FI107053B - Gene cleavage associated with nogalamycin biosynthesis and its use in the production of hybrid antibiotics - Google Patents

Description

107053

Gene clustering associated with nogalamycin biosynthesis and its use in the production of hybrid antibiotics

FIELD OF THE INVENTION The present invention relates to a gene cluster related to nogalamycin biosynthesis derived from Streptomyces nogalator and the use of genes contained therein for the preparation of novel hybrid antibiotics for drug screening.

Background of the Invention

Anthracyclines are anti-tumor antibiotics produced mainly by Strepto-mycesA & yi. Of the anthracyclines, the daunomycin family is the most important commercially, since almost all of the approximately 10 anthracyclines currently in clinical use or in end-stage clinical trials on cytotoxic drugs belong to this family. Despite the long history of anthracyclines for about three decades, their biosynthesis studies are still ongoing, and there is still interest in finding new molecules for the development of chemotherapeutic agents for the treatment of cancer. Gene technology is currently being used to search for 20 new molecules for drug screening. Cloning of genes for anthracycline biosynthesis facilitates the production of hybrid anthracyclines as well as their use in combinatorial biosynthesis to generate new molecules.

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Nogalamycin, first described by Bhuyan and Dietz in 1965, is an anthracycline antibiotic produced by 25 Streptomyces nogalateri. This compound is highly active against tumor cells, but on the other hand its toxic properties have prevented it from progressing into clinical trials (Bhuyan and Smith, 1975). Menogaryl (7-O-methylnogarol) is a semisynthetic derivative of nogalamycin, and its value in cancer treatment has been studied (e.g., Yoshida et al., 1996), currently of interest mainly in Japan. The structure of nogalamycin (Figure 1) differs from most other anthracyclines, such as the daunomycin family, in two important aspects: (i) its stereochemistry at position 9 is reversed and (ii) its 107053 2 sugar moiety, nogalamine, is attached at position 1 with a typical glycosidic bond carbon with a CC bond. Wiley et al. further presented a study on the structure of nogalamycin in 1977. Further, in 1978, Wiley et al. published biosynthetic studies of nogalamycin, which provided information on its constituents: the Aglycon moiety is composed of 5 of the ten acetates; neutral sugar, Nogalose, derived from glucose; and the methyl groups of both sugars, nogalamine and nogalose have been changed from methionine. Wiley did not clearly determine the origin of the Nogalamine, but most probably the Nogalamine is also derived from glucose.

Molecular cloning of anthracycline biosynthetic genes has facilitated molecular genetics studies, providing tools for rational structural alterations while providing surprising combinations with other antibiotics. Of particular interest has been the daunomycin biosynthetic genes as reported in several publications (Lomovskaya et al., 1998; Rajgarhia and Strohl, 1997 and references cited therein). Similarly, some aclasinomycin biosynthesis genes from S. galilae (Fujii and Ebizuka, 1997) and rhodomycin biosynthesis genes from S. purpurascens (Niemi et al, 1994) have been cloned. We have cloned the biosynthesis genes of nogalamycin and have used these genes successfully in the production of hybrid anthracyclines. Most genes are related to the polyketide pathway and are responsible for the formation of the tricyclic intermediate and have been reported in Ylihonko et al.,. 1996a and b, and Torkkell et al., 1997. Despite advances in molecular cloning, the glucose biosynthetic pathway observed for anthracyclines is still mainly hypothetical.

25 Regarding genes in the deoxyhexose pathway, Madduri et al. (1998) reported that a gene from the avermectin biosynthetic cluster caused the production of hybrid anthracyclines when it altered the sugar residue upon transfer to a mutant of S. peucetius. The product obtained was epirubicin, a commercially important anthracycline. In this case, the hydroxy group on the daunosamine moiety had the opposite stereochemistry provided by the avermectin biosynthetic gene. S. galilaeus has been used as a host for the preparation of hybrid anthracyclines using genes derived from the S. purpurascens 3,107053 rhodomycin pathway (Niemi et al., 1994), and genes from the S. nogalaterin nogalamycin biosynthesis cluster (Ylihonko et al., 1996a). The genes of the nogalamycin pathway were used to generate the production of hybrid anthracyclines in S. steffisburgensis, which typically produces steffimycin (Kunnari et al., 5 1997). Previously, actinorodine biosynthetic genes have been expressed in S. galilaeus, leading to aloesaponarin production (Strohl et al., 1991). In these hybrid compounds, the aglycon residue had been modified.

SUMMARY OF THE INVENTION This invention relates to a gene cluster of Streptomyces nogalateri, where most of the genes are derived from the deoxyhexose pathway of nogalamine and nogalose. Expression of the DNA sequence contained in said region in S. galilaeus, which produces aclasino mycins, results in hybrid anthracyclines in which the aglycon moiety is derived from S. galilaeuk-15 s, while the sugar moiety is neither characteristic of S. nogalater nor S.

Galilaeus. Further introduction of the gene encoding nogalamycin cyclase into said cluster into a suitable plasmid construct yields nogalamycinone, which is the aglycon of nogalamycin. Because the stereochemistry of nogalamycin differs from most other anthracyclines, it is possible to produce C-9 stereoisomers of anthracycline molecules using this gene.

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DETAILED DESCRIPTION OF THE INVENTION The test methods used in the present invention are conventional in the art. Techniques not specifically described herein are described in Hopwood et al., "Genetic Manipulation of Streptomyces: A Laboratory Manual" by The John Innes

Foundation, Norwich (1985) and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. The full details of the publications, patents and patent applications cited herein are provided in the list of references.

This invention relates in particular to a gene cluster (5 to 5 clusters) involved in the biosynthesis of nogalamycin, which is capable of producing hybrid antibiotics with a modified sugar moiety. More particularly, the invention relates to the use of genes related to nogalamine / nogalose biosynthesis for the production of hybrid antibiotics modified from the sugar moiety. The invention also relates to the use of a specific cyclase gene contained in a gene cluster according to the invention for the preparation of C-9 stereoisomers of typical anthracyclines.

The gene cluster of the present invention is linked to previously reported clusters of nogalamycin biosynthesis. The starting point for this invention was a gene cluster related to nogalamycin chromophore (International Patent Application WO 96/10581). Subsequently, we found some genes associated with the deoxyhexose pathway in nogalamycin biosynthesis (Torkkell et al., 1997), and part of the fragment containing the said genes was used to clone the genes of this invention.

The genes involved in nogalamycin biosynthesis can be isolated from Sire / tfo / nym species, particularly S. nogalater, which produces nogalamycin. Species that produce nogalamycin-like anthracyclines, e.g., S., may also be used.

20 violaceochromogenesis that produce arugomycin (Kawai et al., 1987), or S. avi, diniit, which produces avidinorubicin (Aoki et al., 1991).

The genomic library is prepared using genomic DNA of a Streptomyces strain containing genes related to nogalamycin biosynthesis. Gene fragments suitable for cloning can be obtained by any of the densely cleaving restriction enzymes.

Typically & zw3AI is used. Isolated fragments could be introduced by ligation into any Escherichia coli vector, such as a plasmid, phagemid, phage or cosmid. The cosmid vector is recommended because it can be used to clone large DNA fragments. A cosmid vector such as 30 pFD666 (ATCC No. 77286) is suitable for this purpose since it can clone fragments of about 40 kb. The fiamHI site of pFD666 can be used for cloning, which yields the cohesive ends of 107053 to Sa3AI fragments. Commercially available kits can be used to package DNA into phage particles. Various E. coli strains can be used to infect packaged DNA. A suitable E. coli strain is, for example, XLIBlue MRF ', which lacks several restriction systems.

5

When E. coli is used as a host strain for a genomic library, hybridization is a preferred screening strategy. Any known fragment derived from the nogalamycin gene cluster can be used as the hybridization probe, but a short fragment of about 1 kb from the other end of the previously cloned biosynthetic region is preferred. The colonies obtained for the genomic library are transferred to membranes, preferably nylon membranes, for filter hybridization. Because the average size of the genomic DNA fragment is 40 kb, 2300 colonies gave a 99.99% probability of finding an expanded region of nogalamycin biosynthesis. Any hybridization method can be used, but the DIG system (Boehringer Mannheim GmbH, Germany) is particularly useful. Because the probe is homologous to the hybridized DNA, it is preferable to perform hybridization limiting washes at 70 ° C at low salt concentration according to Boehringer Mannheim's "DIG System User's Guide for Filter Hybridization". Probably at least 80% homology is required for DNA binding to 20 probes under the washing conditions.

Using this procedure, seven of the approximately 5,000 clones gave a positive signal and were isolated for DNA isolation. Positive clones can be cleaved with appropriate restriction enzymes to demonstrate the physical linkage of DNA fragments. The cosmid used for cloning was a transporter cosmid which replicates in both E. coli and Streptomyces - > eissdi. However, transfer of recombinant cosmids to S. lividans TK24, a typical laboratory strain used in cloning of Streptomyces, led to deletions and this approach was rejected. Instead, we used plasmid pIJ486, which is a highly transcriptional Äre / tfomyces plasmid, for expression studies. However, any plasmid capable of stably replicating in Streptomyces can be used for this purpose.

From one clone, two γ g / II fragments were separately introduced into pIJ486 vectors, and two of the resulting plasmids were transferred to the primary host, S. lividans TK24. The resulting recombinant plasmids (pSY42 and pSY43) containing the 10 kb and 7kb fragments of S. nogalater genomic DNA, respectively, were isolated from the primary host and further introduced into other StreptomycesA110M protoplast transformations. A recombinant plasmid containing the 10 kb fragment induced production of hybrid anthracyclines by S.

10 galilaeus mutant strain H039, which naturally produces aclavinone-rhodinose-rhodinosis-rhodinosis. A few other S. galilaeus strains (H075, H026, H063) whose deoxyhexose pathway had been mutated to alter aclasinomycin sugars were used for transformation and new hybrid compounds were obtained. Because the structure of nogalamycin is almost unique among anthracyclines, plasmids could be transferred to other anthracycline-producing strains, such as S. peucetius, which produces daunomycin, and S. purpurascens, which produces rhodomycins, to alter the structures of the characteristic antibiotics.

Because the cloned cluster was linked to a previously known region of nogalamycin biosynthesis, its ability to effect a change in the sugar moiety indicated that deoxyhexose-throwing genes may be present. However, sequencing is necessary to determine the function of genes in a cloned cluster. The 10 kb and 7 kb DNA fragments were further introduced into pSL1 190 for subcloning. Sequencing strategies such as gradual deletion of a DNA fragment, random cloning of fragments, or sequencing with specific primers can be used, but sequencing; for this purpose, we recommend making subclones from known restriction sites. ABI

With the PRISM system (Perkin-Elmer), 500 to 700 bases per reaction can be obtained for sequencing, which means that approximately 1 kb of overlapping bases are required for sequencing: 27 subclones were constructed for this purpose. .

107053 7

Sequencing of adjacent J3g / II fragments consisting of about 16,000 base pairs yielded 15 complete ORFs. Sequence analysis can be performed using any computer-based program such as GCG (Madison, Wisconsin, USA).

Gene functions implied by the homology of sequences available in libraries according to the present invention are aminotransferase (snogT), not complete 1. dTDP-glucose synthetase (snog) 2. aminomethyltransferase (snogA) 3. polyketide dicyclase (s / zoaM) 10 4. deoxyhex unknown (snogN) 5. hydroxylase, (snoaG) 6. dTDP-4-dehydroramnose reductase (snogC) 7. dTDP-glucose-4,6-dehydratase (snogK) 8. NAME-cyclase (snoaL) 15 9. unknown (jtzöK) 10. glycosyltransferase, GTF (snogD) 11. unknown (snoW) 12. glycosyltransferase, GTF (swogE) 13. unknown (snoL) 20. 14. unknown (snoO) 15. C-7 ketoreductase (s? OF) unknown (snoN) , not perfect

Gene names: g means that the gene was involved in the biosynthesis of the glycosidic moiety, e.g.

25 glycosyltransferases, whereas a means that the gene is required for the formation of the aglycone moiety.

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Given the biosynthetic pathway suggested for nogalamysUn in Figure 3, our ability to identify genes, e.g. snoaL, which is responsible for the cyclization of the fourth ring of the aglycon moiety, to effect multiple alterations in the structures of the antibiotics, while defining the anthracyclinone stereochemistry, and with the genes involved in the production of nogalamine and nogalose (snogJ, snogK, snogN, snog, genes responsible for attaching sugar residues to the aglycone moiety (snogD and snogE).

These genes could be introduced separately into the vector using appropriate restriction sites, or amplified by PCR. The fragments may contain an internal promoter, or the promoter may be cloned separately. It is preferable to use a vector containing a promoter that causes gene expression in the Streptomyces strain. Plasmid pIJE486 contains the ermE promoter of the erythromycin resistance gene, which allows constitutive expression of 10 genes linked in the correct orientation. Particular attention has been paid to the gene encoding aliphatic ring cyclase, but any gene of said cluster can be expressed in Streptomyces hosts. Said cyclase will convert the stereochemistry of auramycinone C9 in the TK24 strain if introduced into a plasmid carrying other auramycinone biosynthetic genes, except for the cyclase corresponding to the typical stereochemistry of anthracyclines.

Strains of Streptomyces containing recombinant plasmids, especially S. galilaeus, are grown in media that produce antibiotics. The hybrid compounds are extracted with organic solvents from the culture medium and the compounds are separated and purified using chromatographic techniques.

In accordance with the present invention, S. galilaeus H039 bearing the plasmid pSY42, designated H039 / pSY42, produces aclavinone-4'-epi-2-deoxifucose in E1 medium supplemented with thiostrepton to exert selection pressure on plasmid-containing strains.

S. lividans TK24 harboring plasmid pSY15c containing the genes of nogalamycin chromophore and the genes for cyclase (snoaL) and ketoreductase (s / zoaF) was grown in E1 medium supplemented with thiostrepton. The compound 9-epi-auramycinone was formed and this structure is now called nogalamycinone. Any DNA fragment of the invention subcloned within the 17 kb region of nogalamycin biosynthesis can be inserted into a vector that replicates in Streptomyces, and the products can be produced by fermentation of plasmid-containing strains.

Brief Description of the Drawings

Figure 1 shows the structures of nogalamycin, daunomycin and aclasinomycin.

Figure 2 is a diagram of a gene cluster (Sno5) associated with nogalamycin biosynthesis.

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Figure 3 illustrates a proposed biosynthetic pathway for nogalamycin.

Figure 4 shows a diagram of plasmid pSY15c. The black genes snozL (aL) and snoaF (aF) are inserted into pSY15 to give pSY15c. aL means-

15 cycloase snoaL and aF represent C-7 ketoreductase snoaF. pSY15 (WO

96/10581) results in the formation of a tricyclic intermediate of nogalamycin biosynthesis in S. lividans. The abbreviations a1, a2 and a3 refer to the minimal PKS genes sno & l, snoa2 and snoa3, respectively. rA is the activator snorA gene, aB is the oxygenase snoaB gene, aC is the methylase snoaC gene, aD is the polyketide-ketoreductase snoaD gene, and aE is the aromatase • snoaE gene. gF (snogF gene) and gG (snogG gene) associated with the deoxyhexose pathway are not functional in this construct, aph is an aminoglycoside phosphotransferase gene and tsr is a thiostrepton resistance gene.

The following are examples which further illustrate the invention.

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EXPERIMENTAL PART

Materials Used 30 The restriction enzymes used were purchased from Promega (Madison, Wisconsin, USA) or

Boehringer Mannheim (Germany), and alkaline phosphatase from Boehringer Mannheim, and were used according to the manufacturers' instructions. Proteinase K was obtained from Promega * and lysozyme from Sigma (St. Louis, USA). Hybond ™ N-Nylon Membranes used for hybridization were purchased from Amersham (Buckinghamshire, England), DIG DNA Stamping Kit and DIG Luminescence Measurement Kit from Boehringer Mannheim.

5 Qiaquick Gel Extraction kits from Qiagen (Hilden, Germany) were used to isolate the DNA from the agarose.

Bacterial strains and their use

Escherichia coli XL1 Blue MRF' strain was used for cloning.

10 Streptomyces nogalater ATCC 27451; The gene cluster associated with nogalamycin biosynthesis was cloned from this strain.

The host strains used to express the cloned genes were:

Streptomyces lividans TK24, which was also used as the primary host for the cloning of the amplified DNA in E. coli.

15 Streptomyces galilaeus H039, which produces aclavinone-rhodinosis-rhodinosis-rhodinosis. Streptomyces galilaeus H026, which produces aclasinomycin N, ACMN (aclavinone-rhodosamine-2-deoxifucose-rhodinosis).

Streptomyces galilaeus H063, which produces aclavinone.

Streptomyces galilaeus H075, which produces aclavinone-rhodosamine-2-deoxifucose-2-20 deoxifucose.

c

A detailed description of mutants H039 and H026 is given in Ylihonko et al. (1994) and mutant H075 in FI patent application No. 981062 (Ylihonko et al., 1998). H063 has not been described in the literature, but was obtained by NTG-25 mutagenesis of S. galilaeus and was selected as the host strain for the production of hybrid compounds because it accumulates no sugar residues.

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Plasmids E. coli - Streptomyces shuttle cosmid pFD666 (ATCC 77286) was used to clone Kro-30 mosomal DNA. E. coli cloning vectors pSL1 190 (Pharmacia) and pUC19 were used to prepare subclones.

107053 11 pIJ486 is a multi-copy plasmid vector obtained by prof. Sir David Hopwood, John Innes Center, UK (Ward et al., 1986).

pIJE486 is a vector containing the ermE gene in the polylinker of pIJ486 (Bibb et al., 1985).

pSY15 is a pIJ486-based plasmid construct in which genes for the polyketide pathway in nogalamycin biosynthesis were cloned (Ylihonko et al., 1996a).

Media and solutions TSB medium was used to grow S. nogalateri for total DNA isolation. A solution of lysozyme (0.3 M sucrose, 25 mM Tris, pH 8 and 25 mM EDTA pH 8) was used to isolate total DNA. TE buffer (10 mM Tris, pH 8.0 and ImM EDTA) was used to dissolve the DNA.

15 TRYPTON SOYA SOYA (TSB)

Per liter: 30 g Oxoid tryptone soybean powder.

ISP4 Bacto ISP Medium 4, Difco; 37 g / l.

Per liter of tap water: Glucose 20 g Soluble starch 20 g 25 Pharmamedia 5 g Yeast extract 2.5 g Κ2ΗΡ04 · 3Η20 1.3 g -. · MgSO4 * 7H20 1 g

NaCl 3g 30 CaCO 3 3g was adjusted to pH 7.4 before autoclaving 10705312

General Methods NMR values were collected on a JEOL JNM-GX 400 spectrometer at ambient temperature. TMS was used as the internal reference for 1H and 13C NMR samples.

Anthracycline metabolites were detected by HPLC (LaChrom, Merck Hitachi, L-7100 pump, L-7400 detector and D-7500 integrator) using a LiChroCART RP-18 column (4.6 x 250 mm). Acetonitrile: potassium hydrogen phosphate buffer (60 mM, pH 3.0 adjusted with citric acid) was used as mobile phase. A gradient system from 65% to 30% potassium dihydrogen-10 phosphate buffer was used to separate the compounds. The flow rate was 1 ml / min and detection was performed at 430 nm.

ISP4 plates supplemented with thiostrepton (50 µg / g) were used to maintain plasmid containing cultures.

15 Example 1. Cloning of a gene cluster related to nogalamycin biosynthesis 1.1 Cosmid library

To isolate total DNA, Streptomyces nogalateria (ATCC 27451) was grown for 20 days in 50 ml of TSB supplemented with 0.5% glycine. Cells were harvested by centrifugation for 15 min at 3900 x g in 12 ml Falcon tubes and stored at -20 ° C. Cells obtained from a 12 ml sample were used to isolate the DNA. 5 ml of lysozyme solution containing 5 mg / ml lysozyme was added to the cells and incubated for 20 min at 37 ° C. To the cells were added 500 μl of 10% SDS containing 0.7 mg of proteinase K and incubated for 80 min at 62 ° C, others were added. 500 µl of 10% SDS containing 0.7 mg proteinase K and incubation was continued for 60 min. The sample was cooled on ice and 600 μΐ 3M NaAc, pH 5.8 was added and the mixture was extracted with balanced phenol (Sigma). The phases were separated by centrifugation at 1400 x g for 10 min. The DNA was precipitated from the aqueous phase with an equal volume of isopropanol 30 and collected on a glass rod, and washed by dipping in 70% ethanol, air-dried and dissolved in 500 µl TE buffer.

107053 13

The chromosomal DNA was partially digested with & ut3AI. The DNA fragments were separated by agarose gel electrophoresis, and the 30-50 kb fragments were cut from 0.3% low temperature gelatinized SeaPlaque® agarose. The DNA bands were isolated from the gel by heating to 65 ° C, extracting an equal volume of equilibrated phenol, and the phases were separated by centrifugation for 15 min at 2500 x g. The phenol phase was extracted with TE buffer, centrifuged and the aqueous phases combined. The DNA was precipitated by adding 0.1 volumes of NaAcc, pH 5.8 and 2 volumes of ethanol at -20 ° C for 30 min, centrifuged for 30 min at 15,000 rpm on a Sorvali RC5C centrifuge using an SS-34 rotor fitted with 10 ml tubes . The pellet was air dried and dissolved in 20 µl of TE buffer. The isolated fragments were ligated into the pFD666 cosmid vector which had been digested with BamHI and dephosphorylated. The DNA was packaged in phage particles and infected with E. coli using the Gigapack® III XL Packing Extract Kit according to the manufacturer's instructions.

15 1.2 Identification of Clones by Hybridization

Infected cells were grown on LB plates containing 50 µg / ml kanamycin and transferred to Hybond ™ -N-nylon membranes (Amersham). The membranes were processed according to the methods described in Boehringer Mannheim's "The DIG System User's Guide to Filter Hybridization". The probe used to screen colonies containing the expanded 20 gene clusters of nogalamycin was a 1.07 kb '5ad fragment of the cluster described previously (Torkkell et al., 1997).

The probe-containing plasmid was digested with Sac I, and the fragment was separated from the vector by agarose gel electrophoresis and isolated from the gel using the Qiaquick Gel Extraction Kit (Qiagen). The probe was labeled with digoxigenin using a short oligo-25 nucleotide primer labeling system according to Boehringer Mannheim's "The DIG System User's Guide to Filter Hybridization". 5000 colonies were screened by hybridization at 70 ° C using the probe described. Positive colonies were detected using the DIG Luminescent Detection Kit (Boehringer Mannheim). Seven colonies gave a positive signal. 30 cosmids from positive clones were isolated from the culture by 5 ml by alkaline lysis method. Restriction analysis showed that the cloned fragments overlapped corresponding to at least 60 kb of 107053 14 fragments were overlap corresponding to at least 60 kb of continuous DNA. The resulting positive clones were named pFDShol - pFD5> 10.

1.3 Subcloning of Fragments for Sequencing 5 Clone # 5, called pFDiSWo5, was cleaved with 2 µg / II, and for sub-cloning, two fragments, about 10 kb and 7 kb, were isolated and ligated into pSL1190, cleaved with Bgl-II. and dephosphorylated. The resulting plasmids were named pSn42 and pSn43, respectively. These two fragments cover a region of DNA immediately adjacent to the previously characterized region of the nogalamycin biosynthetic moiety.

Appropriate restriction sites were used to determine the entire 17 kb nucleotide sequence of the cloned plasmids pSn42 and pSn43 to subclone the fragments into vector pUC19 or pSL1190, yielding 16 subclones from pSn42 insert and 11 subclones from pSn43 insert.

15 E. coli XL1 Blue MRF 'cells were cultured overnight at 37 ° C in 5 ml LB medium supplemented with 50 µg / ml ampicillin. Promegan Wizard Plus Minipreps DNA purification kit or Biometran silica spin Plasmid miniprep kit (Biomedizinische Analytik Gmbh) was used to isolate plasmids for sequence reactions according to the manufacturers' instructions.

20 r DNA sequencing was performed using an ABI automated DNA sequencer (Perkin-Elmer) according to the manufacturer's instructions.

1.4 Sequence Analysis and Gene Inferred Functions Sequence analyzes were performed using the GCG Sequence Analysis Software Kit (Version 8; Genetics Computer Group, Madison, Wisconsin, USA). The translation table was converted to accept GTG as the start codon. Codon usage was analyzed using published data (Wright and Bibb 1992).

The DNA fragment sequenced according to the CODONPREFERENCE program contained 15 complete open reading frames (ORFs), and two other 10,107,053 5'-ends of the ORF of the invention at both ends. The functions of the genes were deduced by comparing the amino acid sequences translated from their base sequences with those of known proteins in the databases. The results are shown in Table 1. The positions given refer to the attached sequence list. The amino acid sequences of the peptides 5 are given in SEQ ID NO: 2 to SEQ ID NO: 18.

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Table 1 __ ^

Gene Position Amino Acids Inferred Function Note.

___ (SEQ ID NO) ___ snogl -1027> 342 (2) 5'-end of aminotransferase __ more ____ snogJ__1192-2073 293 (3) __ dTDP-glucose synthetase__ snogA 2106-2822 238 (4) aminomethyltransferase __ more 28 5 5 38 38 M z 5 38 38 M ) polyketide dicyclase __ more ____ s / zogN 3799-5025 408 (6) <7 «rQ homology (Otten et al., 1995), unknown__ snoaG 5088-6356 422 (7) __ hydroxylase__ snogC 6334-7209 291 (8) dTDPam-4-deh - __more ___ reductase__ snogK 7245-8297 350 (9) dTDP-glucose-4,6-dehydra __ more ___ again__ snoaL 8537-8941 134 (10) NAME-Cyclase (nogalone-h-acid methyl ester) __ snoK__8992-9699 235 (979-9699 235 on 10917 390 (12) glycosyltransferase __more ____ snoW__11057-11884 275 (13) __ unknown__ *; snogE__11928 - * __> 424 (14) __ glycosyltransferase__ Snob 13335-13754 139 (15) unknown __basic ____ snoO 13974-14441 155 (16) homologous to mtmX with the mitral ____ mycine clone__ snoaF 14532-15377 281 (17) C-7- ____etreductase__ STioN__15450 -__> 190 (18) __ unknown__5 'end *, about 100 bp nucleotide sequence, unknown 107053 17 1.5 Expression Cloning A 10 kb Bg / II fragment of pFD5no5 was cloned into plasmid pIJ486 and obtained pSY42. Similarly, the 7 kb 5g / II fragment of pFDSno5 was cloned into pIJE486 and plasmid pSY43 was obtained. Plasmid pSY42 was ligated into S.

5 .. lividans was cut by TK24 protoplast transformation, isolated from it and further linked to S. galilaeus mutzattiw H039 and, when amplified in H039, was transferred to other S. galilaeus mutants whose acacinomycins were characterized by a deoxyhexose , and H063). E1 medium was used for the production of anthracycline and the products were extracted from the culture with toluene: methanol (1: 1) at pH 7.

The anthracycline metabolites were analyzed by HPLC. The products of mutants H039, H026, H063 and H075 contained in pSY42 were different from those of the mutants lacking the plasmid.

By sequence analysis, pSY42 contained a cyclase, termed NAMEC 15 (nogalonic acid methyl ester cyclase), and a ketoreductase gene was identified from pSY43. Expression constructs containing all the genes needed to form the nogalamycin glycone were made. A 1.4 kb 5 amHI-5acI fragment from pSY42 (containing NAMEC) and a 1.1 kb MluI-Kpn1 fragment from pSY43 carrying the C-7 keto group ketoreductase gene were inserted into pSY15 linearized with SacE to form plasmid pSY15c. (Figure 4). Plasmid pSY15c was ligated to S. lividans TK24, and strain TK24 / pSY15c was cultured in E1 medium supplemented with thiostrepton. An aglycone compound was born, and this structure is now called nogalamysmon.

25 Example 2. Compounds produced by s «o5 group • · 2.1 Production and purification of products derived from H039 / pSY42 and TK24 / pSY15c

An inoculum culture of 180 ml of the E1 culture of strain H039 / pSY42 or TK24 / pSY15c containing the plasmid was obtained by culturing the strain in three 250 ml Erlenmeyer flasks containing 60 ml of E1 medium supplemented with thiostreptone (5 Mg / ml). for 30 days at 30 ° C, 330 rpm. The combined culture broths (180 mL) were used to inoculate 13 L of El medium in a fermenter (Biostat E). The fermentation was allowed to proceed for seven days at 28 ° C (330 rpm, aeration: 450 rpm).

Cells were harvested by centrifugation. 2.6 L methanol was used to disrupt the bacterial cells and extract the accumulated anthracycline metabolites. The anthracycline metabolites were extracted with 2 L dichloromethane at pH 6. The organic layer was evaporated to dryness. The viscous residue was flash chromatographed on a polyamide column (11) using water: methanol 1: 9-0: 10 as eluent. The combined fractions containing the compounds were further purified by Merck-Hitachi HPLC using a preparative reverse phase column (LichroCART RP-18, 5 μτα) using acetonitrile: 1% AcOH in water (1: 1) as mobile phase. Evaporation of the acetonitrile gave pure products as yellow vacuum dried powders.

2.2 Determination of Structure of Products Derived from H039 / pSY42 and TK24 / pSY15c NMR analysis was performed using NON, BMC, NOE, DEPT and HMBC techniques.

Protons were determined using NOESY and 2D pTOCSY techniques and carbons were determined using DEPT and HMBC techniques.

Determined from the data shown in Tables 2 and 3, the constructs obtained were aclavinone 4'-epi-2-deoxifucose from H039 / pSY42 culture and 9-epi-auramycinone (= nogalamycinone) from TK24 / pSY15c culture. The chemical structures of the compounds are shown below in formulas I and II, respectively.

25 O COOCH3 OH 0 0H 9 30 ^

We ^ TT ^ J

HO

OH

107055 19 O COOCH3 5 ΟΜΟ ^ λ m

OH O OH OH

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Deposited microorganisms

The following microorganisms were deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), 15 Mascheroder Weg lb, D-38124 Braunschweig, Germany, in accordance with the Budapest Treaty.

Microorganism Deposit Number Deposit Date S. lividans TK24 / pSY42 with plasmid pSY42 DSM 12451 October 14, 1998 «S. lividans TK24 / pSY43 with plasmid pSY43 DSM 12452 October 14, 1998 107053 20

Table 2. The 'H' and '13 C' Transitions of the Compound aklaYinom-4'-epi-2-Deoxifucose (Formula I)

~ Item * H 13C

1 7.74, 1H, dd, 7.5, 1.3 120.1 2 7.68, 1H, dd, 8.4,7.5 137.3 3 7.27, 1H, dd, 8.3 , 1,3,124.6 4 - 161.9 4-OH 11.70, 1H, s 4a - 115.4 5 - 192.3 5a - 114.4 6 - 162.4 6-OH 12.46, 1H , s 6a - 130.9 7 5.18, 1H, dd, 4.3.3.1 71.3 8A 2.51, 1H, dd, 15.0.4.3 33.9 8B 2.32, 1H, dd, 15.0, 3.1 9 - 72.1 9-OH 4.58, 1H, s 10 4.02, 1H, s 56.9 10a - 142.4 11 7.40, 1H, s 120.8 11a - 133.1 12 - 180.7 12a - 132.6 13A 1.73, 1H, dq, 14.2.7.4 32.0 13B 1.51, 1H, dq, 14.2, 7.4. - 14 1.10.3H, t, 7.4 6.7 15 - 171.1 16 3.69.3H, s 52.5 Γ 5.41, 1H, d, 3.5 101.7 2'a 1.75, 1H, ddd, 12.8, 11.2, 3.4 37.7 2'e 2.19, 1H, dd, 12.8, 5.3 3 '3.71, 1H, ddd, 12.0, 9.0, 5.3 69.0 4 '3.14, 1H, dd, 9.1, 9.0 78.1 5' 3.88, 1H, dq, 9.1.6, 2 68.8 6 '1.36, 3H, d, 6.2 17.6 107053 21

Table 3. Compounds of 9-epi-auramycinone Ή and 13C transitions (Formula Π).

Item * H 13C

1 7.76, 1H, dd, 7.5, 1.2 119.8 2 7.67, 1H, dd, 8.3, 7.5, 137.4 3 7.28, 1H, dd, 8.3 , 1.2 124.8 4 - 162.5 4-OH 11.86, 1H, s 4a - 115.6 5 - 192.7 5a - 114.6 6 - 160.9 6-OH 12.76, 1H , s 6a - 134.1 7 5.40, 1H, t, 7.0 64.0 8A 2.66, 1H, dd, 13.9,7.0 40.9 8B 1.89, 1H, dd, 13.9, 7.1 9 - 70.5 9-OH 3.49, 1H, brs 10. 3.93, 1H, d, 0.8 56.0 10a - 142.1 11 7.51, 1H, d , 0.8 120.1 11a - 133.3 12 - 180.9 12a - 132.1 13 1.44.3H, s 28.7 14 - 173.0 15 3.90, 3H, s 52.6 107053 22

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107053 24

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4 25 107053

Sequence Listing

(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Galilaeus Oy (B) STREET: Kairiskulmantie 10 (C) CITY: Piispanristi (E) COUNTRY: Finland (F) POSTAL NUMBER: FIN-20760 (ii) INVENTION OF THE INVENTION : Gene Grouping on Nogalamycin Bisynthesis and Its Use in the Production of Hybrid Antibiotics (iii) NUMBER OF SEQUENCES: 18 (iv) COMPUTER FORMAT: (A) TYPE: Diskette (B) COMPUTER: IBM PC Compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) PROGRAM: Patent # 1.0, Version # 1.25 (EPO) (2) SEQ ID NO: 1 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 16020 base pairs (B) TYPE: Nucleic Acid (C) SURFACE : single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: DNA (genomic) (vi) ORIGIN: (B) STRAIN: Streptomyces nogalater ATCC 27451 (ix) FEATURE: • (A) DESCRIPTION: CDS (= coding sequence) (B) ) LOCATION: Complementary (1..1027) (D) OTHER INFORMATION: / function = "aminotransferase" / gene = "snogl" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 1192..2073! (D) OTHER INFORMATION: / function = "dTDP-glucose synthetase" / gene = "snogJ" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: Complementary (2106..2822) * (D) OTHER INFORMATION: / Function »" aminomethyltransferase "/ gene» "snogA" • 107053 26 (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: Complementary (2826..3800) (D) OTHER INFORMATION: / function = "polyketide dicyclase" / gene = "snoaM" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 3799..5025 (D) OTHER INFORMATION: / function = "unknown" / gene = "snogN" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 5088..6356 (D) OTHER INFORMATION: / function = "hydroxylase" / gene = "snoaG" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: Complementary (6334.7209) (D) OTHER INFORMATION: / function = "dTDP-4-dehydro-rhamnose reductase" / gene = "snogC" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: complementary (7245..8297) (D) OTHER INFORMATION: / function = "dTDP-glucose-4,6-dehydratase" / gene = "snogK" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 8537..8941 (D) OTHER INFORMATION: / function = "NAME-cyclase" / gene = "snoaL" (ix) FEATURE:

'(A) DESIGNATION: CDS

(B) LOCATION: 8992..9699 (D) OTHER INFORMATION: / function® "unknown" / gene = "snoK" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: Complementary (9745..10917). (D) OTHER INFORMATION: / function = "your glycosyltransferase" / gene = "snogD" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 11057..11884 (D) OTHER INFORMATION: / function = "unknown" / gene = "snoW" 107053 27 (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 11928..13200 (D) OTHER INFORMATION: / function = "your glycosyltransferase" / gene = "snogE" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: Complementary (13335..13754) (D) OTHER INFORMATION: / function = "unknown" / gene = "snoL" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 13974..14441 (D) OTHER INFORMATION: / function »" homologous to the jntmX of the mitramycin moiety "/ gene =" snoO "(ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 14532..15377 (D) OTHER INFORMATION: / function = "C-7 ketoreductase" / gene = "snoaF" (ix) FEATURE:

(A) DESIGNATION: CDS

(B) LOCATION: 15450..16020 (D) OTHER INFORMATION: / function »" unknown "/ gene» "snoN" (ix) FEATURE: (A) NAME: miscellaneous feature (B) LOCATION: 3799..3800 (D) ) OTHER INFORMATION: / Note » "the overlapping sequence of the snoaM and snogN genes". (ix) FEATURE: (A) DESCRIPTION: Miscellaneous Feature (B) LOCATION: 6334..6356 (D) OTHER INFORMATION: /note.= "Overlapping sequence of genes snoaG and snogC" (ix) FEATURE: (A) NAME: mixed feature. (B) LOCATION: 13201..13300 * (D) OTHER INFORMATION: / Note » 'Unknown region "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGATCTCGTC CGCCAGTGCC TCGGTGACCG GCAACGAGCC CTTGGCGTAG CCGAGATGGG 60 AGAAACCGGT CATGGTGTGC ACGGGCCAGG GATAACTGAT GTTGAGGGCG ATGTCGTAGG 120 • - 28 107 053 AGGCGCGCAG GGCCTCCAGC ACCGCGTCCC GTCGCGGATG GCGCACCACG TACACGTAGT 180 AGACGTGCTC GTTGCCCTGC GCGGTCCTCG GCAGCAGCAG CCCCGTGTCC GCCAGGCCCT 240 CCTCATAGCG GCGTGCCACC GCCCGGCGGG CCTCGATGTA GGACGGCAAC CGGGACAGCT 300 TGCGCCGCAG GATCTCTGCC TGTACTTCGT CCAGCCGGCT GTTGTGCCCG GGGGTTTCGA 360 CGACGTAGTA GCGGCTCTCC ATGCCGTAGT AGCGCAGCCG CCGCAGCCGG TCCGCCACCC 420 GCTCGTCGTC GGTGAGCACC GCGCCGCCGT CCCCGTACGC GCCCAGCACC TTGGTCGGGT 480 AGAAGGAGAA CGCGGCCGCG TCACCGGTCG AGCCGGCGAG TCGGCCGTGC CGGCGCGCCC 540 CGTGCGCCTG CGCGCAGTCC TCCAGGATCA CCAGGTTGTG CCGGGCGGCC AGATCGCGCA 600 GCGGTGCCAT GTCCACGCAC TGCCCGTAGA GGTGGACCGG CAGCAGACAC CGGGTGCGTG 660 GCGTGAGGAC GGCCTCCACC TGGGACGTGT CCATCAGGTA GTCCTCCTCG CGCACGTCCA 720 CGAAGACGGG CGTGGCACCG GCCGAGTCGA TCGCGACGAC CGTGGGCGCG GCGGTGTTGG 780 ACACGGTGAC GACCTCGT CG CCGGGCCCGA CACCCAAGGC CTGTAACCCC AGCTTGACGG 840 CGTTGGTCCC GTTGTCGACG CCGACGGCAT GTCCGACGCC CTGGAATGAG GCGAACTCGG 900 ACTCGAAGCC GCGCACGCTC TCACCGAGGA CGAGCCGGCC GGAGCGGAAC ACCGTCTCCA 960 CGGCATCGTG GATGTCCTCG CGTTCCAGCT CGTATTCCGG CAGATAGTCC CACACGTGTA 1020 CGGTCATCGA GCCCCTCCGG GATTCTCCCT GCGAAAAGTC ACCACTCTAC GACAACGTTC 1080 ACCACTCGCT TTTTCCTCAA CGTCCGCTTG AGACGGCCCG GCCTGCTGTG GCCCGGGGAA 1140 AGGTGCGGTC GTTATCATCG ACTCCGTCTT CTCATTCGGA GGTTGTTCAG GGTGAAGGGA 1200 ATCATTCTCG CCGGGGGTAC GGGGAGCAGG CTCCACCCGA CGACTCTCGC GGTGTCCAAG 1260 · CAGCTTCTCC CCGTCGGGGA CAAGCCGATG ATCTACTACC CGCTCTCCGT GCTGATGCTG 1320 GCCGGCGTCA CGGACATCCT CATCATCAGC ACACCGCACG AACTCCCCCG AATGCGCCGT 1380 CTGTTCGGCG ACGGCGCACA GCTCGGACTC CGCCTGGCCT ACGCCGAGCA GGAGAAACCC 1440 AGGGGTATCG CCGAGGCGTT CCTGATCGGT GCCGACCACG TGGGAAGCGA TGCCGTTGCG 1500 !! CTGGCGCTGG GCGACAACAT ATTCCACGGG AGTTCTTTTC AGGGGGTGCT GCGCAAGGAA 1560 GCCGAGGAAT TGGACGGGTG TGTCCTGTTC GGTTATCCGG TCAAGGATCC CCAGCGTTAT 1620 GGAGTCGGCG AGGCGAACGC GTCCGGGCGG CTCGTCTCCA TCGAGGAGAA ACCGGTACGC 1680 CCCCGCTCCA ACCGGGCCAT CACCGGACTC TATTTCTACG ACAACGAGGT GGTGGACATC 1740 GCCCGGCGGC TGCGCCCCTC CGCCCGCGGC GAACTCGAAA TCACCGACAT CAACCGTACC 1800 • <107053 29 TACATGGAAC GAGGCCGGGC CCGGCTCGTG GACCTGGGCC GGGGATTCGC CTGGCTCGAC 1860 ACCGGCACAC CCGAGTCACT CCTGCAGGCC TCGCAGTACG TGTCCGCCCT GGAGGAACGC 1920 CAGGGCATCA GGATCGCCTG CATCGAGGAG GTGGCCCTCC GCATGGGCTT CATCAACGCC 1980 CAGGCCTGCT ACGAACTGGG CGCGCGCCTG TCCGGCTCCG GCTACGGGCA GTACGTGATG 2040 GCCATCGCGG AGGAGTGCAC GGGGCGGGTG TGAGCGGCCG TGCCGGGTGG GCGAACGGCC 2100 CGGCCTTACC CGGCCCCGCG CACCCCGACG AACAACCCCC GGCCGGTCAG CCCGTCGTCC 2160 AGGAACTCGG CCGGGCAGCC CGCGTCCTCG AACGCGGCGA GGTACTCCTC CCTGGTGAAC 2220 AGGGTGAGCA GGTCGATCTC CGTGAACTCG CGTATCCCGG TGGCCTCGCC GACCAGGAAC 2280 CGCACCTCCA TGCGGGTCCT GCGGCCCTGC CTGGTGGAGT GGGACACCCG GGCCAC GGTC 2340 CGGCCCTCAC CGCGTGCCAG GTCCCCGGCG ACGTAGCCCT CCAGGAACCG CTCGGGGAAC 2400 CACCAGGGCT CCACCACGAG CACGCCGCCC GGCACCAGGT GCGCGGCCAT CGTGCGCACC 2460 GCCGCCCGCA TGTCCGCGAC GGTCTCCAGA TACCCGATGG AGCAGAACAG GCAGACCACG 2520 GCGTCGAAAC GCCCGCTCAG GGCGAAGTCG CGCATGTCCC CGGGCCGCAC CGGCACCCCC 2580 GGCAGCCGCC GTTCGGCCAG GGCCCGCATC TCGTCCGACA GCTCCAGGCC CTCCGTGTGC 2640 GCGAACAGCC CGCGGAAGGC CTCCAGATGG GCGCCGGTGC CGCAGGCGAC GTCGAGCAGC 2700 GAACGCGCCC CGGGCCGACG GGACCTGATC TCCGCGGTGA CCCGTTCGGC CTCGTCCGCC 2760 CAGCTCTTTC CCCGGCTGCG GTAGACCATC TCGTACACGT CCGCCAGTTC CCGGCCGTAC 2820 ACGCGTCAGT CCTCGTCCAC CAGGGCGACC GCCCGGGTCC ACCCGGCGCC GGCGCCGGCG 2880 ACCTTGACCG GGAAGCAGCA GACGCGGAAC CCGAAGGAGA CCGGCAGGCG GTCGAGGTTC 2940 <GCCAGCCGCT CGATCTGGCA GTACTCCCGC TCCCGGCCCA CCACGTGCGC GGGCCACAGC 3000 ACCGATCGGT CGCCGGTCGC GCGGTACCGG TCGATGATGT GGCCGAAGGG CGCGTCCAGG 3060 CTGAAGGCAT CGGTCCCGAT CACCCGGACC CCGTGGTCGA GAAGCATCCG TACCGCGGGC 3120 CCGTCGAGAC CGGCGAAGTC CGTGAAGTAG CGCGGGGTGC CCGCGTGCCG CTGGGCACCG 3180 *! GTGTGCAGCA GCACGATGTC CCCGGGCCGC AACGCGCACC CGGTCCGGGC CAGTTCCTTC 3240 TCCAGGCGCG CGGCGCTCAC GGTGCCCGTC GGAGCGTCGG TGAGGTCCAG CACCACCCCG 3300 CGCCCGAAGA ACCACTCCAG CGGCATCTGG TCGATGTGGC GGGGGACGCC GTCCCCGTAC 3360 AGCGCGCGCG AACCATAGTG CGACGGCGCG TCGACGTGCG TGCCGGTGTG CGTGGTCAGC 3420. . GTGATCCTGT CCAGTGACAG GAACTCGCCG TCCGGCAGTT CGTCCGGAGA GAACTCGACA 3480 30 107053 CCGAAGTGCT CGCGCATCTC CGCGCACATG TGTTCCGCGC CCTGCCGGGG CGTGAGGACG 3540 TCGTGCACCA CCGGGTCGGG CTCGTACTGT GAGGAATCCA CCGGTGACGA AAGGTCGATG 3600 AGCCGCACGC GCACCTCCGG GTTCGTAGAC GGGCTCGGCT GACGCAGCGC GGGTACGACG 3660 CTGACACGCC CCTCTTGACG TGGCCTGGAA GCTGGTTCGA CGGGCGGGCA CCGCACGCGA 3720 CGGCCGGCGC CGCACCGGCG CCGTCCCGGC CGAGCGGGAA TCCAGGGAGG GTATAGCGGC 3780 GCGCCCCACG CTGCCGTCAT GGTGATGAAA CTGACGGACA GCGAGCTGGG GCGTGCGCTG 3840 CTCTCGCTGC GTGGTTACCA GTGGCTCCGC GGCATCCACC ACGATCCCTA CGCCCTGCTG 3900 CTGCGCGCCG AGAGCGACGA TCCGGCGCAG CTCGGCCGGC TGCTGCGTGA ACGCGGCCGG 3960 CTCCACCGCA GCGACACCGG CACCTGGGTC ACCGCGGACC ATGCGACGGC CTCCCGGCTG 4020 CTCGCCGACC CGCGCTTCGT GCTGCGCCGC CCGCCGGCCG GGCCCGCCAC CGGCACCGGG 4080 GACGTCATGC CGTGGGAAGA GGCCACGCTG AGCGACCTGC TGCCCCTCGA CGAGGCGCGC 4140 CTGACGACCG ACCGGGCACG GTGCCGCCGG CTCGGCGCGA CCGCCGCGCG GATCGCGGCG 4200 GACGGTCCCG TCGCGACGCG ACTCGCGGAC CTGGCCGGGG CCCGAGCCGA ACAGGTGCGC 4 260 TCAACGGGCC ACTTCGACCT CAGGGCCGAC TACGCCCTCC CGTACGCGGT CGAGCCGGCC 4320 TGCGCGCTGC TCGGCCTGCC GGCCGGGCAG TGTTCCCTCT TCGGCGCCTT 'CTCCCCGGCC 4380 GTCCTGCTCG ACGCGACGGT CGTACCGCCC CGCCTTCCGG AGGCGCGCGC CCTGATCGCC 4440 TCCACGGCGG AACTGACCGC CCTCTGGCCG CGGCTGGCCC CGAGCCTGTC GAAGACCGTC 4500 CCGGAGGACG AAGCGCCGGA CCTCTTCCTG CTGACGGCCG TGTTACTCGT ACCGGCCGTC 4560 GTCCACCTGG TCTGCGAGGC GGTCGCCGCC CTGTCGCACG ACCCCGGGCA GGCCGGGCTG 4620 CTCAGGGACG ACCCGGTACT CGCCGCACCG GCGGTCGAGG AGACGCTGCG CCACGCACCG 4680 CCCGCCCGTC TGTTCACCCT CCACGCGACC GGACCGGAGC GCGTCGCGGA CGTCGACCTC 4740 CCCGCGGGCG CCGAGGTCGC CGTCGTCGTG GCGGCGGGGCCGGGCGCGCGCGCGCGCGCGC CTGCCGCTGG GGGCGCTCGC CCCGCTGCTG CGCGTCTGCG CGACCGCGGC CGTCGCGGCC 4920 CTCGCGGCCG GACTCCTCCC GCTGCGGGCC GTCGGCCCGC CCGTACGACG GCTGCGTGCC 4980 CCGGTCACCC GGTCCGTGCT GCGCTTCCCC GTCGCCCCGT GCTGAGCAGC CCCTCCTCAC 5040 GTCATCCCCG GCCCGCCTTC CCCCGCCCGC AACGGAAGGG ACTCTCCATG GACAACCGCG 5100 AGACCGTACG ACCGGTGAGC GTCTGCCGGG TCTGCGGCGG CAACGACTGG CAGGACGTCG 5160 «107 053 31 TGGACTTCGG TGACGTTCCC CTCGCCAACG GCTTCCTGTC CCCGGCCGAC TCCTACGAGA 5220 ACGAGCGCCG CTACCCGCTG GGCGTCCTGT CCTGCCGCGC CTGCCGGCTG ATGAGCCTGA 5280 CCCACGTGGT CGACCCCGAG GTGCTGTACC GCGACTACGC CTACACCACC CCCGACTCCG 5340 AAATGATCAC CCAGCACATG CGGCACATCA CCGCGCTGTG CCGCACCCGT TTCGAGCTTC 5400 CCCCGGACÄG CCTCGTCGTG GAGCTGGGCA GCAATACCGG CCGTCAGCTC ATGGCCTTCC 5460 GCGAAGCGGG GATGCGCACC CTGGGCGTGG ACCCCGCGCG CAACCTCACG GACGTCGCCC 5520 GGCGCAACGG CATCGAGACC TTCCCCGACT TCTTCTCCCA CGACGTGGCC CGCACCATCC 5580 GGCGCGACCA CGGGCAGGCG CGGCTCGTGC TGGGACGGCA TGTCTTCGCC CACATCGACG 5640 ACGTGTCGGA CATCGCGGCC GGCGTACGCG AACTCCTGTC TCCCGACGGG GTGTTCGC GA 5700 TCGAGGTGCC GTACGTTCTG GACCTGCTGG AGAAGGTCGC GTTCGACACC ATCTACCACG 5760 AGCACTTGTC GTACTTCACC ATGCGGTCCT TCGTCACCCT CTTCGCGCGC CACGGGCTGC 5820 GGGTGCTCGA CGTGGAGCGG TTCGGCGTGC ACGGCGGATC GGTCCTCGTC TTCGTGGGCC 5880 ACGAGGACGG CCCCTGGCCC GAACGTCCCT CCGTCCCCGA ACTGCTGCGC GTGGAACGGC 5940 AGCGGGGCCT CTACGACGAC GCCACCTACC GCACGTTCGC GCAGCGGATC GAGCGGGTGC 6000 GCACCGAACT GCCGGAACTG CTGCGCTCCC TCGTGGCCCA GGGCAAGCGC ATCGTCGGCT 6060 ACGGTGCTCC GGCCAAGGGC AACACCATCC TCACGGTGTG CGGGCTCGGC CTGAAGGAGC 6120 TGGAATACTG CACCGACACC ACCGAGCTGA AGCAGGGCAG GGTGCTGCCC GGCACCCACA 6180 TACCGGTGCA CGCTCCCGAG CACGCCAAGG AACACATCCC CGACTACTAC CTGTTGCTCG 6240 CCTGGAACTA CGCCACGGAG ATCCTCGACA AGGAGACGGC CTTCCGGGAC AACGGCGGCC 6300 GGTTGATCGT GCCCATCCCC CGCCCGTCGA TCCTCACGTC CCCGTCAGGT TCCTGAGGCG 6360 CCCGCCGGGC AGCAGCTGAC GCATCGCCTC GCGCAGGGCT GCACGCCAGT CGCGGGGCGG 6420 TGCGACGCCG ACCAGCCGCC AGCGGTCGTG CCCGAGCACC GTGCACGCCG GCCGGGGCGC 6480 CGGGCCCGGC CGGTCGGCCG TCGCCACCGG GCGCACCCGT TCCGGGTCCG CGCCCGCCAG 654 0! CCGGAACACC TCCCGGGCCA GCTCGTACCA GGTGGCCGCC CCGGCGTTGG TGGCGTGGAA 6600 GATCCCGCGC GCCCGGTCTG GCGGCGTGCG GGCCAGCGTC ACCAGCAGCC GGGCCACGTC 6660 ACCGGCCCAC GTCGGCTGCC CCCACTGGTC GTTGACGACG TCGACATGGC CGTCGTCCGG 6720 GGCACGCTCC AGCATCGTGC GCACGAAGCT GCGGCCCTGC CCGCCGTAGA GCCACGCCGT 6780; GCGCACCACG GTGCCCGTAT CCGGCAGCAG CGACAGCACG GCCCGTTCCC CGGCCAGTTT 6840 107053 32 GCTGCGGCCG TACACCGTGC GCGGGCCCGG AGCGTCCGAC TCGCCGTAAG GGCTGCGGGT 6900 GTCGCCCGGG AAGACGTAGT CGGTCGAGAC GTGGATCAGC CGTACGCCGT GGCGCGCACA 6960 GCGGCGGGCC AGCAGCCGGG GCCCGCCGCC GTTGACGCGC ATCGCCTCCG CCCACCGCGA 7020 CTCGGCGCCG TCCACGTCCG TGAAGGCGGC GCAGTTGACC ACCACCCGCG GCCGGTGCGC 7080 GGCGAACGCG GCGTCCACCG CCCGCCCGTC GGTGATGTCC AGCGCGCGCC GCCCGAGTAC 7140 CACCGCCTCG GCGGCGGGCC GGCTCCTGCC GGTCTCCGCC AGGGCCGCGG TCAGGTGCCG 7200 GGCGAGCATG CCTTCTCCTC CGGTGACCAG CACGCGCATC CCGCTCACCG GACCCCGGGG 7260 ACGACGGTGG ACGTACCGCC CGGCGCCGTG ACTCCCCGCT TGAGCGGCTC CCACCAGGAC 7320 CGGTTCTCGC GGTACCACTG GACCGTCGAG CGCAGCCCCG AGGAGAACTC CCGCGCCGGA 7380 CGGTAGCCCA GTTCCTCACG GGCCCTGCCC CAGTCCAGGC TGTAACGCAG GTCGTGCCCC 7440 TTGCGGTCGG GCACGTGCCG GACGCTGCTC CAGTCCGCCC CGCACAGCTC CAGCAACATA 7500 CCCACCAGCT CCCGGTTGGA GAGCTCCCGG CCGCCGCCGA TGTGGTACAC ACCGCCGGGC 7560 CGGCCCGCGG TGCGCACCAG GTCCACGCCC CGGCAGTGGT CCTCCACGTG CAGCCACTCC 7 620 CGCACGTTCC GCCCGTCCCC GTACAGCGGC ACCGGCAGCC CGTCCAACAA GTTGGTGACG 7680 AAGCGCGGGA TGAGCTTCTC CGGGTGCTGA CGCGGGCCGT AGTTGTTGGA ACAGCGGGTC 7740 ACCCGCACGT CCAGGCCGTG CGTGCGGTGG CAGGCGAACG CCATCAGGTC GGCCGACGCC 7800 TTGGAGGCGG CGTACGGGGA GTTGGGGCTC AGCGGGTGCT CCTCCGGCCA GGAACCGGAC 7860 GCGATGGAGC CGTAGACCTC GTCCGTGGAC ACCAGGACGA AGGGCTCCAC GCCGTGGCGC 7920. . AGCGCGGCGT CCAGCAGCCG CTGGGTGCCG ACGACGTTGG TCAGCACGAA GTCGTCGGCC 7980 •> <GCGCGGATGG ACCGGTCGAC GTGCGACTCC GCGGCGAAGT GGACGACCTG GTCGCTGTGT 8040 GCCATCAGCT CGTCGACCAG CTCGGCGTCG AGGATGTCGC CCCGCACGAA GCGCAGCCGG 8100 TCACCGCGTA CCGCGTCCAG GTTCGTGAGG TTGCCCGCGT ACGTCAGTTT GTCGAGGACG 8160 GTGACGCGTA CCGCCGGGGC CCCCGCTCCG GGGGCCCGGT TCTCCAGCAG CATGCGCACA 8220 1 TAGGCCGAGC CGATGAAACC GACCGCGCCG GTGACCAGGA TGTTCACGTC CGTCGTCGCG 8280 GAGGTGTGCG ACGCCATGGG TTCCCTCCAT CCGTCGGGTG CCGTGGGGCG GAGTGCGCCC 8340 CCTCGACCCA GCGTCGGGGG CGGCCGTGGA GGAGCGGTTG AGCTTCGGCG CAGCGGCGGC 8400 TCGACCGGCG GCGGCCGGCG TCGCCGGACT CCAACGGGA CCTGGCGAGA CTGCCCGGAC AGCCCGGCCG AGAGAGGGAG GACCCGTTGA GCCGTCAGAC 8520 107053 33 AGAGATCGTC CGCCGGATGG TGAGCGCCTT CAACACCGGC AGGACCGACG ACGTGGACGA 8580 GTACATCCAC CCCGACTACC TCAATCCGGC CACCTTGGAA CACGGCATCC ACACCGGGCC 8640 CAAGGCGTTC GCCCAGCTGG TCGGCTGGGT GCGGGCGACG TTCTCCGAGG AAGCCCGCCT 8700 GGAGGAGGTG CGGATCGAGG AGCGCGGCCC GTGGGTCAAG GCCTACCTCG TGCTCTACGG 8760 CCGCGACGTC GGCCGGCTTG TCGGTATGCC GCCCACCGAC CGGCGCTTCT CCGGTGAACA 8820 GGTGCACCTG ATGCGCATCG TCGACGGGAA GATCCGCGAC CACCGGGACT GGCCCGACTT 8880 CCAGGGGACG CTGCGCCAGC TCGGCGACCC GTGGCCCGAC GACGAGGGCT GGCGTCCGTG 8940 ACCGTCCCTG AAACCGCACC CGACGAGACA TCAGACCAGG AAGGATGGCT CATGCCGGAT 9000 CCCGGCGGCC CGACCACGGC CGAGAACCTG TCGAAGGAGG CTGTCCGCTT CTACCGCGAG 9060 CAGGGTTACG TGCACATCCC GCGCGTCCTG TCGGAGACGG AGGTGACCGC CTTCCGGGCC 9120 GCCTGTGAGG AGGTCCTGGA GAAGGAGGGC CGCGAGATCT CCGGCATCGC CCTGCGGCTG 9180 GCCGGCGCGC CCCTGCGGGT CTACAGCAGC GACATCCTGG TCAAGGAGCC CAAGCGCACC 9240 CTGCCCACCC TGGTCCACGA CGACGAGACG GGACTGCCGC TGAACGAGCT GAGTGCCACG 9 300 CTGACGGCCT GGATCGCGCT GACGGACGTA CCCGTCGAAC GCGGCTGCAT GAGCTACGTG 9360 CCGGGCTCCC ATCTCAGGGC CCGCGAGGAC CGGCAGGAGC ACATGACCAG CTTCGCCGAG 9420 TTCCGGGACC TCGCGGACGT GTGGCCCGAT TACCCGTGGC AGCCGCGCGT CGCCGTGCCC 9480 GTCCGCGCCG GAGACGTCGT GTTCCACCAT TGCCGTACCG TCCACATGGC CGAAGCCAAC 9540 ACCAGCGACT CGGTCCGCAT GGCGCATGGC GTCGTCTACA TGGACGCGGA CGCCACCTAC 9600 CGGCCGGGCG TCCAGGACGG CCACCTGTCC CGCCTGTCGC CGGGAGATCC ACTCGAAGGC 9660 GAGCTGTTCC CCCTGGTCAC GGCAGGCACA CGGCAGTGAG GTCCGCCGTT CCCGGCGGTC 9720 GCGGGACCGC CGGGGACGGC ACCGTCAGCC GGCCAGCGCC ACGAGCTTGG CGGCCGTCTC 9780 GGCCGGCGGC GGCATCTCGC TCATCTCCTG CCGCACCCGC AGGGCCGCCT CCCGCAACCC 9840 CGCGTCGTCC AGCAGCCGTC GGCACTGCTC GGCACCCAGC GATCCCGCCT CGGCATCGAA 9900 II CCCGATGCCC AGCCCGGTCA GCACATCGCG GTTGGTGTCC TGGTAGGAGC CGTGCGGGAT 9960 GACGCACTGC GGGACGCCGG CGGCCAGGGC CGTCAGCAGT GTGCCGCTGC CCCCGTGATG 10020 GATGATCGCG TCGCACGTCT CCAGCAGCGC GCCCAGCGGA ATCCACTCCA CCACCGGTAC 10080 GTTCGCGGGC AGTTCACCGA GCAGGGCCAG GTCGCCGCCG CCCAGGGTCA GCACGAACTC 10 140,. CGCGTCCACG TCCGCCACTT CGGAGAACAG CGGGGCCAGC TTGGCGATGC CGCCCGACAG 10200 • · 107053 34 CGCGTCGATG GAGCCCAGCG TCACCGCGAT ACGCCGCCGG CCGGCCGCGG GCGGCAGCCA 10260 GTCCGGCAGC ACCGCTCCGC CGTTGTAGGG GACGTACCGC ATCGGCCÄGG CACCCGGGGA 10320 GCGCCGGTCC TCCGGCAGCA GCGCCTCCAC GCTCGGCGGT GTCGTCGTCA GCCGCACGGA 10380 ACCGGTCGGC TCGCCGGTGA CGCCGTGGCG CTCGTAGTCC TTGGACATCG CCCGCCGGAT 10440 GAGCGCGCCG AGCCCCGGCT CGCTGTCCGC GGGACCCAGC GGCAGCTCTA CGCACGGCAG 10500 TTGCAGCGCT GCCGCCGTCA GCGGGCCCGC GCCCTGTGTC GGAGTGTGCA CGACGAGGTC 10560 GGGCCGCCAG CTCCGCGCCG TCCGCAGCGC CCCGTCGACG GCCACCGCCG ATACCCGGGC 10620 GAACATCTCG GCGAAGAAGC CCTCGCCCAG CCCCTCGGAG TGCATCGGGT CGGTGACGTC 10680 GGTGTCGTCG GGCACGAACA GCTTCGCGTA GTTCACGCCG GGCGACACGT CCACGGCGCA 10740 GAGCCCGGCC TCCGCGACGG CGCGGATGTC GCCCCCCGTG GCGTAGCGGA CCTCGTGGCC 10800 GAGAGCGCGC AGCGCCTGTG CCAGCGGCAC CGTCGGCAGG ATGTGGCTGA GCCCGGGTGA 10860 AGTGATGAAC AACGCACGCA TGATGCCCCC TGTTCGACAT GAACCTGGAA CACGCATCCT 10920 GACGGCGCCT TCTGTTGCTC CGGTCGACGC CCGGTCGACA GG CCCTCGTA CAGCCCGCCG 10980 GGGGCCGGTC CGGCCACGAC GCAGGCTCCA GCGGACGTCG ACGGCGGGGA CGCAGCGTGG 11040 TCGCCGGGAG GCATCGATGA CAGTATTGGT AACCGGAGCC ACAGGAAACG TCGGCCGGCA 11100 CGTCGTCACC GGGCTACTGG CCGCCGGCCG CCGGGTGCGG GCGCTGACCC GCACACCCGA 11160 CCGGTCCGGC CTGCCCGGCG GCGCGGAGAT CACAGGCGGC GACCTGACCC GCCCGGAGAC 11220 CTACGAGCGG ATGCTGGACG GTGTCGAAGC CGTCTACCTG TTCCCCGTCC CGGAGACCGC 11280 CGCGGCGTTC GCCGGGGCCG CGCGACGGGC CGGTGTCCGG CGGATCGTGG TGCTCTCCTC 11340 GGACTCCGTC ACCGACGGCA CCGACACCGG AGGACACCGG CGCGTGGAAC TGGCCGTGGA 11400 GGACACGGGG CTCGAGTGGA CCCATGTGCG CCCCGGCGAG TTCGCGCTCA ACAAGGTCAC 11460 CCTGTGGGCG CCGTCGATCC GCGCGGAGGG CGTCGTCCGG TCCGCGTATC CGGACGCCCG 11520 GGTGGCCCCG GTGCACGAGG CCGACGTCGC GGCCGTCGCG GTGACCGCGC TGCTGAAGGA 11580 !! GGGGCACGCC GGCCGCGCCT ACAGCGTGAC CGGACCGCAG GCCCTCACCC AGCGCGAACA 11640 GGTCCGCGCG GTAGGGGAGG GGCTCGGCCG GTCCCTCGCC TTCGTCGAGG TGACCCCCGG 11700 GCAGGCGCGG GCCGACCTGA CCGCCCAGGG GCTGCCCGCG CCCATCGCCG ACTACGTCCT 11760 CGCCTTCCAA GCCGGGTGGA CCGAGCGGCC CGCCCCCGCC CGGCCGACCG TGCGGGAGGT 11820 CACCGGCCGG CCCGCCCGCA CGCTCGCCCA GTGGGCCGCC GACCACCGAG CGGACTTCCG 11880 35 107 053 GTGACCGGAG ACCGCGTCCA CCGCGCCACG ACAGAAAGGC GACGCCCGTG CGCGTACTGC 11940 TGACGTCCTT CGCCATGGAC GCCCACTTCT GCACCGCCGT GCCGCTGGCG TGGGCACTGC 12000 GGTCGGCCGG GCACGAGGTA CGGGTGGCCG GCCAGCCCGC GCTCACCTCC ACCATCACGG 12060 GAGCCGGCCT GACCGCCGTG CCGGTCGGCC GCGACCACAC GCACGGCAGC CTCCTGGGCC 12120 GGGTCGGCAG CGACATCCTC GCCCTGCACG ACGAGGCGGA CTACCTGGAG GCCCGTCACG 12180 ACGCCCTGGG CTTCGAGTTC CTCAAAGGGC ACAACACGGT GATGTCCGCG TTGTTCTACT 12240 CGCAGATCAA CAACGACTCG ATGGTCGACG ACCTGGTGGA CTTCGCCCGT CACTGGCGGC 12300 CCGACCTGGT CGTCTGGGAG CCGTTCACCT TCGCGGGCGC CGTGGCCGCG CGGGCCTCGG 12360 GCGCCGCCCA CGCCCGCCTG CTGTCCTTCC CCGACCTGTT CCTCAGCACG CGCCGCCTCT 12420 TCCTGGAGCG CATGGCGCGC CAGGAGCCCG AGCATCACGA CGACACACTC GCCGAATGGC 12480 TCGACTGGAC CCTTGGCCGG CACGGCCACT CCTTCGACGA GGAGATCGTC ACGGGGCAGT 12540 GGTCCATCGA CCAGACCCCC GCCCCCGTGC GGCTCGACGC CGGCGGTCCC ACCGTGCCGA 12600 TGCGGTACGT CCCCTACAGC GGACTGGTGC CCACAGTGGT GCCCGACTGG CTGCGCAGGC 12660 CGCCCGAGCG GCCACGGGTC CTGGTCACCC TCGGCATCAC CTCACGGCGG GTGAAGTCCT 12720 TCCTCGCCGT CTCCGTGGAC GACCTTTTCG AGGCCGTGGC CGGGCTCGGC GTCGAGGTGG 12780 TCGCCACCCT CGACGCCGAC CAGCGGGAGC TGCTGGGGCG CGTGCCGGAC CACTTCCGCA 12840 TCGTCGAGCA CGTGCCGCTG GACGCCGTTC TGCCGACCTG CTCGGCGATC GTCCACCACG 12900 GCGGAGCCGG CACCTGGTCG ACGGCCGCCG TGTACGGGGT GCCGCAGGTC TCCCTGGGCT 12960 CGATGTGGGA CCACTTCTAC CGGGCCCGTC GCCTGGAGGA ACTCGGGGCG GGGCTGCGGC 13020 • '<TGCCCTCCGG CGAGCTGACT GCCGAGGGGC TGCGCACCCG GCTGGAGAGG GTGCTCGGCG 13080 AGCCCTCCTT CGGCACCGCC GCGCAGGCGC TGAGCGACAC CATCGCGGCG GAACCCAGCC 13140 CCAGCGAGGT CGTGCCGGTC CTGGAGGAGC TGACCGGACG GCACCGTCCC GGCACCCGGG 13200 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNN NNNNN NNNNNNNNNN NNNNNNNNNN 13260 "NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN CCGTCCGGGC CCCTCGCCGG 13320 TGAGGGAGCC CGGATCACAG TCCGTCCGGC ACCACGCCCA GGTCCCGGAA CAGCGGGGAG 13380 AAGTTGAAGA CGTCCCAGTG CTCCACGACC TTGCCGGCTT CGGAGAAGCG CAGCTCCTCC 13440 AAGTAGGTCC AGCGGACCTT GCGGCCGGTG GGGGCGATGC CCATGAACAC GCCCTGGTGC 13500 GTGGCCGAGC AGGTGATCCG CAGCATCACG CGGTCGCCCT CGCCCACGAT GCTCCGCACG 13560 • · 107053 36 TCCAGACGAA GGTCCGGGAA GGCCTCCACC GCGCTGTTCA TACGCCGTAC GACCTCCTCG 13620 GCGCTCACCG GTTTGTCCTC GTCGTCGTAG TGGACGACGT CGGGTGCCCA GTGCGCGACC 13680 ACCCCGGAGA CGTCCCACCG GTTCCATGCG GCCACCATCT CCAGGCAGCG TTCCTTGTTC 13740 GCGGTCGTTG ACATGTCGAC TCCTTGAAGG CCCGGGACTA CTGGTCACGC GCCAGCCTTC 13800 CAACCCGCCC CGGAAAAGCG GTGCACGACC GCTGGAGCCC GCACCGGAAC CTGCGCGGCG 13860 GAGCTGAACG GGGTTTCGAG CCGTTCACCA AGGACCTGCC GCAGCCTGTT ACGGCACACC 13920 CTGACGCCTC GCTCCGCGCG GGACGCGCCC GCCGGGAGGA AGGACÄCACC ACCATGTCGG 13980 TACGCACCGA TCAGACGGCG GCACCGGAAG ACCGAGCGGC GGCCACGGAT CCCGGGTTCG 14040 GGCACCTG TA CGCGCAGGTG CAGCAGTTCT ACGCCCGGCA GATGCAGCTC CTCGACTCCG 14100 GCGCGGCCGA GGAGTGGGCC GCCACCTTCA CCGAGGACGG CACGTTCGCC CGGCCCTCCT 14160 CGCCGGAACC GGCACGCGGQ CACGCCGAAC TGGCCGCCGG CGCCCGCGCC GCCGCCGAAC 14220 GCCTCGCCGC CGAGGGCCTT TCGCACCGGC ACGTCATCGG CATGACCGCG GTACGCCGGG 14280 AACCCGACGG CAGCGTGTTC GTACGCAGCT ACGCCCAGGT CTTCGCCACC CGCCGCGGGG 14340 AAGCTCCCCG GCTGCATCTG ATCTGCGTCT GCGAGGACGT GCTCGTGCGG GAGGGGCCGG 14400 GGCTGAAGGT GCGGGAACGG GTTGTCACGC ACGACGCGTG AGGGCGGTCG ACCCGCCGGC 14460 CGAGCCGCAC CTCTGCCACC CCCTCGGCAC GCCAGCCGGC GTCGAGTCCG CTGCGAGAGG 14520 GCGCACTTAG CGTGCGAGCC ATGACTGACT CGACAGGTCC CCGCCCGGTG CCCGCCATGT 14580 CACCCGCCCC CAGCCCCACG CCTTCCCCCG GCCCCGCCCC CGGGAGCGAA CCCGCGCCGC 14640 TCGCCGTGAT CGTCACCGGC GGCGGTTCGG GTATCGGCCG GGCCACCGCC CGCGCCTTCG 14700 «CCGCTCAGGG TGCGAAGGTG CTCGTCGTCG GCCGTACCGA GGACGCGCTC GCGCAGACCG 14760 CCGAGGGCTG TGCGGACATG CGTGTGCTCG TCGCCGACGT GGCCTCGCCC GACGGGCCGC 14820 AGGCGGTCGT CAACGCCGCC CTGCGGGAGT TCGGGAGGAT CGACGTCCTG GTCAACAACG 148 80 CTGCCGTGGC GGGCATGGAG ACCCTGCAGA CCGTCGACCG GGACGCCGTG GCACGGCAGT 14940 • <, ·· TCGGCACCAA TCTGACGGCT CCCCTCTTCC TCGTCCAGTC CGCACTCGGC GCGCTGGAGA 15000 AGTCGCGCGG CATCGTCGTC AACGTGGGGA CCGCCGCGAC CCTGGGCCTG CGCGCCGCCC 15060 CGACCGGCGC GCTGTACGGG GCGAGCAAGG TGGCCCTCGA CTACCTGACC CGGACCTGGG 15120 CCGTCGAACT GGCCCCCCGG GGCATCCGTG TCGTCGGCGT GGCACCCGGG GTGATCGACA 15180. CGGGCATCGG CGTCCGCATG GGCATGACCC CGGAGGGCTA CCGGGAGTTC CTGACCGGCA 15240 • · 107053 37 TGGGCGGCAG GGTGCCCGTG GGCCGGGTCG GCCGTCCGGA GGACGTGGCC TGGTGGATCG 15300 TCCAGCTCGC CCGCCCGGAG GCCGGCTACG CGACGGGCAT GGTCGTCCCC GTCGACGGCG 15360 GGCTGTCGCT GGTCTGACCG GACAAGGAAG GAAATACCGC AGGAAGGAAG TACCGCAGCA 15420 AGGAAATACC GCAGGAAGGA GATATCGCCG TGCAGGAAAC CGAACCCGGC GTCCCCGCGG 15480 ACCTGCCCGC CGAGAGCGAC CCTGCCGCCC TGGAGCGCCT CGCCGCACGG TACCGGCGGG 15540 ACGGCTACGT CCACGTCCCC GGCGTCCTCG ACGCCGGGGA GGTCGCCGAA TACCTGGCCG 15600 AGGCCCGTCG GCTCCTCGCC CACGAGGAGT CCGTGCGCTG GGGCTCCGGC GCCGGCACCG 15660 TCATGGACTA CGTCGCCGAC GCCCAGCTCG GCAGCGACAC GATGCGCCGC CTTGCCACCC 15720 ACCCGCGCAT CGCCGCCCTC GCCGAGTACC TGGCCGGCTC GCCCCTGAGG CTGTTCAAGC 15780 TGGAGGTGCT GCTCAAGGAG AACAAGGAGA AGGACGCCTC GGTCCCCACC GCCCCGCACC 15840 ACGATGCGTT CGCCTTCCCG. TTCTCCACCG CCGGCACCGC CCTGACGGCG. TGGGTCGCGC 15900 TGGTCGACGT CCCGGTGGAA CGCGGCTGCA TGACCTTCGT CCCCGGATCA CACCTGCTGC 15960 CGGATCCCGA TACCGGCGAC GAGCCGTGGG CCGGGGCCTT CACCCG

(2) DETAILS OF SEQ ID NO: 2

(i) SEQUENCE DETAILS: (A) LENGTH: 342 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER DETAILS: / note.= n Translation Product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Thr Is His Vai Trp Asp Tyr Leu Pro Glu Tyr Glu Leu Glu Arg 15 10 15

Glu Asp Ile His Asp Ala Vai Glu Thr Vai Phe Arg Ser Gly Arg Leu; 20 25 30 • «• (

Will Leu Gly Glu Ser Will Arg Gly Phe Glu Ser Glu Phe Ala Ser Phe 35 40 45

Gin Gly Vai Gly His Bottom Gly Or Asp Asn Gly Thr Asn Bottom 50 55 60

Lys Leu Gly Leu Gin Ala Leu Gly Vai Gly Pro Gly Asp Glu Vai Vai; - 65 70 75 80 ♦ 107053 38

Thr Vai Ser Asn Thr Ala Ala Pro Thr Vai Val Ala lie Asp Ser Ala 85 90 95

Gly Ala Thr Pro Val Phe Val Asp Val Arg Glu Glu Asp Tyr Leu Met 100 105 110

Asp Thr Ser Gin Val Glu Ala Val Leu Thr Pro Arg Thr Arg Cys Leu 115 120 125

Leu Pro Val His Leu Tyr Gly Gin Cys Val Asp Met Ala Pro Leu Arg 130 135 140

Asp Leu Ala Area Arg His Asn Leu Val lie Leu Glu Asp Cys Ala Gin 145 150 155 160

Ala His Gly Ala Arg Arg His Gly Arg Leu Ala Gly Ser Thr Gly Asp 165 170. 175

Ala Ala Ala Phe Ser Phe Tyr Pro Thr Lys Val Leu Gly Ala Tyr Gly 180 185 190

Asp Gly Gly Ala Val Leu Thr Asp Asp Glu Arg Val Ala Asp Arg Leu 195 200 205

Arg Arg Leu Arg Tyr Tyr Gly Met Glu Ser Arg Tyr Tyr Val Val Glu 210 215 220

Thr Pro Gly His Asn Ser Arg Leu Asp Glu Or Gin Lower Glu He Leu 225 230 235 240

Arg Arg Lys Leu Ser Arg Leu Pro Ser Tyr He Glu Ala Arg Arg Ala 245 250 255

Val Ala Arg Arg Tyr Glu Glu Gly Leu Ala Asp Thr Gly Leu Leu Leu 260 265 270, Pro Arg Thr Ala Gin Gly Asn Glu His Val Tyr Tyr Val Val Val '275 280 285

Arg His Pro Arg Arg Asp Ala Val Leu Glu Ala Leu Arg Ala Ser Tyr 290 295 300

Asp He Ala Leu Asn He Ser Tyr Pro Trp Pro Val His Thr Met Thr 305 310 315 320, Gly Phe Ser His Leu Gly Tyr Ala Lys Gly Ser Leu Pro Val Thr Glu 325 330 335

Ala Leu Ala Asp Glu He 340

(2) DETAILS OF SEQ ID NO: 3

(i) SEQUENCE DETAILS: (A) LENGTH: 293 amino acids 107053 39 (B) TYPE: amino acid (C) SURFACE: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide <D) OTHER INFORMATION: /h. snogJ Translation Product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Vai Lys Gly Ile Ile Leu Lower Gly Gly Thr Gly Ser Arg Leu His Pro 15 10 15

Thr Thr Leu Lower Or Ser Lys Gin Leu Leu Pro Or Gly Asp Lys Pro 20 25 30

Met Ile Tyr Tyr Pro Leu Ser Vai Leu Met Leu Ala Gly Vai Thr Asp 35 40 45

Ile Leu Ile Ile Ser Thr Pro His Glu Leu Pro Arg Met Arg Arg Leu 50 55 60

Phe Gly Asp Gly Ala Gin Leu Gly Leu Arg Leu Ala Tyr Ala Glu Gin 65 70 75 80

Glu Lys Pro Arg Gly Ile Ala Glu Ala Phe Leu Ile Gly Ala Asp His 85 90 95

Vai Gly Ser Asp Ala Vai Ala Leu Ala Leu Gly Asp Asn Ile Phe His 100 105 110

Gly Ser Ser Phe Gin Gly Vai Leu Arg Lys Glu Ala Glu Glu Leu Asp 115 120 125

Gly Cys Vai Leu Phe Gly Tyr Pro Vai Lys Asp Pro Gin Arg Tyr Gly 130 135 140

Vai Gly Glu Ala Asn Ala Ser Gly Arg Leu Vai Ser Ile Glu Glu Lys 145 150 155 160

Pro Vai Arg Pro Arg Ser Asn Arg Ala Ile Thr Gly Leu Tyr Phe Tyr 165 170 175

Asp Asn Glu Are Do Asp Ile Ala Arg Arg Leu Arg Pro Ser Ala Arg 180 185 190. Gly Glu Leu Glu Ile Thr Asp Ile Asn Arg Thr Tyr Met Glu Arg Gly 195 200 205

Arg Ala Arg Leu Is Asp Leu Gly Arg Gly Phe Ala Trp Leu Asp Thr 210 215 220

Gly Thr Pro Glu Ser Leu Leu Gin Ala Ser Gin Tyr Vai Ser Ala Leu 225 230 235 240 107053 40

Glu Glu Arg Gin Gly Ile Arg Ile Ala Cys Ile Glu Glu Val Ala Leu 245 250 255

Arg Met Gly Phe lie Asn Alla Gin Alla Cys Tyr Glu Leu Gly Alla Arg 260 265 270

Leu Ser Gly Ser Gly Tyr Gly Gin Tyr Val Met Ala lie Ala Glu Glu 275 280 285

Cys Thr Gly Arg Val 290

(2) DETAILS OF SEQ ID NO: 4

(i) SEQUENCE DETAILS: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER DETAILS: / note.= n Translation Product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Vai Tyr Gly Arg Glu Leu Ala Asp Vai Tyr Glu Met Vai Tyr Arg Ser 15 10 15

Arg Gly Lys Ser Trp Ala Asp Glu Ala Glu Arg Or Thr Ala Glu Ile 20 25 30

Arg Ser Arg Arg Pro Gly Ala Arg Ser Leu Leu Asp Vai Ala Cys Gly 35 40 45

Thr Gly Ala His Leu Glu Ala Phe Arg Gly Leu Phe Ala His Thr Glu 50 55 60

Gly Leu Glu Leu Ser Asp Glu Met Arg Ala Leu Ala Glu Arg Arg Leu 65 70 75 80

Pro Gly Vai Pro Vai Arg Pro Gly Asp Met Arg Asp Phe Ala Leu Ser 85 90 95

Gly Arg Phe Asp Ala Vai Vai Cys Leu Phe Cys Ser Ile Gly Tyr Leu .. 100 105 110 • «

Glu Thr Is Area Asp Met Arg Area Area Ar Ar Thr Met Area Area His 115 120 125

Leu Vai Pro Gly Gly Or Leu Vai Pro Glu Pro Trp Trp Phe Pro Glu 130 135 140

Arg Phe Leu Glu Gly Tyr Vai Ala Gly Asp Leu Ala Arg Gly Glu Gly 145 150 155 160 107053 41

Arg Thr Vai Area Arg Vai Ser His Ser Thr Arg Gin Gly Arg Arg Thr 165 170 175

Arg Met Glu Val Arg Phe Leu Vai Gly Glu Ala Thr Gly He Arg Glu 180 185 190

Phe Thr Glu He Asp Leu Leu Thr Leu Phe Thr Arg Glu Glu Tyr Leu 195 200 205

Ala Ala Phe Glu Asp Ala Gly Cys Pro Ala Glu Phe Leu Asp Asp Gly 210 215 220

Leu Thr Gly Arg Gly Leu Phe Val Gly Val Arg Gly Ala Gly 225 230 235

(2) DETAILS OF SEQ ID NO: 5

(i) SEQUENCE DETAILS: (A) LENGTH: 324 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide (D) OTHER DETAILS: / Note: = n Translation Product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Thr Ala Ala Trp Gly Ala Pro Leu Tyr Pro Pro Trp He Pro Ala 1 5 10 15

Arg Pro Gly Arg Arg Arg Cys Gly Area Gly Arg Arg Arg Cys Pro 20 25 30

Pro Vai Glu Pro Ala Ser Arg Pro Arg Gin Glu Gly Arg Vai Ser Vai 35 40 45

Vai Pro Ala Leu Arg Gin Pro Ser Pro Ser Thr Asn Pro Glu Vai Arg 50 55 60

Vai Arg Leu He Asp Leu Ser Ser Pro Or Asp Ser Ser Gin Tyr Glu 65 70 75 80

Pro Asp Pro Or Do His Asp Or Leu Thr Pro Arg Gin Gly Lower Glu 85 90 95 *

His Met Cys Ala Glu Met Arg Glu His Phe Gly Vai Glu Phe Ser Pro 100 105 110

Asp Glu Leu Pro Asp Gly Glu Phe Leu Ser Leu Asp Arg He Thr Leu 115 120 125

Thr Thr His Thr Gly Thr His Vai Asp Ala Pro Ser His Tyr Gly Ser 130 135 140 107053 42

Arg Ala Leu Tyr Gly Asp Gly Val Pro Arg His He Asp Gin Met Pro 145 150 155 160

Leu Glu Trp Phe Phe Gly Arg Gly Val Val Leu Asp Leu Thr Asp Area 165 170 175

Pro Thr Gly Thr Val Ser Ala Ala Arg Leu Glu Lys Glu Leu Ala Arg 180 185 190

Thr Gly Cys Ala Leu Arg Pro Gly Asp He Val Leu Leu His Thr Gly 195 200 205

Ala Gin Arg His Ala Gly Thr Pro Arg Tyr Phe Thr Asp Phe Ala Gly 210 215 220

Leu Asp Gly Pro Area Val Arg Met Leu Leu Asp His Gly Val Arg Val 225 230 235 240

He Gly Thr Asp Ala Phe Ser Leu Asp Ala Pro Phe Gly His He He 245 250 255

Asp Arg Tyr Arg Ala Thr Gly Asp Arg Ser Val Leu Trp Pro Ala His 260 265 270

Val Val Gly Arg Glu Arg Glu Tyr Cys Gin He Glu Arg Leu Ala Asn 275 280 285

Leu Asp Arg Leu Pro Val Ser Phe Gly Phe Arg Val Cys Cys Phe Pro 290 295 300

Val Lys Val Ala Gly Ala Gly Ala Gly Trp Thr Arg Ala Val Ala Leu 305 310 315 320

Val Asp Glu Asp

(2) DETAILS OF SEQ ID NO: 6

(i) SEQUENCE DATA: (A) LENGTH: 408 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER INFORMATION: /Note.= n Translation Product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Or Met Lys Leu Thr Asp Ser Glu Leu Gly Arg Lower Leu Leu Ser 15 10 15

Leu Arg Gly Tyr Gin Trp Leu Arg Gly He His His Asp Pro Tyr Ala 20 25 30 107053 43

Leu Leu Leu Arg Area Glu Ser Asp Asp Pro Area Gin Leu Gly Arg Leu 35 40 45

Leu Arg Glu Arg Gly Arg Leu Arg Ser Asp Thr Gly Thr Trp Val 50 55 60

Thr Ala Asp His Ala Thr Ala Ser Arg Leu Leu Ala Asp Pro Arg Phe 65 70 75 80

Val Leu Arg Arg Pro Pro Area Gly Pro Area Thr Gly Thr Gly Asp Val 85 90 95

Met Pro Trp Glu Glu Ala Thr Leu Ser Asp Leu Leu Pro Leu Asp Glu 100 105 110

Ala Arg Leu Thr Thr Asp Arg Ala Arg Cys Arg Arg Leu Gly Ala Thr 115 120 125

Area Area Arg lie Area Area Asp Gly Pro Val Thla Arg Leu Area Asp 130 135 140

Leu Ala Gly Ala Arg Ala Glu Gin Val Arg Ser Thr Gly His Phe Asp 145 150 155 160

Leu Arg Ala Asp Tyr Ala Leu Pro Tyr Ala Val Glu Pro Ala Cys Ala 165 170 175

Leu Leu Gly Leu Pro Alla Gly Gin Cys Ser Leu Phe Gly Alla Phe Ser 180 185 190

Pro Ala Val Leu Leu Asp Ala Thr Val Val Pro Pro Arg Leu Pro Glu 195 200 205

Ala Arg Ala Leu lie Ala Ser Thr Ala Glu Leu Thr Ala Leu Trp Pro 210 215 220

Arg Leu Ala Pro Ser Leu Ser Lys Thr Val Pro Glu Asp Glu Ala Pro • 225 230 235 240

Asp Leu Phe Leu Leu Thr Ala Val Leu Leu Val Pro Ala Val Val His 245 250 255

Leu Val Cys Glu Ala Val Ala Ala Leu Ser His Asp Pro Gly Gin Ala 260 265 270

Gly Leu Leu Arg Asp Asp Pro Val Leu Area Ala Pro Area Val Glu Glu 275 280 285

Thr Leu Arg His Ala Pro Pro Ala Arg Leu Phe Thr Leu His Ala Thr 290 295 300

Gly Pro Glu Arg Val Ala Asp Val Asp Leu Pro Ala Gly Ala Glu Val 305 310 315 320 107055 44

Area Or Do Or Area Lower Area His Arg Asp Pro Ser Trp Cys Pro Asp 325 330 335

Pro Asp Arg Phe Asp Leu Thr Arg Asn Glu Arg His Leu Ala Leu Pro 340 345 350

Pro Asp Leu Pro Leu Gly Lower Leu Lower Pro Leu Leu Arg Vai Cys Lower 355 360 365

Thr Area Area Do Area Area Leu Area Area Gly Leu Leu Pro Leu Arg Area 370 375 380

Vai Gly Pro Pro Or Arg Arg Leu Arg Ala Pro Or Thr Arg Ser Vai 385 390 395 400

Leu Arg Phe Pro Vai Ala Pro Cys 405

(2) DETAILS OF SEQ ID NO: 7

(i) SEQUENCE DETAILS: (A) LENGTH: 422 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide (D) OTHER DETAILS: / note.= n translation product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Asp Asn Arg Glu Thr Or Arg Pro Or Ser Or Cys Arg Or Cys 15 10 15

Gly Gly Asn Asp Trp Gin Asp Or Or Asp Phe Gly Asp Or Pro Leu 20 25 30

Ala Asn Gly Phe Leu Ser Pro Ala Asp Ser Tyr Glu Asn Glu Arg Arg 35 40 45

Tyr Pro Leu Gly Or Leu Ser Cys Arg Lower Cys Arg Leu Met Ser Leu 50 55 60

Thr His Vai Or Asp Pro Glu Or Leu Tyr Arg Asp Tyr Ala Tyr Thr 65 70 75 80 • Thr Pro Asp Ser Glu Met Ile Thr Gin His Met Arg His Ile Thr Ala 85 90 95

Leu Cys Arg Thr Arg Phe Glu Leu Pro Pro Asp Ser Leu Or Vai Glu 100 105 110

Leu Gly Ser Asn Thr Gly Arg Gin Leu Met Ala Phe Arg Glu Ala Gly 115 120 125 107055 45

Met Arg Thr Leu Gly Val Asp Pro Area Arg Asn Leu Thr Asp Val Area 130 135 140

Arg Arg Asn Gly lie Glu Thr Phe Pro Asp Phe Phe Ser His Asp Val 145 150 155 160

Ala Arg Thr lie Arg Asp His Gly Gin Ala Arg Leu Val Leu Gly 165 170 175

Arg His Val Phe Ala His lie Asp Asp Val Ser Asp lie Ala Ala Gly 180 185 190

Val Arg Glu Leu Leu Ser Pro Asp Gly Val Phe Ala lie Glu Val Pro 195 200 205

Tyr Val Leu Asp Leu Leu Glu Lys Val Ala Phe Asp Thr lie Tyr His 210 215 220

Glu His Leu Ser Tyr Phe Thr Met Arg Ser Phe Val Thr Leu Phe Ala 225 230 235 240

Arg His Gly Leu Arg Val Leu Asp Val Glu Arg Phe Gly Val His Gly 245 250 255

Gly Ser Val Leu Val Phe Val Gly His Glu Asp Gly Pro Trp Pro Glu 260 265 270

Arg Pro Ser Val Pro Glu Leu Leu Arg Val Glu Arg Gin Arg Gly Leu 275 280 285

Tyr Asp Asp Ala Thr Tyr Arg Thr Phe Ala Gin Arg He Glu Arg Val 290 295 300

Arg Thr Glu Leu Pro Glu Leu Leu Arg Ser Leu Val Ala Gin Gly Lys 305 310 315 320. Arg He Val Gly Tyr Gly Ala Pro Ala Lys Gly Asn Thr He Leu Thr • 325 330 335

Val Cys Gly Leu Gly Leu Lys Glu Leu Glu Tyr Cys Thr Asp Thr Thr 340 345 350

Glu Leu Lys Gin Gly Arg Val Leu Pro Gly Thr His He Pro Val His 355 360 365 i Ala Pro Glu His Ala Lys Glu His He Pro Asp Tyr Tyr Leu Leu Leu 370 375 380

Ala Trp Asn Tyr Ala Thr Glu He Leu Asp Lys Glu Thr Ala Phe Arg 385 390 395 400

Asp Asn Gly Gly Arg Phe He Val Pro He Pro Arg Pro Ser He Leu 405 410 415 107053 46

Thr Ser Pro Ser Gly Ser 420

(2) DETAILS OF SEQ ID NO: 8

(i) SEQUENCE DETAILS: (A) LENGTH: 291 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide (D) OTHER DETAILS: / note.= n translation product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Leu Ala Arg His Leu Thr Ala Leu Ala Glu Thr Gly Arg Ser 15 10 15

Arg Pro Area Ala Glu Area Ala Is Leu Gly Arg Arla Ala Leu Asp Ile 20 25 30

Thr Asp Gly Arg Ala Is Asp Ala Ala Phe Ala Ala His Arg Pro Arg 35 40 45

Or Or Or Asn Cys Lower Area Phe Thr Asp Or Asp Gly Lower Glu Ser 50 55 60

Arg Trp Ala Glu Ala Met Arg Vai Asn Gly Gly Gly Pro Arg Leu Leu 65 70 75 80

Ala Arg Arg Cys Ala Arg His Gly Vai Arg Leu Ile His Vai Ser Thr 85 90 95

Asp Tyr Vai Phe Pro Gly Asp Thr Arg Ser Pro Tyr Gly Glu Ser Asp. 100 105 110

Ala Pro Gly Pro Arg Thr Vai Tyr Gly Arg Ser Lys Leu Ala Gly Glu 115 120 125

Arg Area Or Leu Ser Leu Leu Pro Asp Thr Gly Thr Or Arg Thr 130 135 140

Ala Trp Leu Tyr Gly Gly Gin Gly Arg Ser Phe Vai Arg Thr Met Leu 145 150 155 160

Glu Arg Area Pro Asp Asp Gly His Or Asp Or Or Asn Asp Gin Trp 165 170 175

Gly Gin Pro Thr Trp Area Gly Asp Vai Area Arg Leu Leu Vai Thr Leu 180 185 190

Ala Arg Thr Pro Pro Asp Arg Ala Arg Gly Ile Phe His Ala Thr Asn 195 200 205 107053 47

Ala Gly Ala Ala Thr Trp Tyr Glu Leu Ala Arg Glu Vai Phe Arg Leu 210 215 220

Ala Gly Ala Asp Pro Glu Arg Or Ar Pro Pro Ala Thr Ala Asp Arg 225 230 235 240

Pro Gly Pro Ala Pro Arg Pro Ala Cys Thr Vai Leu Gly His Asp Arg 245 250 255

Trp Arg Leu Do Gly Or Area Pro Pro Arg Asp Trp Arg Leo Area Leu 260 265 270

Arg Glu Ala Met Arg Gin Leu Leu Pro Gly Gly Arg Leu Arg Asn Leu 275 280 285

Thr Gly Thr 290

(2) DETAILS OF SEQ ID NO: 9

(i) SEQUENCE DETAILS: (A) LENGTH: 350 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide (D) OTHER DETAILS: / note.= "snog. n translation product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Ala Ser His Thr Ser Ala Thr Asp Vai Asn Ile Leu Vai Thr 1 5 10 15

Gly Ala Vai Gly Phe Ile Gly Ser Ala Tyr Vai Arg Met Leu Leu Glu. 20 25 30

Asn Arg Area Pro Gly Area Gly Area Pro Area Area Arg Area Thr Vai Leu 35 40 45

Asp Lys Leu Thr Tyr Ala Gly Asn Leu Thr Asn Leu Asp Ala Vai Arg 55 55 60

Gly Asp Arg Leu Arg Phe Vai Arg Gly Asp Ile Leu Asp Ala Glu Leu. 65 70 75 80

Vai Asp Glu Leu Met Ala His Ser Asp Gin Vai As His Phe Ala Ala 85 90 95

Glu Ser His Vai Asp Arg Ser Ile Arg Lower Area Asp Asp Phe Vai Leu 100 105 110

Thr Asn Vai Or Gly Thr Gin Arg Leu Leu Asp Ala Lower Leu Arg His 115 120 125 107053 48

Gly Vai Glu Pro Phe Val Leu Vai Ser Thr Asp Glu Val Tyr Gly Ser 130 135 140 lie Ala Ser Gly Ser Trp Pro Glu Glu His Pro Leu Ser Pro Asn Ser 145 150 155 160

Pro'Tyr Ala Ala Ser Lys Ala Ser Ala Asp Leu Met Ala Phe Ala Cys 165 170 175

His Arg Thr His Gly Leu Asp Val Arg Val Thr Arg Cys Ser Asn Asn 180 185 190

Tyr Gly Pro Arg Gin His Pro Glu Lys Leu lie Pro Arg Phe Val Thr 195 200 205

Asn Leu Leu Asp Gly Leu Pro Val Pro Leu Tyr Gly Asp Gly Arg Asn 210 215 220

Val Arg Glu Trp Leu His Val Glu Asp His Cys Arg Gly Val Asp Leu 225 230 235 240

Val Arg Thr Ala Gly Arg Pro Gly Gly Val Tyr His He Gly Gly Gly 245 250 255

Arg Glu Leu Ser Asn Arg Glu Leu Val Gly Met Leu Leu Glu Leu Cys 260 265 270

Gly Ala Asp Trp Ser Ser Val Arg His Val Pro Asp Arg Lys Gly His 275 280 285

Asp Leu Arg Tyr Ser Leu Asp Trp Gly Arg Ala Arg Glu Glu Leu Gly 290 295 300

Tyr Arg Pro Ala Arg Glu Phe Ser Ser Gly Leu Arg Ser Thr Val Gin 305 310 315 320 -, - Trp Tyr Arg Glu Asn Arg Ser Trp Trp Glu Pro Leu Lys Arg Gly Val 325 330 335

Thr Ala Pro Gly Gly Thr Ser Thr Val Val Pro Gly Val Arg 340 345 350

(2) DETAILS OF SEQ ID NO: 10

(i) SEQUENCE DETAILS:] '(A) LENGTH: 134 amino acids V. (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER INFORMATION: / Note. = "snoaL Translation Product" 107053 49 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Vai Ser Ala Phe Asn Thr Gly Arg Thr Asp Asp Val Asp Glu Tyr 15 10 15 lie His Pro Asp iyr Leu Asn Pro Ala Thr Leu Glu His Gly lie His 20 25 30

Thr Gly Pro Lys Ala Phe Ala Gin Leu Val Gly Trp Val Arg Ala Thr 35 40 45

Phe Ser Glu Glu Ala Arg Leu Glu Glu Val Arg lie Glu Glu Arg Gly 50 55 60

Pro Trp Val Lys Lower Tyr Leu Val Leu Tyr Gly Arg His Val Gly Arg 65 70 75 80

Leu Val Gly Met Pro Pro Thr Asp Arg Arg Phe Ser Gly Glu Gin Val 85 90 95

His Leu Met Arg lie Val Asp Gly Lys lie Arg Asp His Arg Asp Trp 100 105 110

Pro Asp Phe Gin Gly Thr Leu Arg Gin Leu Gly Asp Pro Trp Pro Asp 115 120 125

Asp Glu Gly Trp Arg Pro 130

(2) DETAILS OF SEQ ID NO: 11

(i) SEQUENCE DETAILS: (A) LENGTH: 235 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear * :( ii) MOLECYL TYPE: peptide (D) OTHER INFORMATION: / Note.` snoKm Translation Product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Pro Asp Pro Gly Gly Pro Thr Thr Ala Glu Asn Leu Ser Lys Glu 15 10 15: Ala Vai Arg Phe Tyr Arg Glu Gin Gly Tyr Vai His Ile Pro Arg Vai 20 25 30

Leu Ser Glu Thr Glu Vai Thr Ala Phe Arg Ala Ala Cys Glu Glu Vai 35 40 45

Leu Glu Lys Glu Gly Arg Glu Ile Ser Gly Ile Ala Leu Arg Leu Ala 50 55 60 107053 50

Gly Ala Pro Leu Arg Val Tyr Ser Ser Asp lie Leu Val Lys Glu Pro 65 70 75 80

Lys Arg Thr Leu Pro Thr Leu Val His Asp Asp Glu Thr Gly Leu Pro 85 90 95

Leu Asn Glu Leu Ser Ala Thr Leu Thr Ala Trp lie Ala Leu Thr Asp 100 105 110

Val Pro Val Glu Arg Gly Cys Met Ser Tyr Val Pro Gly Ser His Leu 115 120 125

Arg Ala Arg Glu Asp Arg Gin Glu His Met Thr Ser Phe Ala Glu Phe 130 135 140

Arg Asp Leu Area Asp Val Trp Pro Asp Tyr Pro Trp Gin Pro Arg Val 145 150 155 160

Ala Val Pro Val Arg Ala Gly Asp Val Val Phe His His Cys Arg Thr 165 170 175

Val His Met Ala Glu Ala Asn Thr Ser Asp Ser Val Arg Met Ala His 180 185 190

Gly Val Val Tyr Met Asp Ala Asp Ala Thr Tyr Arg Pro Gly Val Gin 195 200 205

Asp Gly His Leu Ser Arg Leu Ser Pro Gly Asp Pro Leu Glu Gly Glu 210 215 220

Leu Phe Pro Leu Val Thr Ala Gly Thr Arg Gin 225 230 235

(2) DETAILS OF SEQ ID NO: 12

(i) SEQUENCE DETAILS: (A) LENGTH: 390 amino acids • (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER DETAILS: / Note.` snogD translation product ". (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: •.

Met Arg Vai Pro Gly Ser Cys Arg Thr Gly Gly Ile Met Arg Ala Leu 15 10 15

Phe Ile Thr Ser Pro Gly Leu Ser His Ile Leu Pro Thr Vai Pro Leu 20 25 30

Ala Gin Ala Leu Arg Ala Leu Gly His Glu Vai Arg Tyr Ala Thr Gly 35 40 45 107053 51

Gly Asp Ile Arg Area Or Area Glu Area Gly Leu Cys Area Or Asp Vai 50 55 60

Ser Pro Gly Vai Asn Tyr Ala Lys Leu Phe Vai Pro Asp Thp Asp Thr Asp 65 70 75 80

Vai Thr Asp Pro Met His Ser Glu Gly Leu Gly Glu Gly Phe Phe Ala 85 90 95

Glu Met Phe Ala Arg Or Ser Ala Or Ala Or Asp Gly Ala Leu Arg 100 105 110

Thr Ala Arg Ser Trp Arg Pro Asp Leu Or Is His Thr Pro Thr Gin 115 120 125

Gly Ala Gly Pro Leu Thr Ala Ala Leu Gin Leu Pro Cys Vai Glu 130 135 140

Leu Pro Leu Gly Pro Ala Asp Ser Glu Pro Gly Leu Gly Ala Leu Ile 145 150 155 160

Arg Arg Ala Met Ser Lys Asp Tyr Glu Arg His Gly Vai Thr Gly Glu 165 170 175

Pro Thr Gly Ser Vai Arg Leu Thr Thr Thr Pro Pro Ser Vai Glu Ala 180 185 190

Leu Leu Pro Glu Asp Arg Arg Ser Pro Gly Ala Trp Pro Met Arg Tyr 195 200 205

Vai Pro Tyr Asn Gly Gly Lower Vai Leu Pro Asp Trp Leu Pro Pro Lower 210 215 220

Ala Gly Arg Arg Arg Ile Ala Vai Thr Leu Gly Ser Ile Asp Ala Leu 225 230 235 240

Ser Gly Gly Ile Ala Lys Leu Ala Pro Leu Phe Ser Glu Vai Ala Asp '245 250 255

Vai Asp Ala Glu Phe Vai Leu Thr Leu Gly Gly Gly Asp Leu Ala Leu 260 265 270

Leu Gly Glu Leu Pro Area Asn Or Pro Or Glu Trp Ile Pro Leu 275 280 285

Gly Ala Leu Leu Glu Thr Cys Asp Ale Ile Ile His His Gly Gly Ser 290 295 300

Gly Thr Leu Leu Thr Ala Leu Ala Ala Gly Vai Pro Gin Cys Vai Ile 305 310 315 320

Pro His Gly Ser Tyr Gin Asp Thr Asn Arg Asp Vai Leu Thr Gly Leu 325 330 335 «« 107053 52

Gly Ile Gly Phe Asp Ala Glu Ala Gly Ser Leu Gly Ala Glu Gin Cys 340 345 350

Arg Arg Leu Leu Asp Asp Ala Gly Leu Arg Glu Ala Leu Arg h 355 360 365

Arg Gin Glu Met Ser Glu Met Pro Pro Pro Ala Glu Thr Ala Ala Lys 370 375 380

Leu Val Ala Leu Ala Gly 385 390

(2) DETAILS OF SEQ ID NO: 13

(i) SEQUENCE DETAILS: (A) LENGTH: 275 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER DETAILS: / note.= "s. n Translation Product "<xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Thr Vai Leu Vai Thr Gly Lower Thr Gly Asn Vai Gly Arg His Vai 15 10 15

Will Thr Gly Leu Leu Area Gly Arg Arg Will Thr Gla Leu Thr Arg 20 25 30

Thr Pro Asp Arg Ser Gly Leu Pro Gly Gly Ala Glu Ile Thr Gly Gly 35 40 45

Asp Leu Thr Arg Pro Glu Thr Tyr Glu Arg Met Leu Asp Gly Vai Glu 50 55 60

Ala Vai Tyr Leu Phe Pro Vai Pro Glu Thr Ala Ala Phe Ala Gly 65 70 75 80

Area Area Arg Arg Area Gly Is Arg Arg Ile Or Is Leu Ser Ser Asp 85 90 95

Ser Vai Thr Asp Gly Thr Asp Thr Gly Gly His Arg Arg Vai Glu Leu 100 105 110

Ala Vai Glu Asp Thr Gly Leu Glu Trp Thr His Vai Arg Pro Gly Glu 115 120 125

Phe Ala Leu Asn Lys Vai Thr Leu Trp Ala Pro Ser Ile Arg Ala Glu 130 135 140

Gly Does Or Ar Ser Ser Ala Tyr Pro Asp Arg Or Ar Pro Pro His. 145 150 155 160 107053 53

Glu Ala Asp Vai Ala Ala Ala Ala A Thr Ala Leu Leu Lys Glu Gly 165 170 175

His Ala Gly Arg Ala Tyr Ser Vai Thr Gly Pro Gin Ala Leu Thr Gin 180 185 190

Arg Glu Gin Ar Arg Area Ar Gly Glu Gly Leu Gly Arg Ser Leu Area 195 200 205

Phe Vai Glu Vai Thr Pro Gly Gin Ala Arg Ala Asp Leu Thr Ala Gin 210 215 220

Gly Leu Pro Ala Pro Ile Ala Asp Tyr Vai Leu Ala Phe Gin Ala Gly 225 230 235 240

Trp Thr Glu Arg Pro Area Pro Area Arg Pro Thr Do Arg Glu Ar Thr 245 250 255

Gly Arg Pro Area Arg Thr Leu Area Gin Trp Area Area Asp His Arg Area 260 265 270

Asp Phe Arg 275

(2) DETAILS OF SEQ ID NO: 14

(i) SEQUENCE DETAILS: (A) LENGTH: over 424 amino acids (B) type: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear <ii) MOLECYL TYPE: peptide (D) OTHER DETAILS: / note.= "snogE "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: • t

Vai Ar Vai Leu Leu Thr Ser Phe Lower Met Asp Lower His Phe Cys Thr 15 10 15

Ala Vai Pro Leu Ala Trp Ala Leu Arg Ser Ala Gly His Glu Vai Arg 20 25 30

Will Ala Gly Gin Pro Ala Leu Thr Ser Thr Ile Thr Gly Ala Gly Leu 35 40 45

Thr Lower Or Pro Or Gly Arg Asp His Thr His Gly Ser Leu Leu Gly 50 55 60

Arg Vai Gly Ser Asp Ile Leu Ala Leu His Asp Glu Ala Asp Tyr Leu '65 70 75 80

Glu Ala Arg His Asp Ala Leu Gly Phe Glu Phe Leu Lys Gly His Asn: 85 90 95

10705S

54

Thr Vai Met Ser Ala Leu Phe Tyr Ser Gin lie Asn Asn Asp Ser Met 100 105 110

Val Asp Asp Leu Val Asp Phe Area Arg His Trp Arg Pro Asp Leu Val 115 120 125

Val Trp Glu Pro Phe Thr Phe Ala Gly Ala Val Ala Ala Arg Ala Ser 130 135 140

Gly Ala Ala His Ala Arg Leu Leu Ser Phe Pro Asp Leu Phe Leu Ser 145 150 155 160

Thr Arg Arg Leu Phe Leu Glu Arg Met Ala Arg Gin Glu Pro Glu His 165 170 175

His Asp Asp Thr Leu Area Glu Trp Leu Asp Trp Thr Leu Gly Arg His 180 185 190

Gly His Ser Phe Asp Glu Glu lie Val Thr Gly Gin Trp Ser lie Asp 195 200 205

Gin Thr Pro Lower Pro Val Arg Leu Asp Lower Gly Gly Pro Thr Val Pro 210 215 220

Met Arg Tyr Val Pro Tyr Ser Gly Leu Val Pro Thr Val Val Pro Asp 225 230 235 240

Trp Leu Arg Arg Pro Pro Glu Arg Pro Arg Val Leu Val Thr Leu Gly 245 250 255 lie Thr Ser Arg Arg Val Lys Ser Phe Leu Ala Val Ser Val Asp Asp 260 265 270

Leu Phe Glu Ala Val Ala Gly Leu Gly Val Glu Val Ala Thr Leu 275 280 285

Asp Ala Asp Gin Arg Glu Leu Gly Arg Val Pro Asp His Phe Arg 290 295 300 lie Val Glu His Val Pro Leu Asp Ala Val Leu Pro Thr Cys Ser Ala 305 310 315 320 lie Val His His Gly Gly Ala Gly Thr Trp Ser Thr Ala Ala Val Tyr 325 330 335

Gly Val Pro Gin Val Ser Leu Gly Ser Met Trp Asp His Phe Tyr Arg. 340 345 350

Ala Arg Arg Leu Glu Glu Leu Gly Ala Gly Leu Arg Leu Pro Ser Gly 355 360 365

Glu Leu Thr Area Glu Gly Leu Arg Thr Arg Glu Arg Val Leu Gly 370 375 380 55 10705ό

Glu Pro Ser Phe Gly Thr Ala Ala Gin Ala Leu Ser Asp Thr lie Ala 385 390 395 400

Ala Glu Pro Ser Pro Ser Glu Or Val Pro Val Leu Glu Glu Leu Thr 405 410 415

Gly Arg His Arg Pro Gly Thr Arg 420

(2) DETAILS OF SEQ ID NO: 15

(i) SEQUENCE DETAILS: (A) LENGTH: 139 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide (D) OTHER DETAILS: / note.= n translation product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Met Ser Thr Thr Ala Asn Lys Glu Arg Cys Leu Glu Met Vai Ala Ala 15 10 15

Trp Asn Arg Trp Asp Or Ser Gly Or Or His Trp Asla Pro Asp 20 25 30

Vai Or His Tyr Asp Asp Glu Asp Lys Pro Vai Ser Ala Glu Glu Vai 35 40 45

Vai Arg Arg Met Asn Ser Ala Vai Glu Ala Phe Pro Asp Leu Arg Leu 50 55 60

Asp Arg Ser Ile Or Gly Glu Gly Asp Arg Or Met Leu Arg Ile 65 70 75 80

Thr Cys Ser Ala Thr His Gin Gly Vai Phe Met Gly Ile Ala Pro Thr 85 90 95

Gly Arg Lys Or Arg Trp Thr Tyr Leu Glu Glu Leu Arg Phe Ser Glu 100 105 110

Ala Gly Lys Or Or Glu His Trp Asp Or Phe Asn Phe Ser Pro Leu 115 120 125

Phe Arg Asp Leu Gly Vai Vai Pro Asp Gly Leu 130 '135

(2) DETAILS OF SEQ ID NO: 16

(i) SEQUENCE INFORMATION: (A) LENGTH: 155 amino acids. (B) TYPE: amino acid 107053 56 (C) DRAINAGE: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL-TYPE: peptide (D) MOOD DETAILS: / Note: "snoO Translation Product" (xi) SEQ ID NO: NO: 16:

Met Ser Val Arg Thr Asp Gin Thr Area Ala Pro Glu Asp Arg Area Ala 15 10 15

Ala Thr Asp Pro Gly Phe Gly His Leu Tyr Ala Gin Val Gin Gin Phe 20 25 30

Tyr Ala Arg Gin Met Gin Leu Leu Asp Ser Gly Ala Ala Glu Glu Trp 35 40 45

Ala Ala Thr Phe Thr Glu Asp Gly Thr Phe Ala Arg Pro Ser Ser Pro 50 55 60

Glu Pro Area Arg Gly His Area Area Glu Leu Area Area Gly Area Area Area 65 70 75 80

Ala Glu Arg Leu Ala Ala Glu Gly Leu Ser His Arg His Val lie Gly 85 90 95

Met Thr Ala Val Arg Arg Glu Pro Asp Gly Ser Val Phe Val Arg Ser 100 105 110

Tyr Ala Gin Val Phe Ala Thr Arg Arg Gly Glu Ala Pro Arg Leu His 115 120 125

Leu lie Cys Val Cys Glu Asp Val Leu Val Arg Glu Gly Pro Gly Leu 130 135 140

Lys Val Arg Glu Arg Val Val Thr His Asp Ala.

; '145 150 155

(2) DETAILS OF SEQ ID NO: 17

(i) SEQUENCE DETAILS: (A) LENGTH: 281 amino acids (B) TYPE: amino acid (C) DURABILITY: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER DETAILS: / Note: = n translation product "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Vai Arg Lower Met Thr Asp Ser Thr Gly Pro Arg Pro Vai Pro Lower Met: 1 5 10 15 «107053 57

Ser Pro Ala Pro Ser Pro Thr Pro Ser Pro Gly Pro Ala Pro Gly Ser 20 25 30

Glu Pro Ala Pro Leu Ala Vai Ile Val Thr Gly Gly Gly Ser Gly lie 35 40 45

Gly Arg Ala Thr Ala Arg Ala Phe Ala Ala Gin Gly Ala Lys Val Leu 50 55 60

Val Val Gly Arg Thr Glu Asp Ala Leu Ala Gin Thr Ala Glu Gly Cys 65 70 75 80

Ala Asp Met Arg Val Leu Val Ala Asp Val Ala Ser Pro Asp Gly Pro 85 90 95

Gin Ala Val Val Asn Ala Ala Leu Arg Glu Phe Gly Arg lie Asp Val 100 105 110

Leu Val Asn Asn Ala Ala Val Ala Gly Met Glu Thr Leu Gin Thr Val 115 120 125

Asp Arg Asp Ala Val Ala Arg Gin Phe Gly Thr Asn Leu Thr Ala Pro 130 135 140

Leu Phe Leu Val Gin Ser Ala Leu Gly Ala Leu Glu Lys Ser Arg Gly 145 150 155 160 lie Val Val Asn Val Gly Thr Ala Thr Leu Gly Leu Arg Ala Ala 165 170 175

Pro Thr Gly Ala Leu Tyr Gly Ala Ser Lys Val Ala Leu Asp Tyr Leu 180 185 190

Thr Arg Thr Trp Ala Val Glu Leu Ala Pro Arg Gly lie Arg Val Val 195 200 205

Gly Val Ala Pro Gly Val Lie Asp Thr Gly Lie Gly Val Arg Met Gly i: - - 210 215 220

Met Thr Pro Glu Gly Tyr Arg Glu Phe Leu Thr Gly Met Gly Gly Arg 225 230 235 240

Val Pro Val Gly Arg Val Gly Arg Pro Glu Asp Val Ala Trp Trp lie 245 250 255

Val Gin Leu Ala Arg Pro Glu Ala Gly Tyr Ala Thr Gly Met Val Val 260 265 270

Pro Val Asp Gly Gly Leu Ser Leu Val 275 280

(2) DETAILS OF SEQ ID NO: 18

(i) SEQUENCE DETAILS:, (A) LENGTH: 190 amino acids 107053 58 (B) TYPE: amino acid (C) SURFACE: single-stranded (D) TOPOLOGY: linear (ii) MOLECYL TYPE: peptide (D) OTHER INFORMATION: /h. "snoN Translation Product" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Is Gin Glu Thr Glu Pro Gly Is Pro Ala Asp Leu Pro Ala Glu Ser 15 10 15

Asp Pro Area Area Leu Glu Arg Leu Area Area Arg Tyr Arg Arg Asp Gly 20 25 30

Tyr Vai His Vai Pro Gly Vai Leu Asp Ala Gly Glu Vai Ala Glu Tyr 35 40 45

Leu Ala Glu Ala Arg Arg Leu Leu Ala His Glu Glu Ser Vai Arg Trp 50 55 60

Gly Ser Gly Lower Gly Thr Or Met Asp Tyr Or Lower Asp Lower Gin Leu 65 70 75 80

Gly Ser Asp Thr Met Arg Leu Ala Thr His Pro Arg Ile Ala Ala 85 90 95

Leu Ala Glu Tyr Leu Ala Gly Ser Pro Leu Arg Leu Phe Lys Leu Glu 100 105 110

Vai Leu Leu Lys Glu Asn Lys Glu Lys Asp Ala Ser Vai Pro Thr Ala 115 120 125

Pro His His Asp Ala Phe Ala Phe Pro Phe Ser Thr Ala Gly Thr Ala 130 135 140 • - Leu Thr Ala Trp Or Ala Leu Or Asp Vai Pro Or Glu Arg Gly Cys 145 150 155 160

Met Thr Phe Vai Pro Gly Ser His Leu Leu Pro Asp Pro Asp Thr Gly 165 170 175

Asp Glu Pro Trp Ala Gly Ala Phe Thr Arg Pro Gly Glu Ile 180 185 190

Claims (15)

107053 1. Isolerat and Renat DNA fragment, som e en gengruppering, som e för sig account for anthracycline biosynthesis bacterial Streptomyces nogaloter, germinating in succession or lOkb and 7kb BgUl fragment, s. Somal en angivet. 2. An isolated purified DNA fragment, which is a gene cluster related to the anthracycline biosynthetic pathway of Streptomyces nogalater contained in the sequenced 10kb and 7kb Bglil fragments of the S. nogalaterin genome as shown in Figure 2. The DNA fragment enligt patent kravet 1, som innehäller en nucleotide sequence angiven i SEQ ID NO: 1 eller en sequence, som har endinstone up to 80% homologue med avseende this sequence. The DNA fragment of claim 1, comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a sequence having at least 80% homology to said sequence. 10 3. Hybrid DNA, the native fragment of this invention is a fragment of the plasmid som replicase of the Streptomyces. 107053 A recombinant DNA containing a DNA fragment according to claim 1 or 2, cloned in a Streptomyces replicable plasmid. 4. The hybrid DNA enligt 3, the plasmid pSY15c, the plasmid pSY42 and the 1.1 kb A / M-K / wI fragment of ur plasmids pSY43 and the 1.1 kb A / M-K / wI fragment of ur plasmids pSY43. The recombinant DNA of claim 3, which is a plasmid pSY15c, which contains a 1.4 kb SarnHI-AkcI fragment from pSY42 and a 1.1 kb MluI-Kpn1 fragment from pSY43. Plasmiden pSY42, som deponerats to S. lividans-atammen TK24 / pSY42 med deponeringsnumret DSM 12451. 5. Plasmid pSY42 deposited with S. lividans strain TK24 / pSY42 under accession number DSM 12451. 20 6. Plasmiden pSY43, som deponerates to S. lividans-stammen TK24 / pSY43 med deponeringsnumret DSM 12452. 10 Plasmid pSY43 deposited in S. lividans strain TK24 / pSY43 under accession number DSM 12452. 7. Förfarande for att ffamställa hybridföreningar, kännetecknat därav, att that the DNA fragment enligt patentkravet 1 eller 2 transformeras in i en Streptomyces, den erhällna hybridstammen odlas och de bildade föreningarna isoleras. 8. Förfarande enligt patent kravet 7, callus dnav, att Streptomyces galilaeus. A process for the preparation of hybrid compounds, characterized in that the DNA fragment according to claim 1 or 2 is transferred to a host in the heptoreptomyces, the resulting recombinant strain is cultured and the resulting compounds are isolated. The method according to claim 7, characterized in that the Streptomyces host is a Streptomyces galilaeus host. 30, 107055 9. Förfarande enligt patent kravet 8, callus dnav, att Streptomyces galilaeus strain H026, H039, H063 eller H075, mite and mutant strain 20 av S. galilaeus ATCC 31615. • «'· 1« 1 · * Method according to claim 8, characterized in that the host Streptomyces galilaeus is one of the strains H026, H039, H063 or H075 which are mutant strains of S. galilaeus ATCC 31615. 10 OH O OH It was 10. Förfarande enligt patentkravet 8, kännetecknat därav, att man producerar en anthracycline, som har feljande formel I 25. cooch3 in 0 011 9 (I> 30 Me- ^ 7 ~ ° / ηο ^ η X oh 107053 10. A process according to claim 8, characterized in that an anthracycline having the following formula IO COOCH3 Ah 0 0H <? (I) Me-7 ~ 7 OH 15 11. Förfarande enligt patentkravet 8, kännetecknat därav, att man producerar en anthracycline, series har over form II 5 O CÖOCIlj (P) The process according to claim 8, characterized in that anthracyclinone of the following formula II is produced 20. COOCH3 .. Il ATm · OH O OH ÖH 25 107053 12. Förfarande för att framställa hybridföreningar, kännetecknat därav, att man 15 transformerar ätminstone an av genererna snogi, snogA, snoaM, snogN, snoaG, snogC, snogK, snoah, snoK., SnogD, snoW, snogE, snoL, snoL, snoL. oaF in i en Strepto-myces, the generic bovine gene fragment DNA ligand 1 eller 2, odlar den erhällna hybridstammen och isolerar de bildade föreningarna. 13. Förfarande enligt patentkravet 12, kännetecknat därav, att man transformerar in i ... Streptomyces-svden snoäL, som kodar för NAME-cycles. • «• i A method for producing hybrid compounds, characterized by transferring at least one of the genes snog], snogA, snoaM, snogN, snoaG, snogC, snogK, snoaL, snoK, snogO, snoW, snogE, snoL, snoO and inoaF Streptomyces gene, are derived from the DNA fragment of claim 1 or 2, culturing the resulting recombinant strain and isolating the resulting compounds. A method according to claim 12, characterized in that snoaL encoding NAME cyclase is introduced into the Streptomyces host. 14. Förfarande enligt patent kravet 12, kännetecknat därav, att man transformerar in i Streptomyces-arginine sngD och snogE, som codar för 25 glycosyltransferaser. 14. The method of claim 12, wherein at least one of the genes snogL and snogE encoding glycosyltransferases is introduced into the host Streptomyces. Method according to claim 12, characterized in that at least one of the genes snogJ, snogN, snogC, snogK and snogK, which affect the production of nogalamine and nogalose, is introduced into the host Streptomyces-15. 15. Förfarande enligt patent kravet 12, kännetecknat därav, att man transformerar in i streptomyces-arginine åminstone en av genererna snogi, snogN, snogC, snogK och snogA, som inverkar Pä bildning av nogalamin och nogalos. 30 • r «