGCATTCCT GGATAATTGG GAAATGAATG GATATACCAT TCAAACGAAA AATGGCAGTC 600

AAATGGGATG ATTCATTTGC AGAAAAAGGA TATACAAAAT TTGTTTCGAA TCCATATGAA 660

GCCCATACAG CAGGAGATCC TTATACCGAT TATGAAAAAG CAGCAAAAGA TATTCCTTTA 720

TCGAACGCAA AAGAAGCCTT TAATCCTCTT GTAGCTGCTT TTCCATCTGT CAATGTAGGA 780

TTAGAAAAAG TAGTAATTTC TAAAAATGAG GATATGAGTC AGGGTGTATC ATCCAGCACT 840

TCGAATAGTG CCTCTAATAC AAATTCAATT GGTGTTACCG TAGATGCTGG TTGGGAAGGT 900

TTGTTCCCTA AATTTGGTAT TTCAACTAAT TATCAAAACA CATGGACCAC TGCACAAGAA 960

TGGGGCTCTT CTAAAGAAGA TTCTACCCAT ATAAATGGAG CACAATCAGC CTTTTTAAAT 1020

GCAAATGTAC GATAT 1035

345 amino acids

amino acid

single

linear

protein

34

Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr

1 5 10 15

Met Phe Ala Pro Thr Ile Asn Asn Thr Leu Ile Tyr Asp Arg Gln Thr

20 25 30

Ala Asp Thr Leu Leu Asn Lys Gln Gln Gln Glu Phe Asn Ser Ile Arg

35 40 45

Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe Gln

50 55 60

Leu Ser Asp Asp Lys Asn Ala Ile Ile Glu Ile Asp Gly Lys Val Val

65 70 75 80

Ser Arg Arg Gly Glu Asp Lys Gln Thr Ile His Leu Glu Lys Gly Lys

85 90 95

Met Val Pro Ile Lys Ile Glu Tyr Gln Ser Asn Glu Pro Leu Thr Val

100 105 110

Asp Ser Lys Val Phe Asn Asp Leu Lys Leu Phe Lys Ile Asp Gly His

115 120 125

Asn Gln Ser His Gln Ile Gln Gln Asp Asp Leu Lys Ile Leu Asn Leu

130 135 140

Ile Lys Arg Lys Arg Lys Ser Phe Tyr Gln Lys Gln Gln Lys Glu Pro

145 150 155 160

Phe Leu Phe Lys Thr Gly Leu Arg Ser Asp Glu Asp Asp Asp Leu Gly

165 170 175

Tyr Arg Trp Xaa Xaa His Ser Trp Ile Ile Gly Lys Xaa Met Asp Ile

180 185 190

Pro Phe Lys Arg Lys Met Ala Val Lys Trp Asp Asp Ser Phe Ala Glu

195 200 205

Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Tyr Glu Ala His Thr Ala

210 215 220

Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Lys Asp Ile Pro Leu

225 230 235 240

Ser Asn Ala Lys Glu Ala Phe Asn Pro Leu Val Ala Ala Phe Pro Ser

245 250 255

Val Asn Val Gly Leu Glu Lys Val Val Ile Ser Lys Asn Glu Asp Met

260 265 270

Ser Gln Gly Val Ser Ser Ser Thr Ser Asn Ser Ala Ser Asn Thr Asn

275 280 285

Ser Ile Gly Val Thr Val Asp Ala Gly Trp Glu Gly Leu Phe Pro Lys

290 295 300

Phe Gly Ile Ser Thr Asn Tyr Gln Asn Thr Trp Thr Thr Ala Gln Glu

305 310 315 320

Trp Gly Ser Ser Lys Glu Asp Ser Thr His Ile Asn Gly Ala Gln Ser

325 330 335

Ala Phe Leu Asn Ala Asn Val Arg Tyr

340 345

1037 base pairs

nucleic acid

single

linear

DNA (genomic)

35

GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA 60

ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCAG 120

CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGAC 180

TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGTT 240

TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAATC 300

AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCTT 360

AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAAA 420

AATCCTGAAT TTAATAAAAA AGAAACGAAA GAGCTTTTAT CAAAAACAGC AAAAAGRAAC 480

CTTTTCTCTT CAAACGRRGT KGAGAAGCGA TGAGGATGAT RATCYTAGAT ACAGGTGGKG 540

ATAGCATTCC YKGATAATTG GGGAAATGAA WGGRTATACC ATTCAACSGA AAAATGGSAG 600

TCAAATGGGA TGATTCATTT GCGGAAAAAG GATATACAAA ATTTGTTTCG AATCCATATG 660

AAGCCCATAC AGCAGGAGAT CCTTATACCG ATTATGAAAA AGCAGCAAAA GATATTCCTT 720

TATCGAACGC AAAAGAAGCC TTTAATCCTC TTGTAGCTGC TTTTCCATCT GTCAATGTAG 780

GATTAGAAAA AGTAGTAATT TCTAAAAATG AGGATATGAG TCAGGGTGTA TCATCCAGCA 840

CTTCGAATAG TGCCTCTAAT ACAAATTCAA TTGGTGTTAC CGTAGATGCT GGTTGGGAAG 900

GTTTGTTCCC TAAATTTGGT ATTTCAACTA ATTATCAAAA CACATGGACC ACTGCACAAG 960

AATGGGGCTC TTCTAAAGAA GATTCTACCC ATATAAATGG AGCACAATCA GCCTTTTTAA 1020

ATGCAAATGT ACGATAT 1037

1048 base pairs

nucleic acid

single

linear

DNA (genomic)

36

TGGGTTAATT GGGTATTATT TTAAAGGGCA AGAGTTTAAT CATCTTACTT TGTTCGCACC 60

AACACGTGAT AATACCCTTA TTTATGATCA ACAAACAGCG AATTCCTTAT TAGATACCAA 120

GCAACAAGAA TATCAATCTA TTCGCTGGAT TGGTTTAATT CAAAGTAAAG AAACGGGTGA 180

TTTCACATTT AACTTATCAG ATGATCAACA TGCAATTATA GAAATCGATG GCAAAATCAT 240

TTCGCATAAA GGACAGAATA AACAAGTTGT TCACTTAGAA AAAGGAAAGT TAGTCCCGAT 300

AAAAATTGAG TATCAATCAG ATCAACTATT AAATAGGGAT AGTAACATCT TTAAAGAGTT 360

TAAATTATTC AAAGTAGATA GTCAGCAACA CGCTCACCAA GTTCAACTAG ACGAATTAAG 420

AAACCCTGCG TTTAATAAAA AGGAAACACA ACAATCTTAA GAAAAAGCAT CCAAAAACAA 480

TCTTTTTACA CCAGGGACAT TAAAAGGAAG ATACTGATGA TGATGATAAG GATAACAGGA 540

TGGGAGATTC TATTCCTGGA CCTTTTGGGG GAAGAAAATG GGTATACCAA TCCCAAAATA 600

AAATAGCTGG TCCAAGTGGG ATGTTCATTC GCCGCGAAAG GGTATACAAA TTTGTTTCTT 660

AATCCACTTG ATAGTCATAC AGTTGGAGAT CCCTATACGG ATTATGAAAA AGCAGCAAGA 720

GATTTAGACT TGGCCCAATG CAAAAGAAAC ATTTAACCCA TTAGTAGCTG CTTTTCCAAG 780

TGTGAATGTG AATTTGGAAA AAGTCATTTT ATCTAAAGAT GAAAATCTAT CCAATAGTGT 840

AGAGTCACAT TCCTCCACCA ACTGGTCTTA TACGAATACA GAAGGAGCTT CTATCGAAGC 900

TGGGGCTAAA CCAGAGGGTC CTACTTTTGG AGTGAGTGCT ACTTATCAAC ACTCTGAAAC 960

AGTTGCAAAA GAATGGGGAA CATCTACAGG AAATACCTCG CAATTTAATA CAGCTTCAGC 1020

AGGATATTTA AATGCAAATG TACGATAT 1048

1175 base pairs

nucleic acid

single

linear

DNA (genomic)

37

ACCTCTAGAT GCANGCTCGA GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGATTA 60

CTTGGGTATT ATTTTAAAGG GAAAGAGTTT AATCATCTTA CTTTGTTCGC ACCAACACGT 120

GATAATACCC TTATTTATGA TCAACAAACA GCGAATTCCT TATTAGATAC CAAACAACAA 180

GAATATCAAT CTATTCGCTG GATTGGTTTG ATTCAAAGTA AAGAAACAGG TGATTTCACG 240

TTTAACTTAT CTGATGATCA AAATGCAATT ATAGAAATAG ATGGCAAAAT CATTTCGCAT 300

AAAGGACAGA ATAAACAAGT TGTTCACTTA GAAAAAGGAA AGTTAGTCCC GATAAAAATT 360

GAGTATCAAT CAGATCAGAT ATTAACTAGG GATAGTAACA TCTTTAAAGA GTTCAATTAT 420

TCAAAGTAGA TAGTCAAGCA ACACTCTCAC CAAAGTTCAA CTTAGGNCNG AATTAAGNAA 480

CCCTNGGATT TTAANTTNAA AAAAAGGAAC CCNCANCATT CTTTAGGAAA AAGCAGCAAN 540

AACCAAATCC TTTTTTACCA CAGGATATTG AAAAGGAGAT ACGGGNTNGA TGATGGATTG 600

ATACCGGGAT ACCAGTTGGG GNTTCTANTC CCTGACCTTT GGGGAAAGAA AATNGGTATA 660

CCNATCCCAA AANTTAAGCC AGCTGTCCAG GTGGGATGAT TCAATTCGCC CGCGAAAGGG 720

TATACCAAAA TTTGTTTCTT AATCCACTTG AGAGTCATAC AGTTGGAGAT CCCTATACGG 780

ATTATGAAAA AGCAGCAAGA GATTTAGACT TGGCCAATGC AAAAGAAACA TTTAACCCAT 840

TAGTAGCTGC TTTTCCAAGT GTGAATGTGA ATTTGGAAAA AGTAATATTA TCCCCAGATG 900

AGAATTTATC TAACAGTGTA GAATCTCATT CGTCTACAAA TTGGTCTTAT ACGAATACTG 960

AAGGAGCTTC TATCGAAGCT GGGGGTGGTC CATTAGGTAT TTCATTTGGA GTGAGTGCTA 1020

ATTATCAACA CTCTGAAACA GTTGCAAAAG AATGGGGAAC ATCTACAGGA AATACCTCGC 1080

AATTTAATAC AGCTTCAGCA GGATATTTAA ATGCCAATGG TCGATNTAAG CCGAATNCCA 1140

NCACACTGNC GGCCGTTAGT AGTGGCACCG AGCCC 1175

1030 base pairs

nucleic acid

single

linear

DNA (genomic)

38

GGRTTAMTTG GGTATTATTT TAAAGGGAAA GATTTTAATG ATCTTACTGT ATTTGCACCA 60

ACGCGTGGGA ATACTCTTGT ATATGATCAA CAAACAGCAA ATACATTACT AAATCAAAAA 120

CAACAAGACT TTCAGTCTAT TCGTTGGGTT GGTTTAATTC AAAGTAAAGA AGCAGGCGAT 180

TTTACATTTA ACTTATCAGA TGATGAACAT ACGATGATAG AAATCGATGG GAAAGTTATT 240

TCTAATAAAG GGAAAGAAAA ACAAGTTGTC CATTTAGAAA AAGGACAGTT CGTTTCTATC 300

AAAATAGAAT ATCAAGCTGA TGAACCATTT AATGCGGATA GTCAAACCTT TAAAAATTTG 360

AAACTCYTTA AAGTAGATAC TAAGCAACAG TCCCAGCAAA TTCAACTAGA TGAATTAAGA 420

AACCCTGRAA TTTAATAAAA AAGAAACACA AGAATTTCTA ACAAAAGCAA CAAAAACAAA 480

CCTTATTACT CAAAAAGTGA AGAGTACTAG GGATGAAGAC ACGGATACAG ATGGAGATTC 540

TATTCCAGAC ATTTGGGAAG AAAATGGGTA TACCATCCAA AATAAGATTG CCGTCAAATG 600

GGATGATTCA TTAGCAAGTA AAGGATATAC GAAATTTGTT TCAAACCCAC TAGATACTCA 660

CACGGTTGGA GATCCTTATA CAGATTATGA AAAAGCAGCA AGGGATTTAG ATTTGTCAAA 720

TGCAAAAGAA ACATTTAACC CATTAGTTGC GGCTTTTCCA AGTGTGAATG TGAGTATGGA 780

AAAAGTGATA TTGTCTCCAG ATGAGAACTT ATCAAATAGT ATCGAGTCTC ATTCATCTAC 840

GAATTGGTCG TATACGAATA CAGAAGGGGC TTCTATTGAA GCTGGTGGGG GAGCATTAGG 900

CCTATCTTTT GGTGTAAGTG CAAACTATCA ACATTCTGAA ACAGTTGGGT ATGAATGGGG 960

AACATCTACG GGAAATACTT CGCAATTTAA TACAGCTTCA GCGGGGTATT TAAATGCCAA 1020

TRTAMGATAT 1030

23 base pairs

nucleic acid

single

linear

DNA (genomic)

39

CACTCAAAAA ATGAAAAGGG AAA 23

19 base pairs

nucleic acid

single

linear

DNA (genomic)

40

CCGGTTTTAT TGATGCTAC 19

20 base pairs

nucleic acid

single

linear

DNA (genomic)

41

AGAACAATTT TTAGATAGGG 20

20 base pairs

nucleic acid

single

linear

DNA (genomic)

42

TCCCTAAAGC ATCAGAAATA 20

1170 base pairs

nucleic acid

single

linear

DNA (genomic)

43

ATGAAGAAAC AAATAGCAAG CGTTGTAACT TGTACGCTAT TAGCCCCTAT GCTTTTTAAT 60

GGAGATATGA ACGCTGCTTA CGCAGCTAGT CAAACAAAAC AAACACCTGC AGCTCAGGTA 120

AACCAAGAGA AAGAAGTAGA TCGAAAAGGA TTACTTGGCT ATTACTTTAA AGGGAAAGAT 180

TTTAATGATC TTACTGTATT TGCACCAACG CGTGGGAATA CTCTTGTATA TGATCAACAA 240

ACAGCAAATA CATTACTAAA TCAAAAACAA CAAGACTTTC AGTCTATTCG TTGGGTTGGT 300

TTAATTCAAA GTAAAGAAGC AGGCGATTTT ACATTTAACT TATCAGATGA TGAACATACG 360

ATGATAGAAA TCGATGGGAA AGTTATTTCT AATAAAGGGA AAGAAAAACA AGTTGTCCAT 420

TTAGAAAAAG GACAGTTCGT TTCTATAAAA TGATTCAGCT GATGAACCAT TTAATGCGGT 480

AGTAAACCTT TAAAAATTTG AAACTCTTTA AAGTAGATAC TAAGCAACAG TCCCAGCAAA 540

TTCAACTAGA TGAATTAAGA AACCCTGAAT TTAATAAAAA AGAAACACAA GAATTTCTAA 600

CAAAAGCAAC AAAAACAAAC CTTATTACTC AAAAAGTGAA GAGTACTAGG GATGAAGACA 660

CGGATACAGA TGGAGATTCT ATTCCAGACA TTTGGGAAGA AAATGGGTAT ACCATCCAAA 720

ATAAATTGCC GTCAAATGGG ATGATTCATT AGCAAGTAAA GGATATACGA AATTTGTTTC 780

AAACCCACTA GATACTCACA CGGTTGGAGA TCCTTATACA GATTATGAAA AAGCAGCAAG 840

GGATTTAGAT TTGTCAAATG CAAAAGAAAC ATTTAACCCA TTAGTTGCGG CTTTTCCAAG 900

TGTAATTGAG TATGGAAAAA GGATTTGTTC CAGATGAGAA CTTATCAAAT AGTATCGAGT 960

TCATTCATTC CTACAATTGG TCGATACGAA TACAGAAGGG GCTTCTATTG AAGCTGGTGG 1020

GGGAGCATTA GGCCTATCTT TTGGTGTAAG TGCAAACTAT CAACATTCTG AAACAGTTGG 1080

GTATGAATGG GGAACATCTA CGGGAAATAC TTCGCAATTT AATACAGCTT CAGCGGGGTA 1140

TTTAAATGCG AATGTTGCTA CAATAACGTG 1170

348 amino acids

amino acid

single

linear

protein

44

Met Lys Lys Gln Ile Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pro

1 5 10 15

Met Leu Phe Asn Gly Asp Met Asn Ala Ala Tyr Ala Ala Ser Gln Thr

20 25 30

Lys Gln Thr Pro Ala Ala Gln Val Asn Gln Glu Lys Glu Val Asp Arg

35 40 45

Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asp Leu

50 55 60

Thr Val Phe Ala Pro Thr Arg Gly Asn Thr Leu Val Tyr Asp Gln Gln

65 70 75 80

Thr Ala Asn Thr Leu Leu Asn Gln Lys Gln Gln Asp Phe Gln Ser Ile

85 90 95

Arg Trp Val Gly Leu Ile Gln Ser Lys Glu Ala Gly Asp Phe Thr Phe

100 105 110

Asn Leu Ser Asp Asp Glu His Thr Met Ile Glu Ile Asp Gly Lys Val

115 120 125

Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gly

130 135 140

Gln Phe Val Ser Xaa Lys Xaa Xaa Xaa Xaa Ala Asp Glu Pro Phe Asn

145 150 155 160

Ala Xaa Ser Xaa Thr Phe Lys Asn Leu Lys Leu Phe Lys Val Asp Thr

165 170 175

Lys Gln Gln Ser Gln Gln Ile Gln Leu Asp Glu Leu Arg Asn Pro Glu

180 185 190

Phe Asn Lys Lys Glu Thr Gln Glu Phe Leu Thr Lys Ala Thr Lys Thr

195 200 205

Asn Leu Ile Thr Gln Lys Val Lys Ser Thr Arg Asp Glu Asp Thr Asp

210 215 220

Thr Asp Gly Asp Ser Ile Pro Asp Ile Trp Glu Glu Asn Gly Tyr Thr

225 230 235 240

Ile Gln Asn Xaa Ile Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys

245 250 255

Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Asp Thr His Thr Val Gly

260 265 270

Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser

275 280 285

Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val

290 295 300

Asn Xaa Ser Met Glu Lys Xaa Ile Leu Xaa Pro Asp Glu Asn Leu Ser

305 310 315 320

Asn Ser Ile Glu Xaa His Ser Phe Leu Xaa Ile Gly Arg Ile Arg Ile

325 330 335

Gln Lys Gly Leu Leu Leu Lys Leu Val Gly Glu His

340 345

3 base pairs

nucleic acid

single

linear

DNA (genomic)

45

ATG 3

1 amino acids

amino acid

single

linear

protein

46

Met

1

2583 base pairs

nucleic acid

single

linear

DNA (genomic)

47

ATGACATATA TGAAAAAAAA GTTAGTTAGT GTTGTAACTT GCACGTTATT GGCTCCGATA 60

TTTTTGACTG GAAATGTACA TCCTGTTAAT GCAGACAGTA AAAAAAGTCA GCCTTCTACA 120

GCGCAGGAAA AACAAGAAAA GCCGGTTGAT CGAAAAGGGT TACTCGGCTA TTTTTTTAAA 180

GGGAAAGAGT TTAATCATCT TACTTTGTTC GCACCAACAC GTGATAATAC CCTTATTTAT 240

GATCAACAAA CAGCGAATTC CTTATTAGAT ACCAAACAAC AAGAATATCA ATCTATTCGC 300

TGGATTGGTT TGATTCAAAG TAAAGAAACA GGTGATTTCA CGTTTAACTT ATCTGATGAT 360

CAAAATGCAA TTATAGAAAT AGATGGCAAA ATCATTTCGC ATAAAGGACA GAATAAACAA 420

GTTGTTCACT TAGAAAAAGG AAAGTTAGTC CCGATAAAAA TTGAGTATCA ATCAGATCAG 480

ATATTAACTA GGGATAGTAA CATCTTTAAA GAGTTTCAAT TATTCAAAGT AGATAGTCAG 540

CAACACTCTC ACCAAGTTCA ACTAGACGAA TTAAGAAACC CTGATTTTAA TAAAAAAGAA 600

ACACAACAAT TCTTAGAAAA AGCAGCAAAA ACAAATCTTT TTACACAGAA TATGAAAAGA 660

GATACGGATG ATGATGATGA TACGGATACA GATGGAGATT CTATTCCTGA CCTTTGGGAA 720

GAAAATGGGT ATACCATCCA AAATAAAGTA GCTGTCAAGT GGGATGATTC ATTCGCCGCG 780

AAAGGGTATA CAAAATTTGT TTCTAATCCA CTTGAGAGTC ATACAGTTGG AGATCCCTAT 840

ACGGATTATG AAAAAGCAGC AAGAGATTTA GACTTGGCCA ATGCAAAAGA AACATTTAAC 900

CCATTAGTAG CTGCTTTTCC AAGTGTGAAT GTGAATTTGG AAAAAGTAAT ATTATCCCCA 960

GATGAGAATT TATCTAACAG TGTAGAATCT CATTCGTCTA CAAATTGGTC TTATACGAAT 1020

ACTGAAGGAG CTTCTATCGA AGCTGGGGGT GGTCCATTAG GTATTTCATT TGGAGTGAGT 1080

GCTAATTATC AACACTCTGA AACAGTTGCA AAAGAATGGG GAACATCTAC AGGAAATACC 1140

TCGCAATTTA ATACAGCTTC AGCAGGATAT TTGAATGCGA ATGTTCGATA CAATAATGTG 1200

GGAACAGGTG CGATTTATGA GGTGAAACCT ACAACAAGTT TTGTATTAGA TAAAGATACT 1260

GTAGCAACAA TTACCGCAAA ATCGAATTCG ACAGCTTTAA GTATATCTCC AGGAGAAAGT 1320

TATCCCAAAA AAGGACAAAA TGGAATTGCA ATTAATACAA TGGATGATTT TAATTCCCAT 1380

CCGATTACAT TAAATAAACA ACAATTAGAT CAACTATTAA ATAATAAACC TCTTATGTTA 1440

GAAACAAATC AGGCAGATGG TGTTTATAAA ATAAAGGATA CAAGCGGTAA TATTGTGACT 1500

GGTGGAGAAT GGAACGGTGT TATCCAACAA ATTCAAGCAA AAACAGCCTC TATTATCGTT 1560

GATACGGGAG AAAGTGTTTC AGAAAAGCGT GTCGCAGCAA AAGATTATGA TAATCCTGAG 1620

GATAAAACAC CTTCTTTATC TTTAAAAGAG GCACTTAAAC TTGGATATCC AGAAGAAATT 1680

AAAGAAAAAG ATGGATTGTT GTACTATAAG GACAAGCCAA TTTACGAATC TAGTGTTATG 1740

ACTTATCTAG ATGAGAATAC AGCCAAGGAA GTGGAAAAAC AATTACAGGA TACAACCGGA 1800

ATATATAAAG ATATCAATCA TTTATATGAT GTGAAATTAA CACCTACAAT GAATTTTACG 1860

ATTAAATTAG CTTCCTTATA TGATGGAGCT GAAAATAATG ATGTGAAGAA TGGTCCTATA 1920

GGACATTGGT ATTATACCTA TAATACAGGG GGAGGAAATA CTGGAAAACA CCAATATAGG 1980

TCTGCTAATC CCAGTGCAAA TGTAGTTTTA TCTTCTGAAG CGAAAAGTAA GTTAGATAAA 2040

AATACAAATT ACTACCTTAG TATGTATATG AAAGCTGAGT CTGATACAGA GCCTACAATA 2100

GAAGTAAGTG GTGAGAATTC TACGATAACG AGTAAAAAGG TAAAACTAAA CAGTGAGGGC 2160

TATCAAAGAG TAGATATTTT AGTGCCGAAT TCTGAAAGAA ATCCAATAAA TCAAATATAT 2220

GTAAGAGGAA ATAATACAAC AAATGTATAC TGGGATGATG TTTCAATTAC AAATATTTCA 2280

GCTATAAACC CAAAAACTTT AACAGATGAA GAAATTAAAG AAATATATAA AGATTTTAGT 2340

GAGTCTAAAG ACTGGCCTTG GTTCAATGAT GTTACGTTTA AAAATATTAA ACCATTAGAG 2400

AATTATGTAA AACAATATAG AGTTGATTTC TGGAATACTA ATAGTGATAG ATCATTTAAT 2460

AGGATTAAGG ACAGTTACCC AGTTAATGAA GATGGAAGTG TTAAAGTCAA CATGACAGAA 2520

TATAATGAAG GATATCCACT TAGAATTGAA TCCGCCTACC ATTTAAATAT TTCAGATCTA 2580

TAA 2583

860 amino acids

amino acid

single

linear

protein

48

Met Thr Tyr Met Lys Lys Lys Leu Val Ser Val Val Thr Cys Thr Leu

1 5 10 15

Leu Ala Pro Ile Phe Leu Thr Gly Asn Val His Pro Val Asn Ala Asp

20 25 30

Ser Lys Lys Ser Gln Pro Ser Thr Ala Gln Glu Lys Gln Glu Lys Pro

35 40 45

Val Asp Arg Lys Gly Leu Leu Gly Tyr Phe Phe Lys Gly Lys Glu Phe

50 55 60

Asn His Leu Thr Leu Phe Ala Pro Thr Arg Asp Asn Thr Leu Ile Tyr

65 70 75 80

Asp Gln Gln Thr Ala Asn Ser Leu Leu Asp Thr Lys Gln Gln Glu Tyr

85 90 95

Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp

100 105 110

Phe Thr Phe Asn Leu Ser Asp Asp Gln Asn Ala Ile Ile Glu Ile Asp

115 120 125

Gly Lys Ile Ile Ser His Lys Gly Gln Asn Lys Gln Val Val His Leu

130 135 140

Glu Lys Gly Lys Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Gln

145 150 155 160

Ile Leu Thr Arg Asp Ser Asn Ile Phe Lys Glu Phe Gln Leu Phe Lys

165 170 175

Val Asp Ser Gln Gln His Ser His Gln Val Gln Leu Asp Glu Leu Arg

180 185 190

Asn Pro Asp Phe Asn Lys Lys Glu Thr Gln Gln Phe Leu Glu Lys Ala

195 200 205

Ala Lys Thr Asn Leu Phe Thr Gln Asn Met Lys Arg Asp Thr Asp Asp

210 215 220

Asp Asp Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu

225 230 235 240

Glu Asn Gly Tyr Thr Ile Gln Asn Lys Val Ala Val Lys Trp Asp Asp

245 250 255

Ser Phe Ala Ala Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu

260 265 270

Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg

275 280 285

Asp Leu Asp Leu Ala Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala

290 295 300

Ala Phe Pro Ser Val Asn Val Asn Leu Glu Lys Val Ile Leu Ser Pro

305 310 315 320

Asp Glu Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp

325 330 335

Ser Tyr Thr Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Gly Gly Pro

340 345 350

Leu Gly Ile Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr

355 360 365

Val Ala Lys Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn

370 375 380

Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val

385 390 395 400

Gly Thr Gly Ala Ile Tyr Glu Val Lys Pro Thr Thr Ser Phe Val Leu

405 410 415

Asp Lys Asp Thr Val Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala

420 425 430

Leu Ser Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly

435 440 445

Ile Ala Ile Asn Thr Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu

450 455 460

Asn Lys Gln Gln Leu Asp Gln Leu Leu Asn Asn Lys Pro Leu Met Leu

465 470 475 480

Glu Thr Asn Gln Ala Asp Gly Val Tyr Lys Ile Lys Asp Thr Ser Gly

485 490 495

Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Gln

500 505 510

Ala Lys Thr Ala Ser Ile Ile Val Asp Thr Gly Glu Ser Val Ser Glu

515 520 525

Lys Arg Val Ala Ala Lys Asp Tyr Asp Asn Pro Glu Asp Lys Thr Pro

530 535 540

Ser Leu Ser Leu Lys Glu Ala Leu Lys Leu Gly Tyr Pro Glu Glu Ile

545 550 555 560

Lys Glu Lys Asp Gly Leu Leu Tyr Tyr Lys Asp Lys Pro Ile Tyr Glu

565 570 575

Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Glu

580 585 590

Lys Gln Leu Gln Asp Thr Thr Gly Ile Tyr Lys Asp Ile Asn His Leu

595 600 605

Tyr Asp Val Lys Leu Thr Pro Thr Met Asn Phe Thr Ile Lys Leu Ala

610 615 620

Ser Leu Tyr Asp Gly Ala Glu Asn Asn Asp Val Lys Asn Gly Pro Ile

625 630 635 640

Gly His Trp Tyr Tyr Thr Tyr Asn Thr Gly Gly Gly Asn Thr Gly Lys

645 650 655

His Gln Tyr Arg Ser Ala Asn Pro Ser Ala Asn Val Val Leu Ser Ser

660 665 670

Glu Ala Lys Ser Lys Leu Asp Lys Asn Thr Asn Tyr Tyr Leu Ser Met

675 680 685

Tyr Met Lys Ala Glu Ser Asp Thr Glu Pro Thr Ile Glu Val Ser Gly

690 695 700

Glu Asn Ser Thr Ile Thr Ser Lys Lys Val Lys Leu Asn Ser Glu Gly

705 710 715 720

Tyr Gln Arg Val Asp Ile Leu Val Pro Asn Ser Glu Arg Asn Pro Ile

725 730 735

Asn Gln Ile Tyr Val Arg Gly Asn Asn Thr Thr Asn Val Tyr Trp Asp

740 745 750

Asp Val Ser Ile Thr Asn Ile Ser Ala Ile Asn Pro Lys Thr Leu Thr

755 760 765

Asp Glu Glu Ile Lys Glu Ile Tyr Lys Asp Phe Ser Glu Ser Lys Asp

770 775 780

Trp Pro Trp Phe Asn Asp Val Thr Phe Lys Asn Ile Lys Pro Leu Glu

785 790 795 800

Asn Tyr Val Lys Gln Tyr Arg Val Asp Phe Trp Asn Thr Asn Ser Asp

805 810 815

Arg Ser Phe Asn Arg Ile Lys Asp Ser Tyr Pro Val Asn Glu Asp Gly

820 825 830

Ser Val Lys Val Asn Met Thr Glu Tyr Asn Glu Gly Tyr Pro Leu Arg

835 840 845

Ile Glu Ser Ala Tyr His Leu Asn Ile Ser Asp Leu

850 855 860

1356 base pairs

nucleic acid

single

linear

DNA (genomic)

49

ATGGTATCCA AAAAGTTACA ATTAGTCACA AAAACTTTAG TGTTTAGTAC AGTTTTGTCA 60

ATACCGTTAT TAAATAATAG TGAGATAAAA GCGGAACAAT TAAATATGAA TTCTCAAATT 120

AAATATCCTA ACTTCCAAAA TATAAATATC GCTGATAAGC CAGTAGATTT TAAAGAGGAT 180

AAAGAAAAAG CACGAGAATG GGGAAAAGAA AAAGAAAAAG AGTGGAAACT AACTGCTACT 240

GAAAAAGGGA AAATTAATGA TTTTTTAGAT GATAAAGATG GATTAAAAAC AAAATACAAA 300

GAAATTAATT TTTCTAAGAA TTTTGAATAT GAAACAGAGT TAAAACAGCT TGAAAAAATT 360

AATAGCATGC TAGATAAAGC AAATCTAACA AATTCAATTG TCACGTATAA AAACGTTGAG 420

CCTACAACAA TAGGATTCAA TCACTCTTTG ACTGATGGGA ATCAAATTAA TTCCGAAGCT 480

CAACAGAAGT TCAAGGAACA GTTTTTAGGA AATGATATTA AATTTGATAG TTATTTGGAT 540

ATGCACTTAA CTGAACAAAA TGTTTCCGGT AAAGAAAGGG TTATTTTAAA AGTTACAGTA 600

CTTAGTGGGA AAGGTTCTAC TCCAACAAAA GCAGGTGTTG TTTTAAATAA TAAAGAATAC 660

AAAATGTTGA TTGATAATGG ATATATACTA CATGTAGAAA ACATAACGAA AGTTGTAAAA 720

AAAGGACAGG AATGTTTACA AGTTGAAGGA ACGTTAAAAA AGAGCTTGGA CTTTAAAAAT 780

GATAGTGACG GTAAGGGAGA TTCCTGGGGA AAGAAAAATT ACAAGGAATG GTCTGATTCT 840

TTAACAAATG ATCAGAGAAA AGACTTAAAT GATTATGGTG CGCGAGGTTA TACCGAAATA 900

AATAAATATT TACGTGAAGG GGGTACCGGA AATACAGAGT TGGAGGAAAA AATTAAAAAT 960

ATTTCTGACG CACTAGAAAA GAATCCTATC CCTGAAAACA TTACTGTTTA TAGATATTGC 1020

GGAATGGCGG AATTTGGTTA TCCAATTCAA CCCGAGGCTC CCTCCGTACA AGATTTTGAA 1080

GAGAAATTTT TGGATAAAAT TAAGGAAGAA AAAGGATATA TGAGTACGAG CTTATCAAGT 1140

GATGCGACTT CTTTTGGCGC AAGAAAAATT ATCTTAAGAT TGCAGATACC AAAAGGAAGT 1200

TCAGGAGCAT ATGTAGCTGG TTTAGATGGA TTTAAACCAG CAGAGAAGGA GATTCTTATT 1260

GATAAGGGAA GCAAGTATCA TATTGATAAA GTAACAGAAG TAGTTGTGAA AGGTATTAGA 1320

AAACTCGTAG TAGATGCGAC ATTATTATTA AAATAA 1356

451 amino acids

amino acid

single

linear

DNA (genomic)

50

Met Val Ser Lys Lys Leu Gln Leu Val Thr Lys Thr Leu Val Phe Ser

1 5 10 15

Thr Val Leu Ser Ile Pro Leu Leu Asn Asn Ser Glu Ile Lys Ala Glu

20 25 30

Gln Leu Asn Met Asn Ser Gln Ile Lys Tyr Pro Asn Phe Gln Asn Ile

35 40 45

Asn Ile Ala Asp Lys Pro Val Asp Phe Lys Glu Asp Lys Glu Lys Ala

50 55 60

Arg Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr

65 70 75 80

Glu Lys Gly Lys Ile Asn Asp Phe Leu Asp Asp Lys Asp Gly Leu Lys

85 90 95

Thr Lys Tyr Lys Glu Ile Asn Phe Ser Lys Asn Phe Glu Tyr Glu Thr

100 105 110

Glu Leu Lys Gln Leu Glu Lys Ile Asn Ser Met Leu Asp Lys Ala Asn

115 120 125

Leu Thr Asn Ser Ile Val Thr Tyr Lys Asn Val Glu Pro Thr Thr Ile

130 135 140

Gly Phe Asn His Ser Leu Thr Asp Gly Asn Gln Ile Asn Ser Glu Ala

145 150 155 160

Gln Gln Lys Phe Lys Glu Gln Phe Leu Gly Asn Asp Ile Lys Phe Asp

165 170 175

Ser Tyr Leu Asp Met His Leu Thr Glu Gln Asn Val Ser Gly Lys Glu

180 185 190

Arg Val Ile Leu Lys Val Thr Val Leu Ser Gly Lys Gly Ser Thr Pro

195 200 205

Thr Lys Ala Gly Val Val Leu Asn Asn Lys Glu Tyr Lys Met Leu Ile

210 215 220

Asp Asn Gly Tyr Ile Leu His Val Glu Asn Ile Thr Lys Val Val Lys

225 230 235 240

Lys Gly Gln Glu Cys Leu Gln Val Glu Gly Thr Leu Lys Lys Ser Leu

245 250 255

Asp Phe Lys Asn Asp Ser Asp Gly Lys Gly Asp Ser Trp Gly Lys Lys

260 265 270

Asn Tyr Lys Glu Trp Ser Asp Ser Leu Thr Asn Asp Gln Arg Lys Asp

275 280 285

Leu Asn Asp Tyr Gly Ala Arg Gly Tyr Thr Glu Ile Asn Lys Tyr Leu

290 295 300

Arg Glu Gly Gly Thr Gly Asn Thr Glu Leu Glu Glu Lys Ile Lys Asn

305 310 315 320

Ile Ser Asp Ala Leu Glu Lys Asn Pro Ile Pro Glu Asn Ile Thr Val

325 330 335

Tyr Arg Tyr Cys Gly Met Ala Glu Phe Gly Tyr Pro Ile Gln Pro Glu

340 345 350

Ala Pro Ser Val Gln Asp Phe Glu Glu Lys Phe Leu Asp Lys Ile Lys

355 360 365

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

370 375 380

Phe Gly Ala Arg Lys Ile Ile Leu Arg Leu Gln Ile Pro Lys Gly Ser

385 390 395 400

Ser Gly Ala Tyr Val Ala Gly Leu Asp Gly Phe Lys Pro Ala Glu Lys

405 410 415

Glu Ile Leu Ile Asp Lys Gly Ser Lys Tyr His Ile Asp Lys Val Thr

420 425 430

Glu Val Val Val Lys Gly Ile Arg Lys Leu Val Val Asp Ala Thr Leu

435 440 445

Leu Leu Lys

450

47 base pairs

nucleic acid

single

linear

DNA (genomic)

51

GCTCTAGAAG GAGGTAACTT ATGAACAAGA ATAATACTAA ATTAAGC 47

27 base pairs

nucleic acid

single

linear

DNA (genomic)

52

GGGGTACCTT ACTTAATAGA GACATCG 27

2364 base pairs

nucleic acid

single

linear

DNA (genomic)

53

ATGAATATGA ATAATACTAA ATTAAACGCA AGGGCCCTAC CGAGTTTTAT TGATTATTTT 60

AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAATATGAT TTTTAAAACG 120

GATACAGGTG GTAATCTAAC CTTAGACGAA ATCCTAAAGA ATCAGCAGTT ACTAAATGAG 180

ATTTCTGGTA AATTGGATGG GGTAAATGGG AGCTTAAATG ATCTTATCGC ACAGGGAAAC 240

TTAAATACAG AATTATCTAA GGAAATCTTA AAAATTGCAA ATGAACAGAA TCAAGTCTTA 300

AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCATATATA TCTACCTAAA 360

ATCACATCTA TGTTAAGTGA TGTAATGAAG CAAAATTATG CGCTAAGTCT GCAAGTAGAA 420

TACTTAAGTA AACAATTGAA AGAAATTTCT GATAAATTAG ATGTTATTAA CGTAAATGTT 480

CTTATTAACT CTACACTTAC TGAAATTACA CCTGCATATC AACGGATTAA ATATGTAAAT 540

GAAAAATTTG AAGAATTAAC TTTTGCTACA GAAACCACTT TAAAAGTAAA AAAGGATAGC 600

TCGCCTGCTG ATATTCTTGA CGAGTTAACT GAATTAACTG AACTAGCGAA AAGTGTTACA 660

AAAAATGACG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGGA 720

AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCTTCAG AATTAATTGC TAAAGAAAAT 780

GTGAAAACAA GTGGCAGTGA AGTAGGAAAT GTTTATAATT TCTTAATTGT ATTAACAGCT 840

CTACAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGAT 900

ATTGATTATA CATCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGTA 960

AACATCCTTC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAGGA 1020

AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GGTTGGGTTT 1080

GAAATTAGTA ATGATTCAAT GACAGTATTA AAAGTATATG AAGCTAAGCT AAAACAAAAT 1140

TACCAAGTTG ATAAGGATTC CTTATCGGAA GTCATTTATA GTGATATGGA TAAATTATTG 1200

TGCCCAGATC AATCTGAACA AATTTATTAT ACAAATAATA TAGTATTTCC AAATGAATAT 1260

GTAATTACTA AAATTGATTT TACTAAGAAA ATGAAAACTT TAAGATATGA GGTAACAGCT 1320

AATTCTTACG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAGAAAGT AGAATCAAGT 1380

GAAGCGGAGT ATAGGACGTT AAGTGCTAAT AATGATGGAG TATATATGCC GTTAGGTGTC 1440

ATCAGTGAAA CATTTTTGAC TCCAATTAAT GGATTTGGCC TCCAAGCTGA TGAAAATTCA 1500

AGATTAATTA CTTTAACATG TAAATCATAT TTAAGGGAAC TACTACTAGC GACAGACTTA 1560

AGCAATAAAG AAACTAAATT GATTGTCCCG CCTATTAGTT TTATTAGTAA TATTGTAGAA 1620

AATGGGAACT TAGAGGGAGA AAACTTAGAG CCGTGGATAG CAAATAACAA AAATGCGTAT 1680

GTAGATCATA CAGGTGGTAT AAATGGAACT AAAGTTTTAT ATGTTCATAA GGATGGTGAG 1740

TTTTCACAAT TTGTTGGAGG TAAGTTAAAA TCGAAAACAG AATATGTAAT TCAATATATT 1800

GTAAAGGGAA AAGCTTCTAT TTATTTAAAA GATAAAAAAA ATGAGAATTC CATTTATGAA 1860

GAAATAAATA ATGATTTAGA AGGTTTTCAA ACTGTTACTA AACGTTTTAT TACAGGAACG 1920

GATTCTTCAG GGATTCATTT AATTTTTACC AGTCAAAATG GCGAGGGAGC ATTTGGAGGA 1980

AACTTTATTA TCTCAGAAAT TAGGACATCC GAAGAGTTAT TAAGTCCAGA ATTGATTATG 2040

TCGGATGCTT GGGTTGGATC CCAGGGAACT TGGATCTCAG GAAATTCTCT CACTATTAAT 2100

AGTAATGTAA ATGGAACCTT TCGACAAAAT CTTCCGTTAG AAAGTTATTC AACCTATAGT 2160

ATGAACTTTA CTGTGAATGG ATTTGGCAAG GTGACAGTAA GAAATTCTCG TGAAGTATTA 2220

TTTGAAAAAA GTTATCCGCA GCTTTCACCT AAAGATATTT CTGAAAAATT TACAACTGCA 2280

GCCAATAATA CCGGATTATA TGTAGAGCTT TCTCGCTCAA CGTCGGGTGG TGCAATAAAT 2340

TTCCGAGATT TTTCAATTAA GTAA 2364

787 amino acids

amino acid

single

linear

protein

54

Met Asn Met Asn Asn Thr Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asn Leu Thr Leu

35 40 45

Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Glu Ile Ser Gly Lys

50 55 60

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn

65 70 75 80

Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln

85 90 95

Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr

100 105 110

Met Leu His Ile Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val

115 120 125

Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Val Glu Tyr Leu Ser Lys

130 135 140

Gln Leu Lys Glu Ile Ser Asp Lys Leu Asp Val Ile Asn Val Asn Val

145 150 155 160

Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile

165 170 175

Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr

180 185 190

Thr Leu Lys Val Lys Lys Asp Ser Ser Pro Ala Asp Ile Leu Asp Glu

195 200 205

Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val

210 215 220

Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly

225 230 235 240

Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile

245 250 255

Ala Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr

260 265 270

Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Thr

275 280 285

Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr

290 295 300

Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val

305 310 315 320

Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala

325 330 335

Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys

340 345 350

Pro Gly His Ala Leu Val Gly Phe Glu Ile Ser Asn Asp Ser Met Thr

355 360 365

Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp

370 375 380

Lys Asp Ser Leu Ser Glu Val Ile Tyr Ser Asp Met Asp Lys Leu Leu

385 390 395 400

Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe

405 410 415

Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys

420 425 430

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

435 440 445

Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr

450 455 460

Arg Thr Leu Ser Ala Asn Asn Asp Gly Val Tyr Met Pro Leu Gly Val

465 470 475 480

Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala

485 490 495

Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg

500 505 510

Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile

515 520 525

Val Pro Pro Ile Ser Phe Ile Ser Asn Ile Val Glu Asn Gly Asn Leu

530 535 540

Glu Gly Glu Asn Leu Glu Pro Trp Ile Ala Asn Asn Lys Asn Ala Tyr

545 550 555 560

Val Asp His Thr Gly Gly Ile Asn Gly Thr Lys Val Leu Tyr Val His

565 570 575

Lys Asp Gly Glu Phe Ser Gln Phe Val Gly Gly Lys Leu Lys Ser Lys

580 585 590

Thr Glu Tyr Val Ile Gln Tyr Ile Val Lys Gly Lys Ala Ser Ile Tyr

595 600 605

Leu Lys Asp Lys Lys Asn Glu Asn Ser Ile Tyr Glu Glu Ile Asn Asn

610 615 620

Asp Leu Glu Gly Phe Gln Thr Val Thr Lys Arg Phe Ile Thr Gly Thr

625 630 635 640

Asp Ser Ser Gly Ile His Leu Ile Phe Thr Ser Gln Asn Gly Glu Gly

645 650 655

Ala Phe Gly Gly Asn Phe Ile Ile Ser Glu Ile Arg Thr Ser Glu Glu

660 665 670

Leu Leu Ser Pro Glu Leu Ile Met Ser Asp Ala Trp Val Gly Ser Gln

675 680 685

Gly Thr Trp Ile Ser Gly Asn Ser Leu Thr Ile Asn Ser Asn Val Asn

690 695 700

Gly Thr Phe Arg Gln Asn Leu Pro Leu Glu Ser Tyr Ser Thr Tyr Ser

705 710 715 720

Met Asn Phe Thr Val Asn Gly Phe Gly Lys Val Thr Val Arg Asn Ser

725 730 735

Arg Glu Val Leu Phe Glu Lys Ser Tyr Pro Gln Leu Ser Pro Lys Asp

740 745 750

Ile Ser Glu Lys Phe Thr Thr Ala Ala Asn Asn Thr Gly Leu Tyr Val

755 760 765

Glu Leu Ser Arg Ser Thr Ser Gly Gly Ala Ile Asn Phe Arg Asp Phe

770 775 780

Ser Ile Lys

785