DNA COMPOUNDS COMPRISING SEQUENCES ENCODING MANNURONAN C-5-EPIMERASE

Abstract: DNA compounds encompassing sequences coding for enzymes having mannuronan C-5-epimerase activity are disclosed and a process for the preparation of such enzymes. The genetic sequences and enzymes prepared may be used in the production of alginates having a definite G/M ratio and block structure. Alginates having a definite G/M ratio may also be produced by selective inactivation of the genetic sequences.

Description: DNA COMPOUNDS COMPRISING SEQUENCES ENCODING MANNURONAN C-5-EPIMERASE The present invention concerns DNA compounds encompassing sequences coding for enzymes having mannuronan C-5-epimerase activity, a process for the preparation of such enzymes, the use of said genetic sequences in production of alginates having a definite G/M ratio and block structure, and the production of alginates having a definite G/M ratio by inactivating said genetic sequences. Throughout this application, reference is made to publications from the scientific and patent literature. Publications so cited are hereby incorporated in their entirety by reference. In this application the term gene is used to indicate a genetic sequence which encodes a protein, independent of whether the protein encoded by this genetic sequence is expressed or not in the natural host organism under those conditions. Alginates are a family of polysaccharides, which are synthesized in brown algae as well as in bacteria, such as Azotobacter vinelandii and Azotobacter chroococcum. Alginates are also synthesized by some strains of Pseudomonas sp. Chemically, alginates are unbranched binary copolymers of 1-4 linked β-D-mannuronic acid, termed M, and its C-5 epimer α-L-guluronic acid, termed G. Alginates derived from seaweeds and Azotobacter are generally true block copolymers where the monomers are arranged in homopoly eric stretches of M, termed M blocks, and homopolymeric stretches of G, termed G blocks, interspaced with regions containing both monomers, normally termed alternating blocks or MG blocks. The composition and sequential structure of alginates vary widely depending on the source. Alginates produced by Pseudomonas. however, do not have any G blocks. Several functional properties such as the capacity to form gels and the binding of water depend on the M/G ratio and on the length of the various blocks. A relatively high content of G blocks, for instance gives good gelling properties, due to ionic cross linking of chains which takes place when Ca 2+ -ions are added to an alginate solution. The composition and block structure also influence on the immunological properties of alginates. [Otterlei et al, J.of Immunotherapy 10, 286-291, (1991)] have shown that alginates with a high content of mannuronic acid blocks are very potent nontoxic immunostimulants. At present industrial production of alginates rely exclusively on algal sources. The range in composition is however limited as the highest content of guluronic acid to be found is 75% and the lowest 25%. Furthermore there are no suitable sources for alginate with a G content in the range of 42-54%. In the field of biotechnology or biomedicine, polymers with extreme compositions, such as a high G for immobilization of cells, [Martinsen A. , Skjak- Braek G. and Smidsrød O. , Biotechnol. Bioeng. 33, 79-86, (1989)] and a high M (90-100%) as immunostimulants [Otterlei et al, J.of Immunotherapy 10, 286-291, (1991)] are of major interest. The key enzyme responsible for generation of the G blocks is called mannuronan C-5-epimerase. It was previously thought that only one enzyme having a certain amino acid sequence would exhibit this activity. It has now surprisingly been found that there exist at least five genes encoding enzymes having this activity. Some of these enzymes differ in molecular weight and amino acid sequence. The genes were found adjacent to each other in the bacterium Azotobacter vinelandii. It has also been found that the amino acid sequence of the enzyme affects the activity of the enzyme, not only in terms of potency but also in the type of alginate formed, for example, altering the content of guluronic acid and the single/block G content of the alginate. In [Larsen, B. and Haug, A., Carbohydr. Res. 17, (1971) , 287-296 and 297-308] the isolation of mannuronan C-5-epimerase from liquid cultures of Azotobacter vinelandii is reported. In the following, this epimerase will be termed mannuronan C-5-epimerase (2) , and the DNA sequence encoding for it will correspondingly be denominated E2. In [Skjak-Braek, G and Larsen, B, Carbohydr. Res. 103:133- 136, (1982)], the purification of mannuronan C-5-epimerase (2) by affinity chromatography on alginate sepharose is disclosed. In a separate paper [Skjak-Braek, G and Larsen, B., Carbohydrate Research, 139, (1985) 273-283] the characterization of this enzyme is disclosed. Further, the activity of the enzyme is described as an ability to epimerize both bacterial and algal alginate having a wide range in monomer composition and sequence of units. From PCT/ O 86/03781 and Japanese Patent Application J63233797 it is known to produce alginic acid and/or alginate having a high content of guluronic acid by action of the enzyme (E2) on an alginic acid or alginate, whereby the G content increases. In [Chitnis, C.E. and Ohman, D.E., J. Bacteriol., 172, p2894-2900, (1990)] the gene sequences involved in the introduction of guluronic acid into exopolysaccharides from Pseudomonas aeru inosa have been reported. However, the nature of the enzyme responsible for this process has not been identified. Since this genus of bacteria is unable to produce alginate containing G blocks [Skjak-Braek, G. , Larsen, B. and Grasdalen, H. Carbohydr. Res. 54 (1986) 169- 174] it is believed that the epimerization system in alginate producing Pseudomonas is fundamentally different from the epimerase in brown algae and in Azotobacter vinelandii. It seems likely that the Pseudomonas enzyme is a monomer epimerase acting at the sugar nucleotide level, and as such is unable to introduce G-blocks into already polymerized alginates. Production of mannuronan C-5-epimerase from Azotobacter vinelandii culture is difficult due to a very low yield. It is also a major obstacle that the enzyme is secreted together with copious amounts of highly viscous alginate which hampers the purification of the enzyme. Although alginates are secreted by some bacteria, an industrial production based on these microorganisms has not been successful. The main reasons are due to the difficulties in controlling the composition and molecular size of the exopolysaccharides. The content of guluronic acid blocks in the alginate from Azotobacter vinelandii tends to be too low for making a polymer with good gelling properties. Alginates with a high M content having immunogenic properties as reported above, are produced by Pseudomonas aeru inosa, but this organism is unattractive from a production point of view, as it is unstable in the production of the polymer. Further, the organism is known to be a secondary pathogen in patients suffering from cystic fibrosis. Thus, in order to produce medical grade alginates with a defined monomer composition and sequential structure there is a need for improved methods for controlling the biosynthesis of alginate, through controlling the key enzyme, the mannuronan C-5-epimerase. The present invention is directed to cloned DNA fragments encoding mannuronan C-5-epimerase. The invention is encompassed by vectors which contain DNA fragments encoding mannuronan C-5-epimerase linked to DNA elements which direct the expression of mannuronan C-5-epimerase from the cloned DNA encoding the protein. The invention also provides for microorganisms which express the mannuronan C-5-epimerase protein from the cloned DNA as a source of the purified protein and also as a source of alginates of altered composition. Strains in which the expression level of the mannuronan C-5-epimerase gene is altered or in which one, several or all of the mannuronan C-5-epimerase genes have been inactivated are also within the scope of the present invention. The invention further encompasses methods for producing alginates either very efficiently, or having altered composition, or both, by culturing microorganisms having altered levels of expression of a mannuronan C-5-epimerase gene. The invention further features selection of epimerase to achieve a desired level of guluronic acid, and alter the single/block G characteristics of the enzyme. In a further embodiment, the invention features the production of synthetic proteins and DNA encoding such proteins which have mannuronan C-5-epimerase activity. Brief Description of Figures Figure 1 shows the amino acid sequence of the N-terminal end of the 122 kd protein, and the nucleotide sequence of the corresponding oligonucleotide. The DNA probe was synthesized as a mixture (in equal ratios) of the 64 possible combinations that could be deduced from the first seven amino acids in the sequence of the 122 kd protein. N indicates that all four bases were used at this position. Figure 2 is a restriction endonuclase map of the combined inserts in plasmids pHE14, pHE16, pBDl, pHElδ and pMLl. The numbers at the bottom line indicate the molecular sizes in bp. The arrow indicates the localization and orientation of the sequence homologous to the synthetic oligonucleotide used for screening the library. The five genes (open reading frames) found by sequencing are marked by boxes and denoted E4, El, E2, E3 and E5. El corresponds to Epimerase I. Figure 3 shows Mannuronan C-5-epimerase (1) activity of a portion of the El encoded protein as a function of cell growth. *: OD 600 of cell culture, o: Epimerase activity given as dpm/ml of cell culture. The strain used in this experiment was DH5α(pHE5), and the extracts were incubated with the substrate for 23 hours. Figure 4 shows the kinetics of 3 H release. The enzyme activity was assayed by using an extract prepared from IPTG-induced JM105 cells containing pHE5 (see legend to Table 3) . Figure 5 shows the homologies between and within the different genes. Boxes with the same letter are homologous to each other. Gaps are introduced to optimize the alignment. E1-E4 are defined as appears from Figures 2 and 6. Figure 6 shows the nucleotide sequences and corresponding amino acid sequences for E4, El, E2 and part of E3. Figure 7 shows the alignment of the DNA sequences of the A blocks from E4, El, E2 and E3. Figure 8 shows the alignment of the deduced amino acid sequences of the A blocks from E4, El, E2 and E3. Figure 9 shows the alignment of the DNA sequences of the R blocks from E4, El, E2 and E3. Figure 10 shows the alignment of the deduced amino acid sequences from the R blocks of E4, El, E2 and E3. Figure 11 shows 1 H-NMR spectra of alginate epimerized by extracts from A: DH5α(pHE8) (truncated epimerase 1); B: JM109(pBD9) ; C: no extract. The peak to the left gives the signal from G-l; the peak in the centre gives the combined signal from GM-5 and M-1 and the peak to the right gives the signal from GG-5. Figure 12 shows the nucleotide sequence and corresponding amino acid sequence of E2. Now according to the present invention genetic sequences have been found which encode enzymes having mannuronan C-5- epimerase activity, and thus the first aspect of the invention is pure isolated DNA comprising nucleotide sequences encoding mannuronan C-5-epimerase activity. The sequence of amino acids proximal to the amino terminus of purified mannuronan C-5-epimerase protein was determined [G. Skjak Braek et al. , Carbohydr. Res. 103:133-136 (1982)]. This data was used to derive a sequence for an oligonucleotide probe which was used to screen a gene library of Azotobacter vinelandii DNA. One result of this screening experiment was the surprising discovery of a second gene and thereafter three further genes including at least one genetic block A were found. Thus, altogether there appear to be at least five different genes encoding proteins having mannuronan C-5-epimerase activity. Accordingly, it is a second object of the present invention to provide for alternative DNA sequences encoding mannuronan C-5-epimerase. Three different blocks of genetic sequences, designated A, R and S, are found in the genes. These genetic blocks are most commonly found in combinations wherein the A appears one time or two times, the R block appears from 0 to at least 5 times and the S block appears from 0-1 time. There is a high degree of consensus in the nucleotide sequences of each block for the different genes (1-5) . Accordingly, it is a third object of the present invention to provide for DNA sequences encoding mannuronan C-5- epimerase and comprising the DNA blocks A and/or S and/or R, wherein A may appear more than once and R if present may appear singly or in repeats of up to at least 5 or 6 times. The consecutive order of the three blocks if all three blocks are present, is preferably A, R and S. However, it has been shown that a reversed consecutive order, wherein for instance R appears before A also gives a gene encoding a mannuronan C-5-epimerase. Thus, the invention further encompasses genetic sequences having any order and any number of the blocks A, R and S. Another aspect of the present invention concerns the use of said genetic sequences for the preparation of the mannuronan C-5-epimerase in recombinant host cells. It is especially preferred to insert the gene into hosts such as bacteria, for instance Escherichia coli or Bacillus subtilis or in yeast. The cloning and expression of the genetic sequence as described above in E. coli is described in the Examples. The present invention also encompasses recombinant expression plasmids that can be used to produce the mannuronan C-5-epimerase proteins in a host microorganism. Such expression plasmids are made by inserting a DNA fragment encoding mannuronan C-5-epimerase into a vector which contains appropriate expression elements, such as (but not limited to) a promoter, ribosome binding site, translational initiation site and transcription terminator. The expression plasmids can be adapted for transformation into many different commonly used host organisms in which it might be desired to produce the mannuronan C-5- epimerase. The techniques for insertion of foreign genes into commonly employed hosts are known in the art, as described for instance in [METHODS IN ENZYMOLOGY, Vol. 185, Gene Expression Technology, Ed. D.V.Goeddel, Academic Press, Inc. (1990) ] . Further by choice of a broad host range vector and a suitable promoter as known in the art, and described for instance in [J.L.Ramos et al, FEBS Letters, Vol. 226, 2, 241-246] it will be possible to insert and express the mannuronan C-5-epimerase genetic sequences in many different hosts. This will make possible the production of large quantities of one or all of the pure enzymes having this activity, while avoiding the problems of separating the enzymes from the alginate. By inserting a high copy-number vector comprising the genetic sequences encoding the epimerase into a natural alginate producing bacterium such as Azotobacter vinelandii an enhanced production of the enzymes would be possible. Over expression of the epimerases in a natural host could also be achieved by using a promoter which drives high- level expression of the enzymes. By blocking other genetic sequences coding for the alginate production, the production of pure enzymes may be achieved. Yet another aspect of the invention is the selective inactivation of the mannuronan C-5-epimerase genes in the natural host organism so as to provide for bacterial production of alginates having a low content of G blocks or even a pure poly-M alginate. This is accomplished by inserting nucleotides into one, several or all of the mannuronan c-5-epimerase genes in the natural host organism Azotobacter. It is especially preferred to insert a DNA fragment encoding a selectable marker gene, preferably a gene conferring antibiotic resistance. Insertion of a selectable marker allows selection of those bacteria in which the insertion has been successfully accomplished. By choosing different selectable markers, for example providing resistance to different antibiotics, it is possible to select recombinants that have incorporated inserted sequences into some or all of the mannuronan C-5- epimerase genes. Thus, selective production of bacterial strains in which one of the mannuronan C-5-epimerase genes, several or all of them have been inactivated is possible. A second method of inactivating all of the epimerase genes is to transform a cell of the natural host strain, Azotobacter with a vector which expresses an antisense RNA which specifically binds to RNA transcribed from these genes. Use of promoters of varying strength to> drive expression of the antisense RNA in the creation of the vectors used to transform the cells allows production of strains having varying ratios of G-blocks to M-blocks in the alginate produced. Use of inducible promoters to drive expression of the antisense RNA allows the creation of strains which can produce alginates of variable composition, depending on culture conditions. Clearly, if the recombinant host organism is the natural host, Azotobacter. it is possible to enhance production of one epimerase gene while leaving expression of the others at their normal level, thus producing a strain which makes an alginate having an altered ratio of G blocks to M blocks. A strain which makes alginate having 0-25% M blocks is preferred. Alternatively, all but one of the epimerase genes can be inactivated, as described above, and the expression of the remaining epimerase gene can be controlled by a regulated promoter. A strain carrying such a complement of epimerase genes would thus produce alginates having a high content of G-blocks, especially from 75-98%. Another means for making a strain for producing alginates having a high G-block content is to inactivate all but one of the mannuronan C-5- epimerase genes by insertion and control the remaining gene by antisense RNA, using an inducible promoter to regulate transcription of the antisense RNA gene. A still further means for making a strain for producing alginates having a high G-block content is by inactivating all naturally occurring genes and introducing a regulated gene through a vector. Thus the present invention also includes a process for the construction of a recombinant host cell capable of expressing mannuronan C-5-epimerase activity by transforming said host cell with a recombinant DNA expression vector that comprises: (a) a promoter and translational activating sequence that function in said host cell; and (b) a DNA sequence encoding mannuronan C-5- epimerase comprising at least a DNA block A and/or a DNA block S and/or a DNA block R, positioned for expression from said promoter and translational activity sequence. Also the present invention encompasses a process for the bacterial production of pure poly-M alginate or tailored alginates having a lower G block content, preferably in the range from 0-25%, by blocking the DNA sequences encoding the enzymes in a natural host by insertion of a foreign genetic sequence into one, several or all genetic sequences encoding mannuronan C-5-epimerase. Other methods for achieving the same end will be known for persons skilled in the art and are hereby included into the scope of the present invention. A further aspect of the invention are the novel enzymes having mannuronan C-5-epimerase activity. The amino acid sequences and their degree of homology will appear from Figures 6-11. Also as known by a person skilled in the art, variations in the nucleotide sequence which nevertheless encode proteins > having the same activity as the wild-type mannuronan C-5- epimerase are encompassed within this invention. Variations within the amino acid sequence may also encompass deletions, substitutions and additions which do not substantially change the biological activity. Also, it is possible to make a synthetic DNA sequence encodinq a mannuronan C-5-epimerase by techniques well known in the art. See for instance, [Itakura et al.. Science 198:1056 (1977)] and [Crea et al. (Proc. Natl. Acad. Sci. USA 75:5765 (1978)] and also U.S. Patents 4,800,159 and 4,683,202 and also published European patent application EP-A-0258017. Synthetic enzymes may be made by incorporating different combinations of the A, R and S elements, to maintain epimerase activity. The resultant alginate composition can be varied by enzyme selection. Materials and General Methods Bacterial strains, plasmids, and phage. Strains, plasmids, and phages are listed in Table 1. The bacterial strain of A. vinelandii used in these experiments, is freely available from Bjørn Larsen, Inst. of Biotechnology, Lab. for Marine Biochemistry, 7034 Trondheim - NTH, Norway or Svein Valla, Unigen, Center for Molecular Biology, University of Trondheim, 7005 Trondheim, Norway and has been deposited at the Belgian Coordinated Collections of Microorganisms (BCCM) at the Laboratorium voor Microbiologie (LMG) at Universiteit Gent (RUG), K.L. Ledeganckstraat 35, B-9000 Gent. Other strains of A.vinelandii mentioned in Example 9 have the following ATCC numbers: ATCC 478, ATCC 12837 and ATCC 12518. Plasmids/ strains DH5α(pHE14), JM109(pHE16) , JM109(pBDl), JM109(pHE18) and SURE™(pMLl) have been deposited at BCCM at the Laboratoriu voor Moleculaire Bioiogie (LMBP) (same address as LMG) and have the following numbers Growth of bacteria and phages. A. vinelandii was grown at 30°C with shaking in a nitrogen-free medium (9.8 mM K 2 HP0 4 /KH 2 P0 4 , 0.8 mM MgS0 4 7H 2 0, 3.4 mM NaCl, 0.34 mM CaCl 2 , 8.7 μM Na 2 Mo0 4 2H 2 0, 54 μM FeS0 4 7H 2 0, 1% sorbitol, pH 7.4). E. coli was grown in LB-medium [Sambrook J, Fritsch, E.F. and Maniatis T., Molecular cloning, A laboratory manual, 2nd ed. , Cold Spring Harbour Laboratory Press, New York, (1989) ] with shaking at 37°C. When the cells were to be used for growth of phages the LB-medium was supplemented with 2.5 mM CaCl 2 , 10 mM MgCl 2 , and 0.4% maltose. Phages were plated on strain Q359 on L-agar (LB-medium supplemented with 2% agar) . Phage LB-medium supplemented with either 0.8% agar (titrations and gene library amplification) or 0.8% agarose (screening of gene library and preparation of phage lysates) was used for overlaying agar. Standard recombinant DNA technology. Restriction endonuclease digestions, removal of cohesive DNA ends by using the 3' exonuclease activity of T4 DNA polymerase, ligations, agarose gel electrophoresis, and end-labelling with 32 P were performed according to standard protocols [Sambrook J, Fritsch, E.F. and Maniatis T ., Molecular cloning, A laboratory manual, 2nd ed. , Cold Spring Harbour Laboratory Press, New York, (1989)]. Transformations were performed as described by [Chung, C.T. , Nie ela S.L. and Miller R.H. , Proc. Natl. Acad. Sci USA, 86, 2172-2175, (1989)], and DNA sequencing was performed according to [Sanger F. , Nicklen S., and Coulsom, . A.R., Proc. Natl. Acad . Sci USA, 74 , 4563 ( 1977 ) ] . Viscosimetric measurement. The alginate used in this experiment was obtained from Ascophyllum nodosum and had an intrinsic viscosity in 0.1 M NaCl of 17.6 dl/g at 25°C. The viscosity was determined by an Ubbelhode viscosimeter. NMR spectroscopy. The substrate used in these analyses was a low guluronic acid-containing alginate obtained from the brown algae Ascophyllum nodosum. and was prepared as described previously [Larsen, B. , Proceedings of the Tenth International Seaweed Symposium, Ed: Levring, T.Gothenburg, p7-33, (1980)]. For the NMR analyses epimerase was obtained from IPTG-induced E. coli JM105 cells containing pHE5. 250 ml cell culture were harvested by centrifugation and resuspended in 20 ml of 10 mM Tris, 0.34 mM CaCl 2 , pH 7.0. After ultrasonication, the solution was centrifuged at 31.000 x g for 1 hour. The supernatant was stored frozen at 70°C. After thawing the supernatant was filtered through a membrane with pore size 0.22 μm, and the enzyme was further purified on a Mono Q HR515 (Pharmacia) ion exchange column. The enzyme was eluted with a 0-1 M NaCl salt gradient (in the same buffer as the applied solution) , and was collected in 2 ml at approximately 0.6 M NaCl. To each of two tubes was added 0.28 ml of this enzyme solution (0.9 mg/ml total protein), 1 ml alginate (7.5 mg/ml in H 2 0) , and 4.62 ml 2,3,6-trimethylpyridine buffer (see above). CaCl 2 was then added to a total reaction volume of 6 ml such that one tube contained 0.85 mM, and one contained 3.4 mM CaCl 2 . After incubation at 30°C for 20 hours Na 2 EDTA (10 mM) was added to chelate the Ca 2+ -ions, and the solutions were then dialyzed extensively against distilled water. The dialyzed alginate solutions were freeze-dried and then dissolved in D 2 0. NMR spectroscopy of these solutions were finally performed according to [Grasdalen H. , Larsen B. , and Smidsrød 0., Carbohydr. Res., 68, 23-31 (1979)] (Table 4). Further analysis was carried out in a similar fashion for DH5 (pHE8), JM109(pHE16) and JM109(pBD9). The results in Table 4 conclusively demonstrate that the enzymatic activity is mannuronan C-5-epimerase activity. This activity is expressed from a number of the plasmids showing that an entire epimerase gene/protein is not required in order to maintain epimerase activity. The epimerase activity is Ca 2+ dependent. Example 1 Purification of mannuronan C-5-epimerase (1) , partial amino acid sequencing and synthesis of a mixed DNA probe. The enzyme was isolated from liquid cultures of A. vinelandii essentially as described in [Skjak-Braek, G. and Larsen, B. Carbohydrate Research, 103, (1982) 137-140]. The cells were removed by centrifugation and the enzyme was isolated by precipitation with 30% ammoniumsulphate and followed by centrifugation for 20 min. at 10000 rpm. The supernatant was then precipitated with 50% ammonium sulphate .(final concentration) , and the precipitate after centrifugation was dissolved in 0.05 M imidazole/HCl (pH 6.8) containing 0.34 mM CaCl 2 and 0.5 mM dithiothreitol. This crude extract was then desalted on a prepacked column (PD-10) of Sephadex G-25 (Pharmacia) equilibrated with the same buffer. The extract was then applied on an alginate-Sepharose column. Proteins bound by non-specific interactions were eluted with 0.1 M NaCl. The epimerase was eluted as a sharp peak with 0.5 M NaCl.To make the enzyme pure enough for protein sequencing, it was dialyzed against TE-buffer overnight, freezedried, and further purified by SDS-PAGE gel electrophoresis (7.5% polyacrylamide in 25 mM Tris, 192 mM glycine, 0.1% sodium dodecyl sulphate, pH 8.3.) followed by electroblotting (in electrophoresis buffer without sodium dodecyl sulphate) onto a polyvinylidene difluoride membrane, poresize 0.45 μm (Millipore) . The membrane was stained with Coomassie brilliant blue and air dried, and the protein with Mw 122 kd was cut out for N-terminal sequencing on a model 477A protein sequencing apparatus from Applied Biosystems. A DNA oligonucleotide was synthesized on the basis of the amino acid sequence information, and this oligonucleotide was used as a probe for screening of the gene library after end-labelling with 32 P by polynucleotide kinase. Example 2 Isolation of DNA from A. vinelandii and construction of a gene library. A. vinelandii cells were harvested and washed once in 0.9% NaCl. They were then lysed according to [Hansen, J.B. and Olsen, R.H. , J. Bacteriol., 135, 227-238, (1978)], and the lysate was extracted twice with phenol and twice with chloroform. Nucleic acids were precipitated with ethanol, and the DNA was collected on a glass rod and dissolved in TE-buffer (lOmM Tris, lmM Na 2 EDTA, pH 7.9). Further purification was obtained by CsCl/ethidium bromide density gradient centrifugation. After removal of the ethidium bromide by isopropanol extraction, the DNA solution was dialyzed against TE buffer . The DNA (molecular size greater than 60 kb) was subjected to partial Sau3AI digestion under conditions maximizing the generation of 15-20 kb fragments. After ethanol precipitation the DNA was dissolved in 40 μl TE buffer to give a concentration of 0.5 μg/μl. The DNA was then dephosphorylated with calf intestine phosphatase, followed by inactivation of the enzyme by incubation at 75°C for 10 minutes in the presence of 10 mN nitrilotriacetic acid. The dephosphorylated DNA was precipitated with ethanol and dissolved in 40 μl 0.1 x TE buffer. EMBL3 vector DNA was digested with BamHI + EcoRI, followed by an isopropanol precipitation step under conditions leaving the short BamHI/EcoRI oligonucleotides in solution [Frischauf A., Lehrach H. , Poustka A. and Murray N., J. Mol. Biol., 170, 827-842, (1983)]. The Sau3AI-digested and dephosphorylated A. vinelandii DNA (1.75 μg) was then ligated with the BamHI/EcoRI-digested vector DNA (4.75 μg) , using T4 DNA ligase in a total reaction volume of 20 μl. After ligations over night at 10°C, 10 μl of the ligation mixture was subjected to in vitro packaging in a Promega Biotech packaging system. The in vitro constructed phage particles were titrated on the E. coli strain Q359, and the library was finally amplified on Q359 in one cycle by plating on solid medium. Screening of the library was performed according to standard protocols [Sambrook, J. , Fritsch, E.F. and Maniatis, T. , Molecular Cloning, Laboratory Manual, 2nd Ed., Cold Spring Harbour Laboratory Press, (1989)], except that the highest stringency wash was 3.2 M tetramethylammoniumchloride at 50°C. A total of 1.4 x 10 5 primary recombinant phages were constructed, a library complexity far above what is required for obtaining representativity of A.vinelandii genes. Example 3 Measurements of epimerase activity from mannuronan C-5- epimerase (1) . (5- 3 H) alginate was prepared as described in [S jak-Braek, G. and Larsen, B. , Carbohydrate Res., 103, 133-136, (1982) ] . The (5- 3 H)alginate was produced by growing Azotobacter vinelandii in a medium consisting of D-Glucose (20g) , K 2 HP0 4 (lg), MgS0 4 .7 H 2 0 (200mg) , FeS0 4 . 7 H 2 0 (50mg) , NaMo0 4 . 2 H 2 0 (5mg) , NH 4 0Ac (2.3g) and CaCl 2 . 2 H 2 0 (50mg) diluted to one litre with water. The cells were grown at 30°C with vigorous shaking. After 30 hours, D-[5- 3 H]glucose was added to a concentration of 0.6mg/ml (Specific activity, 0.7 μCi/mg) and the cells were allowed to grow for another 72 hours. The culture was cooled in an ice-bath, and the cells were removed by centrifugation. The supernatant solution was dialysed against 0.05M sodium EDTA (3x5 litres) for 24 hours followed by exhaustive dialysis against distilled water. The sodium alginate was then precipitated with ethanol in the presence of 0.2% of sodium chloride. The specific activity of the label was 29 000 dpm/mg alginate. The composition of this alginate was also analyzed by NMR spectroscopy, and it was found to contain 59% mannuronic acid. Phage lysates were prepared by plating 105 phages per plate. Two ml 2,3,6-trimethylpyridine buffer (50 mM, pH 6.9) were added to each plate, and the softagarose/buffer mixture was scraped off, vortexed and centrifuged at 10 000 rpm for 10 min. The supernatant was used for incubations with (5- 3 H)alginate by mixing 0.25 ml (5- 3 H)alginate (2.5 mg/ml), 6,3 μl 0.1 M CaCl 2 , and 1.45 ml phage lysate. The mixture was incubated at 30°C overnight, and the alginate was precipitated by addition of 15 μl 5 M NaCl and 2 ml ethanol. After incubation at -20°C for 30 min. the solution was centrifuged at 10.000 rpm for 30 min. , and 1 ml of the supernatant was used for determination of released 3 H [Skjak-Braek, G. and Larsen, B., Carbohydrate Res., 103, 133-136, (1982)]. in a liquid scintillation counter. For measurements of epimerase activity as release of 3 H in cells containing recombinant plasmids, the cell cultures were harvested by centrifugation and resuspended in 2,3,6 tri ethylpyridine buffer. When IPTG (3 mM) was used for induction of the lac-promoter, the inducer was added to exponentially growing cells and incubations were continued for 3 hours. Cells were disrupted by ultra-sonication, and varying amounts of the lysates were incubated with shaking together with 100 μl (5- 3 H) alginate (2.5 mg/ml) and 400 μl 2,3,6-trimethylpyridine buffer (total volume 0.6 ml) in the presence of 3.3 mM CaCl 2 . The quantities of enzyme- containing cell extracts used were adjusted such that the measurements were performed under conditions where the enzyme represented the limiting factor. After incubation at 30°C at the times indicated in each case, the mixtures were precipitated with ethanol under the conditions described above for phage lysates and 1.0 ml of the supernatant was used for scintillation counting. Controls (using the appropriate host with the pUClδ vector) gave low backgrounds and these numbers were subtracted in the values presented in Table 3. Example 4 Molecular cloning of a DNA fragment expressing a mannuronan C-5-epimerase activity in E. coli. The A.vinelandii gene library was constructed by cloning partially Sau3AI- digested A.vinelandii DNA into the bacteriophage λ vector EMBL3. In order to identify the epimerase gene in this library, we constructed a DNA probe based on the assumption that the previously purified 122 kd protein represented the epimerase [Skjak-Braek, G. and Larsen, B. , Carbohydr. Res., 103, 137-149 (1982)]. Initially we tried to use the corresponding protein solution for determination of the N- terminal amino acid sequence of the 122 kd protein, but the results showed that the preparation was not sufficiently pure for this purpose. We therefore purified the protein further by SDS-polyacrylamide gel-electrophoresis, followed by electroblotting onto a membrane. The band containing the 122 kd protein was cut out from this membrane and subjected to N-terminal amino acid sequence analysis. Based on parts of this sequence, we synthesized the mixed DNA probe shown in Figure 1. The DNA probe synthesized as in Example 1 was labelled with 32 P and then used for screening of the A. vinelandii gene library. Clones which hybridized reproducibly against the labelled probe were identified at a frequency of approximately 10 -3 , and six such clones were selected for further studies. Phage lysates were prepared from each of the six clones, and each lysate was assayed for epimerase activity (Table 2) . As can be seen, the lysates prepared from all six clones appeared to contain a weak enzyme activity that could represent the epimerase. This conclusion was further supported by the observation that control lysates prepared from randomly picked recombinant phages in the library, gave reproducibly lower activity, representing the background activity. Example 5 Subcloning of a DNA fragments encoding the epimerase. DNA from phage EP2 was partially digested with Sau3AI, and fragments ranging from 4 to 9 kb in size were subcloned in the BamHI site of plasmid pUC18. Cell extracts from DH5α transformants containing recombinant plasmids were assayed for epimerase activity, and the corresponding plasmids were also hybridized against the synthetic oligonucleotide used for screening of the gene library. The analysis of the cell extracts showed that one of them contained an enzymatic activity consistent with the assumption that a polypeptide having epimerase activity was expressed from the plasmid (pHEl) in this clone (see Table 3) . We have also tried to centrifuge the extract at 30000 g for 3.5 hours, and. found that the activity was not significantly reduced in the supernatant. Since we were unable to detect any significant activity in the culture medium, we conclude that the epimerase is localized intracellularly in E.coli. The insert in pHEl also hybridized against the synthetic oligonucleotide used for screening, and pHEl was therefore selected for further analysis. Example 6 Characterisation of the cloned DNA required for expression of the epimerase, and stability of the enzyme in vivo and in vitro. The insert in pHEl is approximately 4 kb in size, and Figure 2 shows the restriction map of this insert. Hybridization analysis of pHEl with the original synthetic oligonucleotide showed that the sequence hybridizing to the oligonucleotide was localized downstream of the SphI site. The hybridizing sequence was further characterized by DNA sequencing, and this analysis showed that one of the potential reading frames of the sequence was in 100% agreement with the original N-terminal amino acid sequence of the 122 kd protein. Surprisingly, however, the orientation of the sequence was such that it would be transcribed out of the cloned fragment (see Figure 2) . This result thus indicated that the observed epimerase activity was not correlated with the sequence encoding the 122 kd protein, a conclusion that was further confirmed by the observation that the terminal 0.5 kb SphI fragment could be deleted (generating plasmid pHE7) without loss of the epimerase activity from the corresponding cell extract. In addition to the SphI deletion, we deleted (from pHE7) the 0.7 kb Kpnl fragment at the opposite terminus of the insert, generating plasmid pHE5. As shown in Table 3, pHE5 (in DH5α) expressed the epimerase at a level approximately 27 times higher than the level of expression from pHEl. During the expression studies described above we found that the measurements were quantitatively difficult to reproduce unless the time of harvesting the cells were kept as constant as possible. We have analyzed this problem more carefully by measuring the enzyme activity at different stages of growth of the E. coli cells. The results of such an analysis are shown in Figure 3, and indicate that the enzymatic activities in the cell extracts are drastically reduced shortly after the cells have entered the stationary phase. To obtain optimal enzyme yields it is therefore important to harvest the cells at the end of the exponential phase or at the beginning of the stationary phase. The reason for the reduction of epimerase activity might potentially be due to proteolysis of the epimerase in stationary phase cells. To study the stability of the enzyme in vitro we have also analyzed the kinetics of 3 H release in the DH5 (pHE5) extracts. As can be seen from Figure 4, the enzyme activity is linear over at least 30 hours, demonstrating that the enzyme is very stable in vitro. A critical parameter for obtaining reproducible results is thus the time of harvesting of the cells. Example 7 Stimulation of the epimerase activity by induction of the lac promoter. The results described above showed that the levels of expression of the epimerase from pHE5 was significantly higher than in pHEl. The reasons for this could potentially be that the lac promoter was important for the expression, and we have therefore analysed this problem more closely. The analyses were performed in the E. coli strain JM105, a strain which expresses high levels of lac repressor, thus allowing a more repressed state of the promoter under uninduced conditions. When cell extracts prepared from uninduced and induced (with IPTG) cells of JM105(pHEl), a significant stimulation of enzyme activity was observed in the induced cells (Table 3) . A similar experiment using JM105(pHE5) showed even greater stimulation of the expression of the epimerase upon addition of IPTG in this case. These experiments thus showed that the lac promoter probably is a key element, although not necessarily the only element, involved in the expression of the epimerase from pHE5. The experiments in addition showed that the direction of transcription is from the Kpnl towards the SphI site in the insert. The epimerase gene is therefore transcribed in the same direction as the gene encoding the 122 kd protein, whose N-terminal amino acid sequence was used for the isolation of the cloned DNA. Preliminary experiments on deleting more DNA from the SphI side of the insert indicated that very little could be deleted without loss of the epimerase activity. At the Kpnl side, on the other hand, we found that significant deletions were tolerated. Table 3 shows the results of analysis of expression of the epimerase from a plasmid (pHE8) constructed by deleting the 0.8 kb KpnI/SacII fragment from pHE5. As can be seen, this deletion resulted in a very strong stimulation of the epimerase activity both in uninduced and induced cells. The expression from pHE8 is presumably based on initiation of translation from the Shine-Dalgarno sequence in the vector (localized between the lac promoter and the polylinker) . Similarly, high levels of expression were obtained from pHE22 also due to the coding sequences being in frame with the Shine-Dalgano sequence. So far we have not obtained expression of the epimerase in constructs where deletions have extended beyond the SacII site. Example 8 Use of a different promotor than the lac promotor. The insert in pHE5 (EcoRI-Hindlll) was sub-cloned into plasmid pT7-3 (a derivative of pT7-l described by [Tabor, S., and C.C. Richardson (1985). Proc. Natl. Acad. Sci. 82, 1074- 1078]) , and the new plasmid was designated pLBl. The insert in pLBl is localized downstream of the φlO promoter in the vector. This promoter is only recognized by the bacteriophage T7 RNA polymerase, and expression of genes downstream of this promoter thereby becomes dependent on expression of this polymerase activity in the cells. The 442 bp (see Figure 2) Sad- Spol fragment was finally deleted from the insert in pLBl, generating plasmid pLB2. pLB2 was transformed into E. coli K38 (pGPl-2) . Plasmid pGPl-2 encodes the gene for T7 RNA polymerase, and the expression of the gene is controlled by a temperature inducible repressor. K38(pLBl, pGPl-2) was grown in exponential phase at 30°C for 4 1/2 hours. One of two parallel cell cultures was then transferred to 42°C for 30 minutes to induce the T7-polymerase. The other parallel cell culture was grown at 3C°C for 5 hours. The epimerase activities in the cells were measured as described in example 3, and the results of the measurements are shown in Table 3. Example 9 Cloning of mannuronan C-5-epimerase (2) . Plasmid pHE12 was constructed by inserting a 6.2 kb Xhol fragment from the recombinant bacteriophage lambda derivative EP2 into pUC128. As can be seen from Figure 2 the insert in pHE12 is partly overlapping with the insert in pHEl. Analysis of extracts prepared from cells containing pHE12 (as described for pHEl) , showed that they expressed mannuronan C-5- epimerase activity (Table 3) . Further analysis showed that the 2.5 kb Spol-Xhol fragment could be deleted from the insert in pHE12 without affecting the expression of mannuronan C-5-epimerase. Further plasmids were constructed (see Figure 2) and the activity analysed (see Table 3) . This demonstrated that both the genes and gene fragments were able to express epimerase activity. The nucleotide sequences of the inserts were determined by the method of Sanger [Sanger,F., S. Nicklen, and A.R. Coulson. 1977. Proc. Natl. Acad. Sci. 74, 5436]. The nucleotide sequences are shown in Figure 6. Example 10 Sequence Comparison. Five genes have been identified as shown in Figure 2. The insert containing E5 is located about 5-10 kilobases away from the other genes. Figure 6 shows the nucleotide sequence for the complete genes of E4, El, E2 and a large portion of E3. Detailed analysis of the nucleotide and amino acid sequences revealed highly homologous regions within each gene and between the various genes. Figure 5 characterises each of the genes by reference to the homologous blocks. Each of the genes has at least one A-element and at least one R-element. El, E2 and E4 all end with a reasonably homologous sequence termed the S-element (not shown in Figure 5) . The last 14 amino acids of the S-element of El and E2 are identical with one exception. Figures 7-10 show detailed analysis of the A- and R- elements within each gene by reference to the consensus sequence (con). Each A-element is approximately 1,150 base pairs long and each R-element is approximately 450 base pairs long. Short oligonucleotides are present in El, E2 and E3 between the second and third R-elements. Gaps have been introduced where necessary to maximise alignment (see in particular the third R-element of E2) . Hybridization with a probe made from the first part of the A-element to a Southern blot of A. vinelandii digested with restriction endonuclease Bglll gave 5 distinct bands. One of these bands contained two A blocks, and another of these bands contained two different fragments with the same size. The number of bands were the same when other strains (ATCC 478, ATCC 12837 and ATCC 12518) of the same species were used. This implies that the bacterium contains at least 5 copies of the A-element, and that this is common to several independently isolated strains of A. vinelandii. The first part of each R-element contains six perfect and imperfect repeats of a nonapeptide with the consensus sequence LXGGAGXDX, except for the third R-element of E2 which lacks two of these repeats. Figure 12 shows the complete nucleotide and corresponding amino acid sequence of E2. The nonapeptides have been marked with double lines for a good match with the consensus sequence and single lines for less good matches. This nonapeptide motif is characteristic of the haemolysin family of secreted proteins (Suh, Y. and Benedik, M.J., J. Bacteriol 174, (1992) 2361-2366). These proteins are all calcium dependent and are secreted by a pathway which does not involve cleavage of an N terminal signal peptide. For haemolysin secreted from E.coli it has been proposed that the nonamers are responsible for the binding of calcium ions (Ludwig, A. et al, Mol. Gen. Genet. 214, (1988) 553- 561, Boehm, D.F. et al. Infect. Immun. 58 (1990) 1959- 1964) . It appears that the R-elements are involved in calcium ion binding, calcium being necessary for both enzyme activity and gel formation. Example 11 Making an altered epimerase. As can be seen from Table 3, various elements may be deleted from the gene, while maintaining the expression of a protein having epimerase activity. Clearly, the latter portion of El having the sequence ARS has epimerase activity (see plasmid pHE8) , although deletions in A2 of El are not tolerated (see Example 7) . Additionally, E2 having the sequence ARRRRS also demonstrates epimerase activity. Additionally, fragments of E3 lacking a carboxy terminal and having the sequences ARRR and ARRRARR have expressed epimerase activity. (See plasmids pH18 and pBD6) . Accordingly, it appears that an S-element is not essential for epimerase activity, although the presence of this element may affect activity. We therefore postulated that an epimerase may need at least one A-element and at least one R-element, and that it should be possible to make altered epimerases by combining these elements in different ways. To show this, we constructed a plasmid encoding an epimerase with the sequence RARS: The insert in pHEl (EcoRI-Hindlll) was subcloned into plasmid pTrc99A (Pharmacia), generating plasmid pHE21. This plasmid contains a trc-promoter in front of the epimerase I gene, a strong transcription termination signal downstream of the gene, and the lacl q -gene allowing induction with IPTG. pHE21 was digested with Kpnl and Spol, made blunt-ended with SI nuclase and religated. The resulting plasmid, pHE22, expresses a protein having the carboxy terminal of epimerase 1, RARS. The epimerase activity was measured as in Example 3, see Table 3. Given that epimerase activity is expressed from a number of the constructs, it seems likely that a number of synthetic enzymes may be produced having epimerase activity including differing numbers of A, R and S blocks. The presence of activity in pHE22 implies that it is not essential to have an amino-terminal A block, and so block order may also be altered. Example 12 The 1 H-NMR spectra of alginate epimerased by extracts from plasmids pHE8 and pBD9 show that the proteins encoded by these plasmids have different enzyme activity, pHE8 producing epimerase with single G activity while pBD9 producing epimerase with G block activity. pHE8 encodes the carboxy terminal ARS of El whereas pBD9 encodes ARRRRS of E2. The naturally encoded epimerases may therefore have differing activity particularly in the distribution patterns of Gs. The different activity of the various epimerases encoded within the 5 genes could be used to create alignates having a desired structure, by selectively expressing a desired gene or genes. Alternatively, it may be possible to construct synthetic enzymes varying the A, R and S block content of each epimerase to provide enzymes having altered activity providing a further level of control in the production of desired alginates. Table 1 -1 Bacterial strains , phages , and plasmids . Strain/phage/ plasmid Remarks References Bacterial strains A. vinelandii Strain E Larsen and Haug (1971) E. coli Q359 supE hsdR <£80 r P2 Karn et al. (1980) DH5α supE44ΔlacU169 (tf>80 lacZAM15) Bethesda Research hsdR17 recAl endAl qyrA96 Laboratories (1986) thi-1 relAl JM105 supE endA sbcB15 hsdR4 rpsL Yanisch-Perron thiA(lac-proAB) et al (1985) JM109 recAl supE44 endAl hsd 17 Yanisch-Perron gγrA96 relAl thiA(lac-proAB ) et al (1985) * F[traD36 proAB + lac!^lacZΔM151 SURE TM el4 ~ (mcrA), A(mcrCB-hsdSMR-mrr) Greener (1990) 171 endAl, supE44, thi-1, qyrA96, relAl, lac, recB, recJ, sbcC, umuC:Tn5( an r ) , uyrC, [F' proAB, lacI^ΔM15, TnlO, (tet r ) ] . Phages EMBL3 Bacteriophage λ vector used for Frischauf et al. construction of A. vinelandii (1983) gene library EPx Randomly picked phage from - vinelandii gene library See examples EP2, -3, -6, Phages isolated from A. -1 , -8 and -9 vinelandii gene library See examples Identified by hybridization and expresses mannuronan C-5-epimerase Plasmids pUCl8 Ampicillin resistance, ColEl Norrander et al. replicon pUC128 Ampicillin resistance, ColEl Keen et al. (1988) replicon pTrc99A Ampicillin resistance, ColEl Pharmacia replicon pT7-3 Ampicillin resistance, ColEl Tabor & Richardson replicon (1985) pGPl-2 Kanamycin resistance, P15A Tabor & Richardson replicon (1985) pBluescript II Ampicillin resistance, ColEl SK(+) replicon pHEl Derivative of pUC18 where a See examples 4 kb Sau3Al DNA fragment from phage EP2 was subcloned into the BamHI polylinker Table 1-2 pHE7 Derivative of pHEl where a 0.5 kb See examples SphI DNA fragment was deleted pHE5 Derivative of pHE7 where a 0.7 kb See examples Kpnl DNA fragment was deleted pHE8 Derivative of pHE5 where a 0.8 kb See examples Kpnl/Sac I DNA fragment was deleted. Cohesive ends were removed ;.rior to ligation by using the 3' exonuclease activity of T4 DNA polymerase pLBl Derivative of pT7-3, where the See examples 2,7 kb insert from pHE5 was cloned into the EcoRI/Hindlll polylinker pLB2 Derivative of pLBl, where a 0.4 kb See examples Sacl/Spol fragment was deleted pHE12 Derivative of pUC128, where a See examples 6.2 kb Xhol fragment from phage EP2 was subcloned into the Xhol polylinker pBDl Derivative of pHE12, where a See examples 2.0 kb Spol/Nsil fragment was deleted pHE21 Derivative of pTrc99A, where the See examples 4.0 kb insert of pHEl was cloned into the EcoRI/Hindlll polylinker pHE22 Derivative of pHE21, where a See examples 1.2 kb KpnI/Spol fragment was deleted pHE2 Derivative of pUC18 where a See examples 6.0 kb SphI DNA fragment from phage EP6 was cloned into the SphI site in the polylinker of the vector pHE16 Derivative of pHE2 where a 1.5 kb EcoRI-Smal DNA fragment See examples was deleted pBD9 Derivative of pBDl where a See examples 0.4 kb Xhol-Fspl DNA fragment was deleted pBD6 Derivative of pHE12 where a See examples 3.4 kb Xhol-Espl DNA fragment was deleted pHE18 Derivative of pUC128 where an See examples 5.1 kb Notl-PvuII DNA fragment from EP6 was cloned into the Notl-EcoRV sites in the polylinker of the ver. J ~ T pHE14 Derivative of pUC128 where a See examples 3.0 kb Bglll DNA fragment from EP6 was cloned into the BamHI site of the polylinker pMLl Derivative of pBluescript II See examples SK(+) where a 4.3 kb KpnI-SacII DNA fragment was cloned into the corresponding sites in the polylinker References to Table 1 Larsen, B., and A. Haug. 1971. Biosynthesis of alginate. Carbohydr. Res. 17:287-296. Karn, J. , S. Brenner., L. Barnett, and G. Cesareni. 1980. Proc. Natl. Acad. Sci. U.S.A. 77:5172. Bethesda Research Laboratories. 1986. Bethesda Res. Lab. Focus 8(2) :9. Yanisch-Perron, C. , J. Vieira, and J. Messing. 1985. Gene 33:103-119. Frischauf, A., H. Lehrach, A. Poustka, and N. Murray. 1983. J. Mol. Biol. 170:827-842. Norrander, J. , T. Kempe, J. Messing. 1983. Gene 26:101-106. Keen N.T, S. Tamaki, D. Kobayashi and D. Trollinger (1988) . Gene 79: 191-197. Tabor S. and C.C. Richardson (1985) . Proc Natl Acad Sci USA 82: 10741078. Greener, A. (1990) Strategies 3: 5-6. Table 2. Putative mannuronan C-5-epimerase activity in recombinant phage lysates. Recombinant phage 3 H release (dp ) EPx 39 EP2 91 EP3 107 EP6 74 EP7 75 EP8 245 EP9 75 EPx originated from the A.vinelandii gene library as a randomly picked plaque, while the other six phages were selected on the basis of the hybridization between their DNA and the labelled oligonucleotide used for screening of the library. Table 3. Activity of the mannuronan C-5-epimerase expressed from the plasmids. The extracts were incubated with the alginate for 16 hours, and the numbers are given in dpm. nd = not determined. * The culture was not induced by IPTG, but by raising the temperature from 30°C to 42°C. Table 4. NMR analysis of the reaction product after incubation of the recombinant epimerase with a low guluronic acid containing substrate. *This spectrum was obtained at a 400 MHz instrument to be able to get relatively correct figures in spite of the lower conversion. SEQUENCE LISTING SEQUENCE LISTING NO: 1 SEQUID Nθ:l SEQUENCE TYPE: Nucleotide with corresponding proteins. SEQUENCE LENGTH: 12411 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA ORIGINAL SOURCE ORGANISM: AzotoJacter vinelandii strain E FEATURES: from 290 to 1951 bp epimerase 4 from 2227 to 6438 bp epimerase 1 from 6702 to 9695 bp epimerase 2 from 9973 to 12411 bp first part of epimerase 3 PROPERTIES: Azotobacter vinelandii mannuronan C-5-epimerase .genes 1 GATCCGGCCG TCTGAGACGG CGCCTCCGGC CGTCGGCGAG TGCGCCGTTC GCCGACGGCC 1 GGGCGAACGG ATGAGGACTG CTCCACTCTC ACCCAGATAA GCGCGTGGGC CGTTTCATCC 1 GAGCGCCTTT CCGGGCCGCT TCGAAAGACC GCCACGAGGC ACTCTGTGCA AGGGCCAGGC 1 AGTCGCGTTG CAACCGGAGA CGGGACCGGC CCGTTCGGGC GTCGTCTCTT CCCGCTCCAC 1 TTTTTCCAGG CAGCTTCGGC TGCTCCACTC GGAACCGGGA AGCGGAGAT 0 ATG GAT TAC AAC GTC AAG GAT TTC GGT GCA TTG GGC GAC GGC GTC AGC Met Asp Tyr Asn Val Lys Asp Phe Gly Ala Leu Gly Asp Gly Val Ser 8 GAC GAC CGG GCC TCC ATC CAG GCG GCG ATC GAT GCC GCC TAC GCC GCC Asp Asp Arg Ala Ser lie Gin Ala Ala lie Asp Ala Ala Tyr Ala Ala 6 GGT GGC GGT ACC GTC TAC CTG CCG GCC GGC GAG TAC CGG GTC AGC GCC Gly Gly Gly Thr Val Tyr Leu Pro Ala Gly Glu Tyr Arg Val Ser Ala 34 GCC GGG GAG CCG GGC GAC GGC TGC CTG ATG CTC AAG GAC GGC GTC TAC Ala Gly Glu Pro Gly Asp Gly Cys Leu Met Leu Lys Asp Gly Val Tyr 82 CTG GCC GGT GCC GGC ATG GGC GAG ACG GTG ATC AAG CTG ATC GAC GGC Leu Ala Gly Ala Gly Met Gly Glu Thr Val He Lys Leu He Asp Gly 30 TCC GAC CAG AAG ATC ACC GGC ATG GTC CGC TCG GCC TAC GGC GAG GAA Ser Asp Gin Lys He Thr Gly Met Val Arg Ser Ala Tyr Gly Glu Glu 78 ACC AGC AAC TTC GGC ATG CGC GAC CTG ACC CTC GAC GGC AAC CGC GAC Thr Ser Asn Phe Gly Met Arg Asp Leu Thr Leu Asp Gly Asn Arg Asp 26 AAC ACC AGC GGC AAG GTC GAC GGC TGG TTC AAC GGC TAT ATC CCC GGC Asn Thr Ser Gly Lys Val Asp Gly Trp Phe Asn Gly Tyr He Pro Gly 74 GGG GAC GGC GCC GAC CGC GAC GTG ACC ATC GAG CGG GTG GAG GTC CGC Gly Asp Gly Ala Asp Arg Asp Val Thr He Glu Arg Val Glu Val Arg 22 GAG ATG TCC GGC TAC GGC TTC GAC CCC CAC GAG CAG ACC ATC AAC CTG Glu Met Ser Gly Tyr Gly Phe Asp Pro His Glu Gin Thr He Asn Leu 70 ACG ATC CGC GAC AGC GTG GCC CAC GAC AAC GGC CTC GAC GGC TTC GTC Thr He Arg Asp Ser Val Ala His Asp Asn Gly Leu Asp Gly Phe Val 18 GCC GAC TAC CTG GTC GAC AGC GTG TTC GAG AAC AAC GTC GCC TAC GCC Ala Asp Tyr Leu Val Asp Ser Val Phe Glu Asn Asn Val Ala Tyr Ala 66 AAC GAC CGC CAC GGC TTC AAC GTG GTC ACC AGC ACC CAC GAT TTC GTC Asn Asp Arg His Gly Phe Asn Val Val Thr Ser Thr His Asp Phe Val 14 ATG ACC AAC AAC GTC GCC TAC GGC AAC GGC AGC AGC GGC CTG GTG GTG Met Thr Asn Asn Val Ala Tyr Gly Asn Gly Ser Ser Gly Leu Val Val 62 CAG CGG GGT CTG GAG GAC CTC GCG CTG CCC AGC AAC ATC CTG ATC GAC Gin Arg Gly Leu Glu Asp Leu Ala Leu Pro Ser Asn He Leu He Asp 1010 GGC GGC GCC TAC TAC GAC AAC GCC CGC GAA GGC GTG CTG CTC AAG ATG Gly Gly Ala Tyr Tyr Asp Asn Ala Arg Glu Gly Val Leu Leu Lys Met 1058 ACC AGC GAC ATC ACC CTG CAG AAC GCC GAT ATC CAC GGC AAC GGC TCC Thr Ser Asp He Thr Leu Gin Asn Ala Asp He His Gly Asn Gly Ser 1106 TCC GGG GTG CGC GTC TAC GGC GCC CAG GAC GTG CAG ATC CTC GAT AAC Ser Gly Val Arg Val Tyr Gly Ala Gin Asp Val Gin He Leu Asp Asn 1154 CAG ATC CAC GAC AAC GCG CAG GCG GCC GCC GTG CCC GAS GTC CTG CTG Gin He His Asp Asn Ala Gin Ala Ala Ala Val Pro Glu Val Leu Leu 1202 CAG TCC TTC GAC GAT ACC GCC GGG GCG TCC GGC ACC TAC TAC ACG ACC Gin Ser Phe Asp Asp Thr Ala Gly Ala Ser Gly Thr Tyr Tyr Thr Thr 1250 CTG AAC ACC CGG ATC GAG GGC AAC ACC ATC AGC GGC TCG GCC AAC TCC Leu Asn Thr Arg He Glu Gly Asn Thr He Ser Gly Ser Ala Asn Ser 1298 ACC TAC GGC ATC CAG GAG CGC AAC GAC GGC ACC GAC TAC AGC AGC CTG Thr Tyr Gly He Gin Glu Arg Asn Asp Gly Thr Asp Tyr Ser Ser Leu 1346 ATC GAC AAC GAC ATC GCC GGG GTG CAA CAG CCC ATC CAA CTG TAC GGA He Asp Asn Asp He Ala Gly Val Gin Gin Pro He Gin Leu Tyr Gly 1394 CCT CAC TCG ACG GTA TCC GGC GAA CCC GGC GCG ACA CCG CAA CAG CCG Pro His Ser Thr Val Ser Gly Glu Pro Gly Ala Thr Pro Gin Gin Pro 1442 TCC ACG GGA AGC GAC GGC GAG CCA CTG GTC GGC GGC GAC ACG GAC GAC Ser Thr Gly Ser Asp Gly Glu Pro Leu Val Gly Gly Asp Thr Asp Asp 1490 CAG CTC CAG GGC GGC TCC GGC GCC GAT CGC CTG GAC GGC GGG GCC GGC Gin Leu Gin Gly Gly Ser Gly Ala Asp Arg Leu Asp Gly Gly Ala Gly 1538 GAC GAC ATC CTC GAC GGC GGC GCC GGG CGC GAC CGG CTG AGC GGC GGC Asp Asp He Leu Asp Gly Gly Ala Gly Arg Asp Arg Leu Ser Gly Gly 1586 GCG GGC GCC GAC ACC TTC GTG TTC TCC GCC CGC GAG GAC AGC TAC CGT Ala Gly Ala Asp Thr Phe Val Phe Ser Ala Arg Glu Asp Ser Tyr Arg 1634 ACC GAC ACG GCG GTG TTC AAC GAC CTG ATC CTC GAC TTC GAG GCC AGC Thr Asp Thr Ala Val Phe Asn Asp Leu He Leu Asp Phe Glu Ala Ser 1682 GAG GAT CGC ATC GAC CTG TCC GCG CTG GGC TTT TCC GGC CTG GGC GAC Glu Asp Arg He Asp Leu Ser Ala Leu Gly Phe Ser Gly Leu Gly Asp 1730 GGC TAT GGC GGC ACC CTG CTC CTG AAG ACC AAC GCC GAG GGC ACG CGC Gly Tyr Gly Gly Thr Leu Leu Leu Lys Thr Asn Ala Glu Gly Thr Arg 1778 ACC TAC CTG AAA AGC TTC GAG GCG GAT GCC GAG GGA CGG CGC TTC GAG Thr Tyr Leu Lys Ser Phe Glu Ala Asp Ala Glu Gly Arg Arg Phe Glu 1826 GTC GCC CTG GAC GGC GAC CAC ACG GGC GAT CTT TCC GCC GCC AAT GTG Val Ala Leu Asp Gly Asp His Thr Gly Asp Leu Ser Ala Ala Asn Val 1874 GTC TTC GCC GCG ACC GGG ACG ACC ACC GAA CTC GAA GTi' CTC GGC GAC Val Phe Ala Ala Thr Gly Thr Thr Thr Glu Leu Glu Va. Leu Gly Asp 1922 AGC GGC ACG CAG GCC GGG GCG ATC GTC TAG CGCGTCCCGC TCCGACACAT Ser Gly Thr Gin Ala Gly Ala He Val 1972 AGCCGGTCGT CGGCAAGGCG GCCGGCCGCC GGCTGCCCCG AAGTTTCCAA TCTAATCTCA 2032 CCTACAGACA GGCGCGTTCC GGTGCGCCCG AGCGCCGCCC CCGGGAACGA CCGGCAGGGC 2092 GTGTTTGTGC GCAAGGTGCA GGCGGTCGCG CTCGAAGCCA GAGGCAGGGA AAACCTTTTC 2152 CGGCAGTCGT CTCTTCCTTC TCCACTTCCC AGGCAGCCCT GGGCCGAGCA ACACGACGGG 2212 ATTAGGAAGC GGATC ATG GAT TAC AAC GTC AAG GAT TTC GGA GCA Met Asp Tyr Asn Val Lys Asp Phe Gly Ala 2257 CTG GGC GAT GGC GTC AGC GAC GAC ACG GCG GCC ATC CAG GCG GCG ATC Leu Gly Asp Gly Val Ser Asp Asp Thr Ala Ala He Gin Ala Ala He 2305 GAC GCC GCC CAC GCG GCG GGC GGC GGC ACC GTC TAC CTG CCG GCC GGC Asp Ala Ala His Ala Ala Gly Gly Gly Thr Val Tyr Leu Pro Ala Gly 2353 GAA TAT CGG GTC AGC GGC GGC GAG GAG CCT TCC GAT GGT TGT CTG ACC Glu Tyr Arg Val Ser Gly Gly Glu Glu Pro Ser Asp Gly Cys Leu Thr 2401 ATC AAG AGC AAC GTC CAT ATC GTC GGC GCC GGG ATG GGC GAG ACG GTG He Lys Ser Asn Val His He Val Gly Ala Gly Met Gly Glu Thr Val 2449 ATC AAG ATG GTC GAC GGC TGG ACG CAG AAC GTC ACC GGC ATG GTG CGC He Lys Met Val Asp Gly Trp Thr Gin Asn Val Thr Gly Met Val Arg 2497 TCG GCC TAC GGC GAG GAA ACC AGC AAC TTC GGC ATG AGC GAC CTG ACC Ser Ala Tyr Gly Glu Glu Thr Ser Asn Phe Gly Met Ser Asp Leu Thr 2545 CTC GAC GGC AAC CGC GAC AAC CTG TCC GCC AAG GTC GAC GGC TGG"-TTC Leu Asp Gly Asn Arg Asp Asn Leu Ser Ala Lys Val Asp Gly Trp Phe 2593 AAC GGC TAC ATC CCC GGC CAG GAC GGC GCC GAT CGC GAC GTG ACC CTG Asn Gly Tyr He Pro Gly Gin Asp Gly Ala Asp Arg Asp Val Thr Leu 2641 GAG CGG GTG GAA ATC CGC GAG ATG TCC GGC TAC GGT TTC GAC CCC CAC Glu Arg Val Glu He Arg Glu Met Ser Gly Tyr Gly Phe Asp Pro His 2689 GAG CAG ACC ATC AAC CTG ACG ATC CGC GAC AGC GTG GCC CAC GAC AAC Glu Gin Thr He Asn Leu Thr He Arg Asp Ser Val Ala His Asp Asn 2737 AGC CTC GAC GGC TTC GTC GCC GAC TAC CAG GTC GGC GGG GTG TTC GAG Ser Leu Asp Gly Phe Val Ala Asp Tyr Gin Val Gly Gly Val Phe Glu 2785 AAC AAC GTC TCG TAC AAC AAC GAC CGC CAC GGC TTC AAC ATC GTC ACC Asn Asn Val Ser Tyr Asn Asn Asp Arg His Gly Phe Asn He Val Thr 2833 AGC ACC AAC GAC TTC GTC CTG AGC AAC AAC GTC GCC TAC GGC AAC GGC Ser Thr Asn Asp Phe Val Leu Ser Asn Asn Val Ala Tyr Gly Asn Gly 2881 GGC GCC GGC CTG GTG GTG CAG CGC GGC TCG TAC GAC CTG CCC CAT CCC Gly Ala Gly Leu Val Val Gin Arg Gly Ser Tyr Asp Leu Pro His Pro 2929 TAC GAC ATC CTG ATC GAC GGC GGC GCC TAC TAC GAC AAC GCC TTG GAA Tyr Asp He Leu He Asp Gly Gly Ala Tyr Tyr Asp Asn Ala Leu Glu 2977 GGC GTG CAG CTC AAG ATG GCC CAC GAC GTC ACC CTG CAG AAC GCC GAG Gly Val Gin Leu Lys Met Ala His Asp Val Thr Leu Gin Asn Ala Glu 3025 ATC TAC GGC AAC GGC CTG TAC GGG GTG CGC GTC TAC GGC GCC CAG GAC He Tyr Gly Asn Gly Leu Tyr Gly Val Arg Val Tyr Gly Ala Gin Asp 3073 GTG CAG ATC CTC GAC AAC CAG ATC CAC GAC AAT TCG CAG AAC GGC GCC Val Gin He Leu Asp Asn Gin He His Asp Asn Ser Gin Asn Gly Ala 3121 TAT GCC GAA GTC CTG CTG CAC TCC TAC GAC GAC ACC GCC GGG GTG TCC Tyr Ala Glu Val Leu Leu Gli Ser Tyr Asp Asp Thr Ala Gly Val Ser 3169 GGC AAC TTT TAC GTC ACC ACC GGC ACC TGG CTC GAA GGC AAC GTC ATC Gly Asn Phe Tyr Val Thr Thr Gly Thr Trp Leu Glu Gly Asn Val He 3217 AGC GGC TCG GCC AAT TCC ACC TAC GGC ATC CAG GAG CGC GCC GAC GGC Ser Gly Ser Ala Asn Ser Thr Tyr Gly He Gin Glu Arg Ala Asp Gly 3265 ACC GAC TAC AGC AGC CTC TAC GCC AAC AGC ATC GAC GGT GTG CAG ACC Thr Asp Tyr Ser Ser Leu Tyr Ala Asn Ser He Asp Gly Val Gin Thr 3313 GGG GCG GTA CGG CTG TAT GGC GCC AAC TCG ACG GTT TCC AGC CAG TCC Gly Ala Val Arg Leu Tyr Gly Ala Asn Ser Thr Val Ser Ser Gin Ser 3361 GGC AGT GGC CAG CAG GCG ACC CTC GAA GGC AGC GCG GGC AAC GAT GCG Gly Ser Gly Gin Gin Ala Thr Leu Glu Gly Ser Ala Gly Asn Asp Ala 3409 CTG AGC GGG ACC GAG GCC CAC GAG ACG CTG CTC GGC CAG GCC GGC GAC Leu Ser Gly Thr Glu Ala His Glu Thr Leu Leu Gly Gin Ala Gly Asp 3457 GAC CGC CTG AAC GCC GAT GCC GGC AAC GAC ATC CTC GAC GGC GGG GCA Asp Arg Leu Asn Gly Asp Ala Gly Asn Asp He Leu Asp Gly Gly Ala 3505 GGG CGC GAC AAC CTG ACC GGC GGC GCG GGC GCC GAC ACC TTC CGC TTC Gly Arg Asp Asn Leu Thr Gly Gly Ala Gly Ala Asp Thr Phe Arg Phe 3553 TCC GCG CGC ACC GAC AGC TAC CGC ACC GAC AGC GCC AGC TTC AAC GAC Ser Ala Arg Thr Asp Ser Tyr Arg Thr Asp Ser Ala Ser Phe Asn Asp 3601 CTG ATC ACC GAC TTC GAC GCC GAC GAG GAC AGC ATC GAC CTG TCC GCG Leu He Thr Asp Phe Asp Ala Asp Glu Asp Ser He Asp Leu Ser Ala 3649 CTG GGC TTC ACC GGC CTG GGC GAC GGC TAC AAT GGC ACC CTG CTS. CTG Leu Gly Phe Thr Gly Leu Gly Asp Gly Tyr Asn Gly Thr Leu Leu Leu 3697 AAG ACC AAC GCC GAG GGT ACG CGC ACC TAC CTG AAG AGC TAC GAA GCG Lys Thr Asn Ala Glu Gly Thr Arg Thr Tyr Leu Lys Ser Tyr Glu Ala 3745 GAC GCC CAG GGC CGG CGC TTC GAG ATC GCC CTG GAC GGC AAC TTC ACC Asp Ala Gin Gly Arg Arg Phe Glu He Ala Leu Asp Gly Asn Phe Thr 3793 GGT CTG TTC AAC GAC AAC AAC CTG TTG TTC GAC GCC GCT CCG GCC ACC Gly Leu Phe Asn Asp Asn Asn Leu Leu Phe Asp Ala Ala Pro Ala Thr 3841 GGT ACC GAG GGC AGC GAC AAC CTG CTC GGC ACC GAC GCC GGG GAA ACC Gly Thr Glu Gly Ser Asp Asn Leu Leu Gly Thr Asp Ala Gly Glu Thr 3889 CTC CTG GGC TAC GGC GGC AAC GAC ACC CTC AAC GGC GGG GCC GGC GAC Leu Leu Gly Tyr Gly Gly Asn Asp Thr Leu Asn Gly Gly Ala Gly Asp 3937 GAC ATC CTG GTC GGC GGC GCC GGG CGC GAC AGC CTG ACC GGC GGC GCC Asp He Leu Val Gly Gly Ala Gly Arg Asp Ser Leu Thr Gly Gly Ala 3985 GGG GCG GAC GTG TTC CGC TTC GAC GCG CTG TCC GAC AGC CAG CGC AAC Gly Ala Asp Val Phe Arg Phe Asp Ala Leu Ser Asp Ser Gin Arg Asn 4033 TAC ACC ACC GGC GAC AAC CAG GCC GAC CGC ATT CTC GAC TTC GAC CCG Tyr Thr Thr Gly Asp Asn Gin Ala Asp Arg He Leu Asp Phe Asp Pro 4081 ACC CTG GAC AGG ATC GAC GTG TCG GCG CTG GGC TTC ACC GGG CTG GGC Thr Leu Asp Arg He Asp Val Ser Ala Leu Gly Phe Thr Gly Leu Gly 4129 AAC GGC CGC AAC GGC ACC CTC GCC GTG GTG CTC AAC AGC GCC GGC GAC Asn Gly Arg Asn Gly Thr Leu Ala Val Val Leu Asn Ser Ala Gly Asp 4177 CGC ACC GAT CTG AAG AGC TAC GAC ACC GAC GCC AAC GGC TAC AGC TTC Arg Thr Asp Leu Lys Ser Tyr Asp Thr Asp Ala Asn Gly Tyr Ser Phe 4225 GAG CTT TCC CTC GCG GGC AAC TAC CAG GGG CAG CTC AGC GCC GAG CAG Glu Leu Ser Leu Ala Gly Asn Tyr Gin Gly Gin Leu Ser Ala 3lu Gin 4273 TTC GTT TTC GCG ACG TCT CAG GGG GGA CAG ATG ACG ATT ATC 3AA GGC Phe Val Phe Ala Thr Ser Gin Gly Gly Gin Met Thr He He Glu Gly 4321 ACC GAC GGC AAC GAT ACC TTG CAG GGC ACC GAG GCC AAC GAG CGG CTC Thr Asp Gly Asn Asp Thr Leu Gin Gly Thr Glu Ala Asn Glu Arg Leu 4369 CTC GGC CTG GAC GGC CGG GAC AAC CTG AAC GGC GGC GCC GGC GAC GAC Leu Gly Leu Asp Gly Arg Asp Asn Leu Asn Gly Gly Ala Gly Asp Asp 4 17 ATC CTC GAC GGC GGA GCG GGG CGC GAC ACC CTG ACC GGC GGC ACG GGG He Leu Asp Gly Gly Ala Gly Arg Asp Thr Leu Thr Gly Gly Thr Gly 4465 GCC GAC ACC TTC CTG TTC TCC ACG CGT ACC GAC AGC TAC CGC ACC GAC Ala Asp Thr Phe Leu Phe Ser Thr Arg Thr Asp Ser Tyr Arg Thr Asp 4513 AGC GCC AGC TTC AAC GAC CTG ATC ACC GAC TTC GAT CCC ACC CAG GAC Ser Ala Ser Phe Asn Asp Leu He Thr Asp Phe Asp Pro Thr Gin Asp 4561 CGC ATC GAC CTG TCC GGC CTG GGC TTC AGC GGT TT- GGC AAC GGC TAC Arg He Asp Leu Ser Gly Leu Gly Phe Ser Gly Ph ; Gly Asn Gly Tyr 4609 GAC GGC ACC CTG CTG CTG CAG GTC AAC GCC GCG GGC ACC CGC ACC TAC Asp Gly Thr Leu Leu Leu Gin Val Asn Ala Ala Gly Thr Arg Thr Tyr 4657 CTG AAG AGT TTC GAG GCC GAT GCC AAC GGC CAG CGC TTC GAG ATC GCC Leu Lys Ser Phe Glu Ala Asp Ala Asn Gly Gin Arg Phe Glu He Ala 4705 CTG GAC GGC GAC TTC AGC GGC CAA TTG GAC AGC GGC AAC GTG ATC TTC Leu Asp Gly Asp Phe Ser Gly Gin Leu Asp Ser Gly Asn Val He Phe 4753 GAG CCC GCC GTG TTC AAT GCC AAG GAC TTC GGC GCG CTG GGC GAC GGC Glu Pro Ala Val Phe Asn Ala Lys Asp Phe Gly Ala Leu Gly Asp Gly 4801 GCC AGC GAC GAC CGG CCG GCC ATC CAG GCG GCG ATC GAC GCC GCC TAC Ala Ser Asp Asp Arg Pro Ala He Gin Ala Ala He Asp Ala Ala Tyr 4849 GCG GCC GGT GGC GGC ACC GTC TAC CTG CCG GCC GGC GAG TAC CGG GTC Ala Ala Gly Gly Gly Thr Val Tyr Leu Pro Ala Gly Glu Tyr Arg Val 4897 AGC CCC ACC GGG GAG CCG GGC GAC GGC TGC CTG ATG CTC AAG GAC GGC Ser Pro Thr Gly Glu Pro Gly Asp Gly Cys Leu Met Leu Lys Asp Gly 49 5 GTC TAC CTG GCC GGC GAC GGC ATA GGC GAA ACG GTC ATC AAG CTG ATC Val Tyr Leu Ala Gly Asp Gly He Gly Glu Thr Val He Lys Leu He 4993 GAC GGC TCC GAC CAG AAG ATC ACC GGC ATG GTG CGC TCG GCC TAT GGC Asp Gly Ser Asp Gin Lys He Thr Gly Met Val Arg Ser Ala Tyr Gly 5041 GAA GAG ACC AGC AAC TTC GGC ATG AGC GAC CTG ACC CTC GAC GGC AAC Glu Glu Thr Ser Asn Phe Gly Met Ser Asp Leu Thr Leu Asp Gly Asn 5089 CGC GAC AAC ACC AGC GGC AAG GTC GAC GGC TGG TTC AAC GGC TAC ATC Arg Asp Asn Thr Ser Gly Lys Val Asp Gly Trp Phe Asn Gly Tyr He 5137 CCC GGC CAG GAC GGC GCC GAC CGC AAC GTG ACC ATC GAG CGG GTG GAA Pro Gly Gin Asp Gly Ala Asp Arg Asn Val Thr He Glu Arg Val Glu 5185 ATC CGC GAG ATG TCC GGC TAT GGC TTC GAT CCG CAC GAG CAG ACC ATC He Arg Glu Met Ser Gly Tyr Gly Phe Asp Pro His Glu Gin Thr He 5233 AAC CTG ACG ATC CGC GAC AGC GTG GCC CAC GAC AAC GGC CTC GAC GGC Asn Leu Thr He Arg Asp Ser Val Ala His Asp Asn Gly Leu Asp Gly 5281 TTC GTC GCC GAC TAC CTG GTC GAC AGC GTG TTC GAG AAC AAC GTC GCC Phe Val Ala Asp Tyr Leu Val Asp Ser Val Phe Glu Asn Asn Val Ala 5329 TAC AAC AAC GAC CGC CAC GGC TTC AAC ATC GTC ACC AGC ACC TAC GAT Tyr Asn Asn Asp Arg His Gly Phe sn He Val Thr Ser Thr Tyr Asp 5377 TTC GTC ATG ACC AAC AAC GTC GCC TAC GGC AAC GGC GGC GCC GGC CTG Phe Val Met Thr Asn Asn Val Ala Tyr Gly Asn Gly Gly Ala Gly Leu 5425 ACG ATC CAG CGG GGC TCG GAG GAC CTG GCC CAG CCG ACC GAT ATC CTG Thr He Gin Arg Gly Ser Glu Asp Leu Ala Gin Pro Thr Asp He Leu 5473 ATC GAC GGC GGC GCC TAC TAC GAC AAC GCC CTG GAA GGC GTG CTG TTC He Asp Gly Gly Ala Tyr Tyr Asp Asn Ala Leu Glu Gly Val Leu Phe 5521 AAG ATG ACC AAC AAC GTC ACC CTG CAG AAC GCC GAG ATC TAC GGC AAC Lys Met Thr Asn Asn Val Thr Leu Gin Asn Ala Glu He Tyr Gly Asn 5569 GGC TCC TCC GGC GTG CGC CTG TAC GGC ACG GAG GAC GTG CAG ATC CTC Gly Ser Ser Gly Val Arg Leu Tyr Gly Thr Glu Asp Val Gin He Leu 5617 GAC AAC CAG ATC CAC GAC AAT TCG CAG AAC GGC ACC TAT CCG GAA GTC Asp Asn Gin He His Asp Asn Ser Gin Asn Gly Thr Tyr Pro Glu Val 5665 CTG CTG CAG GCC TTC GAC GAC AGC CAG GTC ACC GGT GAG CTG TAC GAG Leu Leu Gin Al a Phe Asp Asp Ser Gin Val Thr Gly Glu Leu Tyr Glu 5713 ACC CTG AAC ACC CGG ATC GAA GGC AAT CTC ATC GAC GCT TCG GAC AAC Thr Leu Asn Thr Arg He Glu Gly Asn Leu He Asp Ala Ser Asp Asn 5761 GCC AAC TAT GCG GTG CGC GAG CGC GAC GAC GGC AGC GAC TAC ACC ACG Ala Asn Tyr Ala Val Arg Glu Arg Asp Asp Gly Ser Asp Tyr Thr Thr 5809 CTC GTG GAC AAC GAC ATC AGC GGC GGC CAG GTC GCC TCG GTG CAG CTT Leu Val Asp Asn Asp He Ser Gly Gly Gin Val Ala Ser Val Gin Leu 5857 TCC GGC GCC CAT TCG AGT CTT TCC GGC GGC ACC GTC GAA GTG CCG CAG Ser Gly Ala His Ser Ser Leu Ser Gly Gly Thr Val Glu Val Pro Gin 5905 GGG ACC GAC GGC AAC GAC GTG CTG GTC GGC AGC GAT GCC AAC GAC CAG Gly Thr Asp Gly Asn Asp Val Leu Val Gly Ser Asp Ala Asn Asp Gin 5953 CTC TAC GGC GGA GCC GGC GAC GAC CGC CTG GAC GGC GGC GCC GGT GAC Leu Tyr" Gly Gly Ala Gly Asp Asp Arg Leu Asp Gly Gly Ala Gly Asp 6001 GAC CTG CTC GAC GGC GGA GCG GGG CGC GAC GAC CTG ACC GGC GGC ACG Asp Leu Leu Asp Gly Gly Ala Gly Arg Asp Asp Leu Thr Gly Gly Thr 6049 GGT GCC GAC ACC TTC GTG TTC GCC GCG CGT ACC GAT AGC TAC CGC ACC Gly Ala Asp Thr Phe Val Phe Ala Ala Arg Thr Asp Ser Tyr Arg Thr 6097 GAC GCG GGG GTG TTC AAC GAC CTG ATC CTC GAC TTC GAC GCC AGC GAG Asp Ala Gly Val Phe Asn Asp Leu He Leu Asp Phe Asp Ala Ser Glu 6145 GAC CGC ATC GAC CTG TCC GCC CTG GGT TTC AGC GGC TTC GGC GAC GGC Asp Arg He Asp Leu Ser Ala Leu Gly Phe Ser Gly Phe Gly Asp Gly 6193 TAC AAC GGC ACC CTG CTG GTG CAG CTC AGC AGC GCC GGA ACC CGT ACC Tyr Asn Gly Thr Leu Leu Val Gin Leu Ser Ser Ala Gly Thr Arg Thr 6241 TAC CTC AAG AGC TAC GAG GAG GAC CTC GAG GGC CGG CGC TTC GAG GTC Tyr Leu Lys Ser Tyr Glu Glu Asp Leu Glu Gly Arg Arg Phe Glu Val 6289 GCC CTG GAC GGC GAC CAC ACG GGC GAT CTT TCC GCC GCC AAT GTG GTT Ala Leu Asp Gly Asp His Thr Gly Asp Leu Ser Ala Ala Asn Val Val 6337 TTC GCC GAC GAC GGC TCG GCC GCC GTG GCG AGC AGC GAT CCC GCC GCC Phe Ala Asp Asp Gly Ser Ala Ala Val Ala Ser Ser Asp Pro Ala Ala 6385 ACA CAG TTG GAG GTG GTC GGC AGC AGC GGC ACC CAG ACC GAT CAA CTC Thr Gin Leu Glu Val Val Gly Ser Ser Gly Thr Gin Thr Asp Gin Leu 6433 GCC TGA.TCCGACCCCG CCCATACCCG CCCGGCCATT CCGGCCGGGC GAACCAATGG Ala 6489 TCTTCAGCCC AGTCTCAGGC ACAGCAGCGC GCGAGCCGCT TCGCTTTGTC CGCCCCCCGC 6549 TTTTCTCGCT GAACGCGACG ATCGCCGGGC GCCGGGGAAG GGTTCGCCGC ATGCCGAGCC 6609 GGGGACGGGA AAAGCCTGTT CGACCAGTCG ACTCTTCCTC CCTTCACTTT CCAGGCAGCC 6669 TGCGGGCTGC GCAGTAACGG AACAGGAAGC AGC ATG GAT TAC AAC GTC AAA Met Asp Tyr Asn Val Lys 6720 GAT TTC GGG GCG CTG GGC GAT GGC GTC AGC GAC GAT ACG GCC GCC ATC Asp Phe Gly Ala Leu Gly Asp Gly Val Ser Asp Asp Thr Ala Ala He 6768 CAG GCG GCG ATC GAT GCC GCC TAC GCG GCC GGC GGC GGC ACC GTC TAC Gin Ala Ala He Asp Ala Ala Tyr Ala Ala Gly Gly Gly Thr Val Tyr 6816 CTG CCG GCC GGC GAA TAC CGG GTC AGC GGC GGC GAG GAG CCT TCC GAT Leu Pro Ala Gly Glu Tyr Arg Val Ser Gly Gly Glu Glu Pro Ser Asp 6864 GGT TGC CTG ACC ATC AAG AGC AAC GTC CAT ATC GTC GGC GCG GGG ATG Gly Cys Leu Thr He Lys Ser Asn Val His He Val Gly Ala Gly Met 6912 GGC GAG ACG GTC ATC AAG CTG GTC GAC GGC TGG GAT CAG GAC GTC ACC Gly Glu Thr Val He Lys Leu Val Asp Gly Trp Asp Gin Asp Val Thr 6960 GGC ATC GTC CGC TCG GCC TAC GGC GAG GAG ACC AGC AA'C TTC GGC ATG Gly He Val Arg Ser Ala Tyr Gly Glu Glu Thr Ser Asn Phe Gly Met 7008 AGC GAC CTG ACC CTC GAC GGC AAC CGC GAC AAC ACC AGC GGC AAG GTC Ser Asp Leu Thr Leu Asp Gly Asn Arg Asp Asn Thr Ser Gly Lys Val 7056 GAC GGC TGG TTC AAC GGC TAC ATT CCC GGC GAG GAC GGC GCC GAC CGC Asp Gly Trp Phe Asn Gly Tyr He Pro Gly Glu Asp Gly Ala Asp Arg 7104 GAC GTG ACC CTG GAG CGG GTG GAA ATC CGT GAA ATG TCC GGT TAC GGT Asp Val Thr Leu Glu Arg Val Glu He Arg Glu Met Ser Gly Tyr Gly 7152 TTC GAT CCG CAC GAG CAG ACC ATC AAC CTG ACG ATC CGC GAC AGC GTG Phe Asp Pro His Glu Gin Thr He Asn Leu Thr He Arg Asp Ser Val 7200 GCC CAC GAC AAC GGC CTC GAC GGC TTC GTC GCC GAT TTC CAG ATC GGC Ala His Asp Asn Gly Leu Asp Gly Phe Val Ala Asp Phe Gin He Gly 7248 GGG GTG TTC GAG AAC AAC GTC TCG TAC AAC AAC GAC CGC CAC GGC TTC Gly Val Phe Glu Asn Asn Val Ser Tyr Asn Asn Asp Arg His Gly Phe 7296 AAC ATC GTC ACC AGC ACC AAC GAC TTC GTC CTG AGC AAC AAC GTC GCC Asn He Val Thr Ser Thr Asn Asp Phe Val Leu Ser Asn Asn Val Ala 7344 TAC GGC AAC GGC GGC GCC GGC CTG GTG GTG CAG CGC GGC TCG TCC GAC Tyr Gly Asn Gly Gly Ala Gly Leu Val Val Gin Arg Gly Ser Ser Asp 7392 GTG GCG CAC CCC TAC GAC ATC CTG ATC GAC GGC GGC GCC TAC TAC GAC Val Ala His Pro Tyr Asp He Leu He Asp Gly Gly Ala Tyr Tyr Asp 7440 AAC GGC CTG GAA GGC GTG CAG ATC AAG ATG GCC CAC GAC GTC ACC CTG Asn Gly Leu Glu Gly Val Gin He Lys Met Ala His Asp Val Thr Leu 7488 CAG AAC GCC GAG ATC TAC GGC AAC GGC CTA TAC GGG GTG CGC GTC TAC Gin Asn Ala Glu He Tyr Gly Asn Gly Leu Tyr Gly Val Arg Val Tyr 7536 GGC GCC GAG GAT GTG CAG ATC CTC GAC AAC TAC ATC CAC GAC AAT TCG Gly Ala Glu Asp Val Gin He Leu Asp Asn Tyr He His Asp Asn Ser 7584 CAG AAC GGT TCC TAC GCG GAA ATC CTC CTG CAG TCC TAC GAC GAT ACC Gin Asn Gly Ser Tyr Ala Glu He Leu Leu Gin Ser Tyr Asp Asp Thr 7632 GCC GGG GTG TCC GGC AAT TTC TAC ACC ACC ACC GGC ACC TGG ATC GAA Ala Gly Val Ser Gly Asn Phe Tyr Thr Thr Thr Gly Thr Trp He Glu 7680 GGC AAC ACC ATC GTC GGC TCG GCC AAC TCC ACC TAT GGC ATC CAG GAG Gly Asn Thr He Val Gly Ser Ala Asn Ser Thr Tyr Gly He Gin Glu 7728 CGC GAC GAC GGC ACC GAC TAC AGC AGC CTC TAC GCC AAC AGC GTC AGC Arg Asp Asp Gly Thr Asp Tyr Ser Ser Leu Tyr Ala Asn Ser Val Ser 7776 AAT GTG CAG AAC GGC TCG GTG CGC CTC TAC GGC GCC AAC TCC GTC GTC Asn Val Gin Asn Gly Ser Val Arg Leu Tyr Gly Ala Asn Ser Val Val 7824 TCC GAC CTG CCC GGC ACC GGC CAG CAG GCG ACC CTC GAA GGC ACG GCC Ser Asp Leu Pro Gly Thr Gly Gin Gin Ala Thr Leu Glu Gly Thr Ala 7872 GGC AAC GAC ACG CTT GGC GGC AGC GAC GCC CAC GAG ACG CTG CTC GG:j Gly Asn Asp Thr Leu Gly Gly Ser Asp Ala His Glu Thr Leu Leu Gly 7920 CTG GAC GGC AAC GAC CGC CTG AAC GGC GGC GCC GGC AAC GAC ATC CTC Leu Asp Gly Asn Asp Arg Leu Asn Gly Gly Ala Gly Asn Asp He Leu 7968 GAC GGC GGC GCC GGG CGC GAC AAC CTG ACC GGC GGC GCG GGC GCC GAC Asp Gly Gly Ala Gly Arg Asp Asn Leu Thr Gly Gly Ala Gly Ala Asp 8016 CTG TTC CGC GTC TCC GCG CGC ACC GAC AGC TAC CGC ACC GAC AGC GCC • Leu Phe Arg Val Ser Ala Arg Thr Asp Ser Tyr Arg Thr Asp Ser Ala 8064 AGC TTC AAC GAC CTG ATC ACC GAC TTC GAC GCC AGC CAG GAC CGC ATC Ser Phe Asn Asp Leu He Thr Asp Phe Asp Ala Ser Gin Asp Arg He 8112 GAC CTG TCC GCG CTG GGC TTC ACC GGG CTG GGC GAC GGC TAT AAC GGC Asp Leu Ser Ala Leu Gly Phe Thr Gly Leu Gly Asp Gly Tyr Asn Gly 8160 ACC CTG CTG CTG CAG GTC AGC GCC GAC GGC AGC CGC ACC TAT CTG AAG Thr Leu Leu Leu Gin Val Ser Ala Asp Gly Ser Arg Thr Tyr Leu Lys 8208 AGC CTG GAG GCG GAT GCC GAG GGG CGG CGT TTC GAG ATC GCC CTG GAC Ser Leu Glu Ala Asp Ala Glu Gly Arg Arg Phe Glu He Ala Leu Asp 8256 GGC AAC TTC GCC GGC CTG CTC GGT GCC GGC AAC CTG CTC TTC GAG CGC Gly Asn Phe Ala Gly Leu Leu Gly Ala Gly Asn Leu Leu Phe Glu Arg 8304 ACC GCC ATC GAG GGG GAT GCC GGC GAC AAC GCC CTG CTC GGT ACC TCG Thr Ala He Glu Gly Asp Ala Gly Asp Asn Ala Leu Leu Gly Thr Ser 8352 GCC GCC GAG ACA TTG CTC GGC CAC GCC GGC AAC GAC ACG CTC GAC GGC Ala Ala Glu Thr Leu Leu Gly His Ala Gly Asn Asp Thr Leu Asp Gly 8400 GGG GCC GGC GAC GAC ATC CTG GTC GGC GGC GCC GGG CGC GAC AGC CTC Gly Ala Gly Asp Asp He Leu Val Gly Gly Ala Gly Arg Asp Ser Leu 8448 ACC GGC GGC GCC GGA GCG GAC GTG TTC CGC TTC GAC GCG CTG TCC GAC Thr Gly Gly Ala Gly Ala Asp Val Phe Arg Phe Asp Ala Leu Ser Asp 8496 AGC CAG CGC AAC TAC GAC ATC GGC GAC AAC CAG GGC GAC CGC ATC GCC Ser Gin Arg Asn Tyr Asp He Gly Asp Asn Gin Gly Asp Arg He Ala 8544 GAC TTC GCG GTG GGC GAA GAC AAG CTC GAC GTA TCG GCG CTG GGC TTC Asp Phe Ala Val Gly Glu Asp Lys Leu Asp Val Ser Ala Leu Gly Phe 8592 ACC GGG CTG GGC GAC GGC TAC AAC GGC ACC CTC GCC CTG GTG CTC AAC Thr Gly Leu Gly Asp Gly Tyr Asn Gly Thr Leu Ala Leu Val Leu Asn 8640 AGC GCC GGC GAC CGC ACC TAC GTG AAA AGC TAC GAG AAC GGC GCC GAC Ser Ala Gly Asp Arg Thr Tyr Val Lys Ser Tyr Glu Asn Gly Ala Asp 8688 GGC TAC CGC TTC GAG TTT TCC CTC GAC GGC AAC TAT CTG GAG CTA CTC Gly Tyr Arg Phe Glu Phe Ser Leu Asp Gly Asn Tyr Leu Glu Leu Leu 8736 GGC AAC GAG GAT TTC ATC TTC GCC ACG CCC AGC GGC CAG CAA CTC CTC Gly Asn Glu Asp Phe He Phe Ala Thr Pro Ser Gly Gin Gin Leu Leu 8784 GAA GGC AGC GCC GGC AAC GAC AGC CTG CAG GGC ACG GCC GCC GAC GAG Glu Gly Ser Ala Gly Asn Asp Ser Leu Gin Gly Thr Ala Ala Asp Glu 8832 GTG ATC CAC GGC GGC GGC GGG CGC GAC ACG CTG GCC GGA GGG GCC GGG Val He His Gly Gly Gly Gly Arg Asp Thr Leu Ala Gly Gly Ala Gly 8880 GCC GAC GTG TTC CGC TTT AGC GAA CTG ACC GAC AGC TAC CGA GAC AGT Ala Asp Val Phe Arg Phe Ser Glu Leu Thr Asp Ser Tyr Arg Asp Ser 8928 GCC AGC TAT GCC GAT CTG ATC ACT GAC TTC GAT GCC AGC GAG GAT CGT Ala Ser Tyr Ala Asp Leu He Thr Asp Phe Asp Ala Ser Glu Asp Arg 8976 ATC GAC CTG TCC GGC CTC GGC TTC AGC GGT CTG GGC AAC GGC TAC GGC He Asp Leu Ser Gly Leu Gly Phe Ser Gly Leu Gly Asn Gly Tyr Gly 9024 GGT ACC CTG GCG CTG CAG GTG AAC AGC GCC GGT ACC CGC ACC TAC CTG Gly Thr Leu Ala Leu Gin Val Asn Ser Ala Gly Thr Arg Thr Tyr Leu 9072 AAG AGC TTC GAG ACC AAC GCC GCC GGC GAG CGT TTC GAG ATC GCC CTG Lys Ser Phe Glu Thr Asn Ala Ala Gly Glu Arg Phe Glu He Ala Leu 9120 GAC GGC GAC CTG TCC GCG CTC GGC GGG GCC AAC CTG ATC CTC GAC GCG Asp Gly Asp Leu Ser Ala Leu Gly Gly Ala Asn Leu He Leu Asp Ala 9168 CGT ACC GTA CTG GCG GGC GGC GAC GGC AAC GAC ACG CTT TCC GGC AGC Arg Thr Val Leu Ala Gly Gly Asp Gly Asn Asp Thr Leu Ser Gly Ser 9216 AGC GCG GCC GAG GAA CTG CTC GGC GGG GTC GGC AAC GAC AGC CTG GAC Ser Ala Ala Glu Glu Leu Leu Gly Gly Val Gly Asn Asp Ser Leu Asp 9264 GGC GGC GCC GGC AAC GAC ATC CTC GAC GGC GGG GCG GGG CGC GAC ACC Gly Gly Ala Gly Asn Asp He Leu Asp Gly Gly Ala Gly Arg Asp Thr 9312 CTG AGT GGC GGC AGC GGC AGC GAC ATC TTC CGC TTC GGC GGC GCG CTC Leu Ser Gly Gly Ser Gly Ser Asp He Phe Arg Phe Gly Gly Ala Leu 9360 GAC AGC TTC CGC AAC TAC GCC AGC GGG ACG AAC GGC ACC GAC AGC ATC Asp Ser Phe Arg Asn Tyr Ala Ser Gly Thr Asn Gly Thr Asp Ser He 9408 ACC GAC TTC ACC CCC GGC GAG GAT CTG ATC GAC CTC TCC GTG CTC GGC Thr Asp Phe Thr Pro Gly Glu Asp Leu He Asp Leu Ser Val Leu Gly 9456 TAC ACC GGG CTG GGC GAC GGC TAC AAC GGT ACC CTG GCG ATA GTG CTG Tyr Thr Gly Leu Gly Asp Gly Tyr Asn Gly Thr Leu Ala He Val Leu 9504 AAC GAC GCC GGC ACC AAG ACC TAC CTG AAA AAC CGC GAG AGC GAC GCC Asn Asp Ala Gly Thr Lys Thr Tyr Leu Lys Asn Arg Glu Ser Asp Ala 9552 GAA GGC AAC CAG TTC GAG ATC GCC CTG GAG GGC AAC CAC GCC GAC CAG Glu Gly Asn Gin Phe Glu He Ala Leu Glu Gly Asn His Ala Asp Gin 9600 CTC GAT GCG AGC GAC TTC ATC TTC GCC ACG GCG GCC GCG ACC ACC GGA Leu Asp Ala Ser Asp Phe He Phe Ala Thr Ala Ala Ala Thr Thr Gly 9648 ATC GAG GTG GTC GGC GGC AGC GGC ACC CAG ACC GAT CAG CTC GCC TGA He Glu Val Val Gly Gly Ser Gly Thr Gin Thr Asp Gin Leu Ala 9696 TCCGACCCCG CCCGCACCCG CCCGGCCATT CCGGCCGGGC GAACCAATGG CCTTTTGATC 9756 AGTCTCAGGC ACAGCAACGT GTGCGCCGCT TCGCTTGTTC GCCCTCCCGG CCTTGTTTCT 9816 CGCTGAAAGC GACGATCGCC GGGGGCGTGC CGGGCGCGAG AAAAGGTTCG CCGTGTGCAA 9876 AGCCGGGGAC GGGAAAAGCC TGTTCAAGTA GTCGACTCTT CCTTCTCCTT TTTCCTAGAC 9936 GGCCTCT TGG CTG AGC ATT AAC GGA ACA GGA AGC AGC ATG GAC TTC AAC Met Asp Phe Asn 9985 GTC AAA GAT TTC GGG GCA CTG GGC GAT GGC GCC AGC GAC GAC ACG GCG Val Lys Asp Phe Gly Ala Leu Gly Asp Gly Ala Ser Asp Asp Thr Ala 10033 GCC ATC CAG GCG GCG ATC GAT GCC GCC CAC GCG CCG GGC GGC GGC ACC Ala He Gin Ala Ala He Asp Ala Ala His Ala _ la Gly Gly Gly Thr 10081 GTC TAC CTG CCG GCT GGC GAG TAT CGG GTC AGC ( GC GGC GAG GAG CCT Val Tyr Leu Pro Ala Gly Glu Tyr Arg Val Ser Gly Gly Glu Glu Pro 10129 TCC GAC GGC GCG CTG ACC ATC AAG AGC AAC GTC TAT ATC GTC GGC GCC Ser Asp Gly Ala Leu Thr He Lys Ser Asn Val Tyr He Val Gly Ala 10177 GGG ATG GGC GAG ACG GTG ATC AAG ATG GTC GAC GGC TGG ACG CAG AAC Gly Met Gly Glu Thr Val He Lys Met Val Asp Gly Trp Thr Gin Asn 10225 GTC ACC GGC ATG GTG CGC TCG GCC TAT GGC GAG GAG ACC AGC AAC TTC Val Thr Gly Met Val Arg Ser Ala Tyr Gly Glu Glu Thr Ser Asn Phe 10273 GGC ATG AGC GAC CTG ACC CTC GAC GGC AAC CGC GAC AAC CTG TCC GCC Gly Met Ser Asp Leu Thr Leu Asp Gly Asn Arg Asp Asn Leu Ser Ala 10321 AAG GTC GAC GGC TGG TTC AAC GGC TAC ATT CCC GGC CAG GAC GGT GCC Lys Val Asp Gly Trp Phe Asn Gly Tyr He Pro Gly Gin Asp Gly Ala 10369 GAT CGC GAC GTG ACC CTG GAG CGG GTG GAA ATC CGC GAA ATG TCC GGT Asp Arg Asp Val Thr Leu Glu Arg Val Glu He Arg Glu Met Ser Gly 10417 TAC GGT TTC GAT CCG CAC GAG CAG ACC ATC AAC CTG ACG ATC CGC GAC Tyr Gly Phe Asp Pro His Glu Gin Thr He Asn Leu THr He Arg Asp 10465 AGC GTG GCC CAC GAC AAC GGC CTC GAC GGC TTC GTC GCC GAC TAC CAG Ser Val Ala His Asp Asn Gly Leu Asp Gly Phe Val Ala Asp Tyr Gin 10513 GTC GGC GGG GTG TTC GAG AAC AAC GTC TCG TAC AAC AAC GAC CGC CAC Val Gly Gly Val Phe Glu Asn Asn Val Ser Tyr Asn Asn Asp Arg His 10561 GGC TTC AAC ATC GTC ACC AGC ACC AAC GAC TTC GTC CTG AGC AAC AAC Gly Phe Asn He Val Thr Ser Thr Asn Asp Phe Val Leu Ser Asn Asn 10609 GTC GCC TAC GGC AAC GGC GGC GCC GGC CTG GTG GTG CAG CGC GGC TCG Val Ala Tyr Gly Asn Gly Gly Ala Gly Leu Val Val Gin Arg Gly Ser 10657 TAC GAC CTG CCC CAT CCC TAC GAC ATC CTG ATC GAC GGC GGC GCC TAC Tyr Asp Leu Pro His Pro Tyr Asp He Leu He Asp Gly Gly Ala Tyr 10705 TAC GAC AAC GCC TTG GAA GGC GTG CAG CTC AAG ATG ACC CAC GAC GTC Tyr Asp Asn Ala Leu Glu Gly Val Gin Leu Lys Met Thr His Asp Val 10753 ACC CTG CAG AAC GCC GAG ATC TAT GGC AAC GGC CTG TAC GGG GTG CGC Thr Leu Gin Asn Ala Glu He Tyr Gly Asn Gly Leu Tyr Gly Val Arg 10801 GTC TAC GGC GCC CAG GAC GTG CAA CTC CTC GAT AAC CAG ATC CAC GAC Val Tyr Gly Ala Gin Asp Val Gin Leu Leu Asp Asn Gin He His Asp 10849 AAT TCG CAG AAC GGC GCC TAT GCC GAA GTC CTG CTG CAG TCC TAC GAC Asn Ser Gin Asn Gly Ala Tyr Ala Glu Val Leu Leu Gin Ser Tyr Asp 10897 GAC ACC GCC GGG GTG TCC GGC AAC TTT TAC GTC ACC ACC GGC ACC TGG Asp Thr Ala Gly Val Ser Gly Asn Phe Tyr Val Thr Thr Gly Thr Trp 10945 CTC GAA GGC AAC GTC ATC AGC GGC TCG GCC AAT TCC ACC TTC GGC ATC Leu Glu Gly Asn Val He Ser Gly Ser Ala Asn Ser Thr Phe Gly He 10993 CAG GAG CGC GCC GAC GGC ACC GAC TAC AGC AGC CTT TAC GCC AAT ACC Gin Glu Arg Ala Asp Gly Thr Asp Tyr Ser Ser Leu Tyr Ala Asn Thr 11041 ATC GAC GGC GTG CAG AAC GGG ACG GTA CGG CTG TAT GGC GCC AAC TCC He Asp Gly Val Gin Asn Gly Thr Val Arg Leu Tyr Gly Ala Asn Ser 11089 ACG GTT TCC GAG CAG CCC AGC AGC GGC CAG CAG GCG ACC CTC GAA GGC Thr Val Ser Glu Gin Pro Ser Ser Gly Gin Gin Ala Thr Leu Glu Gly 11137 ACC GCG GGC AAC GAC GTG CTC AGC GGA ACG GGT GCC CAC GAG CTG ATT Thr Ala Gly Asn Asp /al Leu Ser Gly Thr Gly Ala His Glu Leu He 11185 CTC GGC CTG GCC GGC VAC GAT CGC CTG GAC GGT GGC GCC GGC GAC GAC Leu Gly Leu Ala Gly Asn Asp Arg Leu Asp Gly Gly Ala Gly Asp Asp 11233 ACC CTC GAC GGC GGC GCG GGG CGC GAT ACC CTG ACC GGC GGC GCG GGC Thr Leu Asp Gly Gly Ala Gly Arg Asp Thr Leu Thr Gly Gly Ala Gly 11281 GCC GAT ACC TTC CGC TTC TCT GCC CGC GAG GAC AGT CAC CGC ACC GAC Ala Asp Thr Phe Arg Phe Ser Ala Arg Glu Asp Ser His Arg Thr Asp 11329 AGC GCC AGC TTC ACC GAC CTG ATC ACC GAC TTC GAC GCC AGC CAG GAC Ser Ala Ser Phe Thr Asp Leu He Thr Asp Phe Asp Ala Ser Gin Asp 11377 CGC ATC GAC CTC TCC GCG CTG GGC TTC ACC GGT CTG GGC AAC GGT TAT Arg He Asp Leu Ser Ala Leu Gly Phe Thr Gly Leu Gly Asn Gly Tyr 11425 GAC GGC ACC CTG GCG GTG ACC ACC GGT TCC GGC GGC ACC CGC ACC TAC Asp Gly Thr Leu Ala Val Thr Thr Gly Ser Gly Gly Thr Arg Thr Tyr 11473 CTG AAG AGC TAC GAG GTG GAC GCC CAG GGC CGG CGT TTC GAA ATC GCC Leu Lys Ser Tyr Glu Val Asp Ala Gin Gly Arg Arg Phe Glu He Ala 11521 CTG GAC GGC AAC TTC GTC GGC CAG TTC AAC GAT GGC AAC CTG TTG TTC Leu Asp Gly Asn Phe Val Gly Gin Phe Asn Asp Gly As : n Leu Leu Phe 11569 GAC GCC GCT CCG GTC ACC GGT ACC GAG GGC AAC GAC AAC CTG TCC GGC Asp Ala Ala Pro Val Thr Gly Thr Glu Gly Asn Asp Asn Leu Ser Gly 11617 ACC GAT GCC GGG GAA ACC CTC CTG GGC TAC GGC GGC AAC GAC ACC CTC Thr Asp Ala Gly Glu Thr Leu Leu Gly Tyr Gly Gly Asn Asp Thr Leu 11665 AAC GGC GGG GCC GGC AAC GAC ATC CTG GTC GGC GGC GCC GGG CGC GAC Asn Gly Gly Ala Gly Asn Asp He Leu Val Gly Gly Ala Gly Arg Asp 11713 ACC CTG ACC GGC GGC GCC GGG GCG GAC GTG TTC CGC TTC GAG GCG CTG Thr Leu Thr Gly Gly Ala Gly Ala Asp Val Phe Arg Phe Glu Ala Leu 11761 TCC GAC AGC CAG CGC AAC TAC ACC GCC GGC GAC AAC CAG GGC GAT TAC Ser Asp Ser Gin Arg Asn Tyr Thr Ala Gly Asp Asn Gin Gly Asp Tyr 11809 ATC ATC GAC TTC GCC GTG GGC GAA GAC AGG ATC GAC GTA TCG GCG CTG He He Asp Phe Ala Val Gly Glu Asp Arg He Asp Val Ser Ala Leu 11857 GGT TAC ACC GGG CTG GGC AAC GGC CGC AAC GGC ACC CTC GCC GTG GTG Gly Tyr Thr Gly Leu Gly Asn Gly Arg Asn Gly Thr Leu Ala Val Val 11905 CTC AAC AGC GCC GGC GAC CGC ACC TAC GTG AAG AGC TAC GAC ACT GAC Leu Asn Ser Ala Gly Asp Arg Thr Tyr Val Lys Ser Tyr Asp Thr Asp 11953 GCC AAC GGC TAT AAC TTC GAG CTT TCC CTC GCG GGC AAC TAC CAG GGG Ala Asn Gly Tyr Asn Phe Glu Leu Ser Leu Ala Gly Asn Tyr Gin Gly 12001 CTG CTC GGC GCC GAA CAG TTC GTC TTC GCC ACG CCC CCG GAA CAG GCG Leu Leu Gly Ala Glu Gin Phe Val Phe Ala Thr Pro Pro Glu Gin Ala 12049 ACC ATC GAG GGA ACC GAC GGC AAC GAC AGC TTG CAA GGG ACC GGG GCC Thr He Glu Gly Thr Asp Gly Asn Asp Ser Leu Gin Gly Thr Gly Ala 12097 GAC GAA CTG CTC CTC GGT CTG GGC GGC CGG GAC AGC CTG AAC GGC GGC Asp Glu Leu Leu Leu Gly Leu Gly Gly Arg Asp Ser Leu Asn Gly Gly 12145 GCC GGC GAC GAT GTC CTG GAT GGC GGG GCG GAG CGC GAC ACC CTG ACC Ala Gly Asp Asp Val Leu Asp Gly Gly Ala Glu Arg Asp Thr Leu Thr 12193 GGC GGC ACG GGG GCC GAC ACC TTC CTG TTC TCC GCG CGT ACC GAC AGC Gly Gly Thr Gly Ala Asp Thr Phe Leu Phe Ser Ala Arg Thr Asp Ser 122^1 TAC CGC ACC GAC AGC GCC AGC TTC ACC GAC CTG ATC ACC"GAC TTC GAT Tyr Arg Thr Asp Ser Ala Ser Phe Thr Asp Leu He Thr Asp Phe Asp 122? 9 CCC GCC CAG GAT CGC ATC GAC CTG TCC GGC CTG GCC TTC AGC GGT TTC Pro Ala Gin Asp Arg He Asp Leu Ser Gly Leu Gly Phe Ser Gly Phe 12337 GCC AAC GGC TAC GAC GGC ACC CTG CTG CTG CAG GTC AAC GCC GCG GGC Gly Asn Gly Tyr Asp Gly Thr Leu Leu Leu Gin Val Asn Ala Ala Gly 12385 ACC CGC ACC TAC CTG AAG AGC CTC GAG GCC GAT GCC GAC GGC CAG CGC Thr Arg Thr Tyr Leu Lys Ser Leu Glu Ala Asp Ala Asp Gly Gin Arg 12433 TTC GAG ATC GCC CTG GAC GGC GAC TTC AGC GGC CAG TTG GAC AGC GGC Phe Glu He Ala Leu Asp Gly Asp Phe Ser Gly Gin Leu Asp Ser Gly 12481 AAC GTG ATC TTC GAG GCC GGC GTG TTC AAT GCC AAG GAC TTC GGC GCG Asn Val He Phe Glu Ala Gly Val Phe Asn Ala Lys Asp Phe Gly Ala 12S29 CTG GGC GAC GGC GCC AGC GAC GAC CGG CCG GCC ATC CAG GCG GCG ATC Leu Gly Asp Gly Ala Ser Asp Asp Arg Pro Ala He Gin Ala Ala He 12577 GAC GCC GCC TAC Asp Ala Ala Tyr SEQUENCE LISTING NO 2 SEQUENCE LISTING NO 3 E4A 349 E1A1 2286 E1A2 4818 E2A 6761 E3A1 10032 E3A2 12559 Con E4A 409 E1A1 2346 E1A2 4878 E2A 6821 E3A1 10092 E3A2 12589 Con GCCATCCAGGCGGCGATCGATGCCGCCTACGCGGCCGGCGGCGGCACCGTCTACCTGCCG E4A C.C..G GGG TGC 469 E1A1 A..T T..T.GT 2406 E1A2 .....................CC.AC..G......GGG..............TGC..... 4938 E2A A T..T 6881 E3A1 . -T T GCG 10152 Con GCCGGCGAGTACCGGGTCAGCGGCGGCGAGGAGCCTTCCGACGGCTGCCTGACCATCAAG E4A GA.GG CC.G.C ..T C A 529 E1A1 2466 E1A2 GA.GG CC.G.C. .C 4998 E2A C ..G .c 6941 E3A1 10212 Con AGCAACGTCTATATCGTCGGCGCCGGGATGGGCGAGACGGTGATCAAGCTGGTCGACGGC E4A .GA. .A. 589 E1A1 ...AC ■A. 2526 E1A2 .CCC GA T A 5058 E2A T G C .C 7001 E3A1 ...AC T 10272 Con TGGGAGCAGAACGTCACCGGCATGGTGCGCTCGGCCTACGGCGAGGAGACCAGCAACTTC E4A ... , ..C 649 E1A1 CTGTC .C 2586 E1A2 5118 E2A 7061 E3A1 CTGTC.C 10332 Con GGCATGAGCGACCTGACCCTCGACGGCAACCGCGACAACACCAGCGGCAAGGTCGACGGC E4A T..C GG A.C 709 E1A1 C T 2646 E1A2 C A A.C 5178 E2A G 7121 E3A1 T T 10392 Con TGGTTCAACGGCTACATTCCCGGCCAGGACGGCGCCGACCGCGACGTGACCCTGGAGCGG E4A GG C C.C 769 E1A1 C.C 2706 E1A2 T..C 5238 E2A T..A T 7181 E3A1 A T 10452 Con GTGGAAATCCGCGAGATGTCCGGCTACGGTTTCGATCCGCACGAGCAGACCATCAACCTG E4A T. 829 E1A1 A 2766 E1A2 T. 5298 E2A T.T 7241 E3A1 10512 Con ACGATCCGCGACAGCGTGGCCCACGACAACGGCCTCGACGGCTTCGTCGCCGACTACCAG 4/09124 48 E4A .A.A.C. .G.C. .GC. .G . G 889 E1A1 2826 EIA2 .A.A.C. . G . C . 5358 E2A 7301 E3A1 10572 Con GTCGGCGGGGTGTTCGAGAACAACGTCTCGTACAACAACGACCGCCACGGCTTCAACATC E4A C T.. A...C A..AG. 949 E1A1 2886 E1A2 T T A.. -C 5418 E2A 7361 E3A1 10632 Con GTCACCAGCACCAACGACTTCGTCCTGAGCAACAACGTCGCCTACGGCAACGGCGGCGCC E4A G..TCT.G.G C .G.T AG.A ,. 1009 E1A1 C T - 2946 E1A2 AC.A.C G G.G GAC ..T 5478 E2A C G G..C 7421 E3A1 C T 10692 Con GGCCTGGTGGTGCAGCGCGGCTCGTACGACCTGGCCCAGCCCTACGACATCCTGATCGAC E4A GC T AG A.. 1069 E1A1 T G 3006 E1A2 .T.T A. -A 5538 E2A G A G 7481 E3A1 T 10752 Con GGCGGCGCCTACTACGACAACGCCCTGGAAGGCGTGCAGCTCAAGATGACCCACGACGTC E4A T...C TC..C ' 1129 E1A1 G 3066 E1A2 TC.C..C CG A.G 5598 E2A A 7541 E3A1 T G 10812 Con ACCCTGCAGAACGCCGAGATCTACGGCAACGGCCTCTACGGGGTGCGCGTCTACGGCGCC E4A .CG GCG.C GTGC . 1189 E1A1 3126 E1A2 G A CG 5658 E2A G T T.C TT CG 7601 E3A1 AC T 10872 Con CAGGACGTGCAGATCCTCGACAACCAGATCCACGACAATTCGCAGAACGGCGCCTATGCC E4A ..G T T C C .A A.G... 1249 E1A1 T T 3186 E1A2 G...T G. CA...CA TG.GC.G AG... 5715 E2A ...A C T T A 7661 E3A1 T T 10932 Con GAAGTCCTGCTGCAGTCCTACGACGACACCGCCGGGGTGTCCGGCAACTTCTACGCCACC E4A CTGAA 1309 E1A1 ■ •G • 3246 E1A2 CTGAA .TC. ..GA..CT A G...A...T.CGG.G 5775 E2A C GT T 7721 E3A1 C G T T 10992 Con ACCGGCACCTGGATCGAAGGCAACATCATCAGCGGCTCGGCCAACTCCACCTACGGCATC E4A A GAT. -A G CG... 1369 E1A1 C G A 3306 E1A2 .GC G C .CG...GTG.A G A CGC 5835 E2A CG..A..AA 7781 E3A1 C T T.C A...C... 11052 Con CAGGAGCGCGACGACGGCACCGACTACAGCAGCCTCTACGCCAACAACATCGGCGGTGTG E4A ..ACG CCA.CAA AC.TC A G.A G.G 1426 E1A1 C...GG T A T GT 3366 E1A2 .. -GT. -C G.A...T.C C.T GTC GGCA...T.GAA 5895 E2A G..C .C CGTC .C A..T 7841 E3A1 GA T C AG A.. -G. 11112 Con CAGAACGGCTCGGTACGGCTGTACGGCGCCAACTCGACGGTTTCCGGCCAGCCCGGCACC E4A ACA.C...ACA.CGTC.ACGGGAAGCGACGGCGAGCCA 1 65 E1A1 3380 EIA2 .TG.C 5904 E2A 7856 E3A1 ll 1 7 Con GGCCAGCAGGCGACCxxCxxx SEQUENCE LISTING NO 4 E4A ..Y V...R.S AAG..G... -ML. 469 E1A1 ..Y V H 2406 E1A2 V-..A RP PTG. -G... -ML. 4938 E2A . -Y V 6881 E3A1 H A 10152 E3A2 V-..A RP 12589 Con MDFNVKDFGALGDGASDDTAAIQAAIDAAYAAGGGTVYLPAGEYRVSGGEEPSDGCLTIK E4A DG..LA I..S..KI R 589 E1A1 .. -H M T L.A 2586 E1A2 DG..LA.D.I I..S..KI 5118 E2A ...H D...1 7061 E3A1 M T L.A 10332 Con SNVYIVGAGMGETVIKLVDGWIQNVTGMVRSAYGEETSNFGMΞDLTLDGNRDNTSGKVDG E4A G I V L 769 E1A1 S 2766 E1A2 N..I L 5298 E2A E F. 7241 E3A1 10512 Con WFNGYIPGQDGADRDVTLERVEIREMSGYGFDPHEQTINLTIRDSVAHDNGLDGFVADYQ E4A .DS A.A V H...MT SS L L.SN 949 E1A1 Y..P 2946 E1A2 .DS A Y...MT TI Q.T 5478 E2A I S.V 7421 E3A1 Y..P 10692 Con VGGVFENNVSYNNDRHGFNIVTSTNDFVLSNNVAYGNGGAGLWQRGSEDLAHPYDILID E4A R.. -L S.I D.H.. -SS A.AA.VP 1129 E1A1 A 3126 E1A2 LF...NN SS...L..TE T.P 5658 E2A G I..A E Y S.. 7601 E3A1 L 10872 Con GGAYYDNALEGVQLKMTHDVTLQNAEIYGNGLYGVRVYGAQDVQILDNQIHDNSQNGAYA E4A F A..TY...LN.R N ID...A.. 1309 E1A1 V L...V S 3306 E1A2 .V.. -AF..SQ-.T.EL.E.LN.R L.DA.D.AN.AVR..D. -S..TT.VD.. -S.G 5835 E2A .1 V D SVSN. 7781 E3A1 V L...V F T 11052 Con EVLLQSYDDTAGVSGNFYTTTGTWIEGNTISGSANSTYGIQERADGTDYSSLYANDIDGV E4A .-QPIQ...PH E..ATP.QPSTGSDGEP 1465 E1A1 .T.A S.S 3381 E1A2 .VA..Q.S. -H.SL..GTVEVP. 5904 E2A V..DL..T 7856 E3A1 ...T E..S 11127 Con QNGSVRLYGANSTVSGQPGSGQQATxxxxxxxx SEQUENCE LISTING NO 5 E4R1 ..G.TC.. -GG....ACGG CA. .AG 1498 E1R1 G..CG TG A....G 3441 E1R2 GCACC..T G... -G AC ...T C. .GG. .A. -C. 3894 E1R3 A T. -CT...AG ..CG.. 4371 E1R4 -- ■ -. -G.GC GT GT G...T. .A. CCA....TA. 5958 E2R1 .. G.C TG G . . .C . -C G 7916 E2R2 A. G..GGAT.C G..A..G.C T...T...TC.. .C AT.G... 8369 E2R3 .. G..C GCT..AG CC GT. 8834 E2R4 ..G.CG...GG TTC G.AGC C.C GAA. -G... 9237 E3R1 .............CG.........GT...CA....A..G.GT. .C CT.A.T... 11187 E3R2 G..ACC ,T G AC...TC T. ..G.G.A..C G 11640 E3R3 A G..A GCT.-.AA..G G ACTΛ 12111 Con CTCGAAGGCACCGACGGCAACGACACGCTGCGCGGCACCGAGGCCGACGAGACGCTCCTC 1558 3501 3954 4431 6018 ~ 7976 8429 8846 9297 11247 11700 E3R3 ..T.T..G CGG...A TG G. -T G 12171 Con GGCCAGGCCGGCAACGACCGCCTGAACGGCGGCGCCGGCGACGACATCCTCGACGGCGGC E4R1 CGG. -GTG. C -C 1618 E1R1 ..A A.. C 3561 E1R2 G.. C .G..G...GTG GA T. 4014 E1R3 ..G A G TG A T 4491 E1R4 ..G GA.. A T GTG...G T 6078 E2R1 A.. CTG G C 8036 E2R2 G.. C .A. -G GTG GA T. 8489 E2R3 .G G. -G A. -G..C .G GTG TAG..AA-T. 8906 E2R4 . -G . -GT AGC AG T GG..GCGC 9357 E3R1 ..G T T T..C..C 11307 E3R2 C .G. -G GTG GAG T. 11760 E3R3 . -G.A A G TG T 12231 Con GCCGGGCGCGACACCCTGACCGGCGGCGCGGGCGCCGACACCTTCCGCTTCTCCGCGCGG E4R1 GAG T CG. -G GTG CT 1672 E1R1 3615 E1R2 T CG A.T...CA A.CAGGC GC ..TCT .4074 E1R3 4545 E1R4 T GCG.GG GTG CT 6132 E2R1 8090 E2R2 T CG....A.T..GA.AT A.CAGGG GC...G 8549 E2R3 A T... ..C.ATGC ..T T 8957 E2R4 CT T A.T..GC.AC ..GACG.A.GG..C ...AGC 9417 E3R1 GAG TC ..* C 11361 E3R2 T CG A.T...C A.CAGGG...TTAC T 11820 E3R3 C 12285 Con ACCGACAGCTACCGCACCGACAGCGCCGGCGACAGCTTCAACGACCTGATCACCGACTTC E4R1 . -G .TΓ. .C 1732 E1R1 GA. .. .A... .C 3675 E1R2 ...CG.C.CT. .A.G. 4134 E1R3 ..TC ..C.C GC C..TT.C..A. 4605 E1R4 C T G...CT.C 6192 E2R1 C 8150 E2R2 .CG.TGG A.. -AAGC G.A..G 8609 E2R3 ..T T. -T .GC .C 9017 E2R4 AC.C.G T.TG C .T...C 9477 E3R1 C C T. 11421 E3R1 .C .TGG....A...A.G. .G.A. .T.A. 11880 E3R3 ..TC .GCC ...T.... .GC ..G. .TT.C ..A. 12345 Con GACGCCAGCGAGGACCGCATCGACCTGTCCGCGCTGGGCTTCACCGGGCTGGGCGACGGC SEQUENCE LISTING NO 6 E4R1 .V.G.TD.Q -Q.GS.A...D R.S V.... 1620 E1R1 ...SA...A E Q..D D..N N 3561 E1R2 AT..E.S.N.L G YG...T V S V...D.L 4014 E1R3 I Q..E.N.R D.R.N T L..T. 4491 E1R4 — V.V.S..NDQ.Y.G..D D L D T V.A.. 6078 E2R1 A G.S D N N L..V... 8036 E2R2 I.. DA. DNA. L..S.A H T.D V S V...D.L 8489 E2R3 ...SA...S.Q..A.D.V IH. -G A V EL 8906 E2R4 -A.G SS.A.E...GV...S.D N S..S.S.I.. -GGA 9356 E3R1 A.. -V C .LI D T 11307 E3R2 VT. -E...N G YG...T N.. -V V...E.L 11760 E3R3 I S.Q..G. D.L GGR.S V E T. Λ ..L 12231 Con LEGTDGNDTLSGTDAHETLLGLAGNDRLNGGAGDD ILDGGAGRDTLTGGAGADTFRFS AR E4R1 E T.-- V L..E S G KT.AE 1734 E1R1 -- D..S KT.AE 3735 E1R2 S..Q.NYTT..NQA.R.L...PTL V N.R AW D..D... 4194 E1R3 -- PTQ C.S.F.N..D V.A 4665 E1R4 AG--V L S.F V..S 6252 E2R1 — Q VSAD.S . ' . " 8110 E2R2 S..Q.NYDI..NQG.R.A..AVG. -KL.V A.V D.. -V.. 8669 E2R3 -...--.YA C .S...N. -G...A..V 9077 E2R4 L..F.NYAS.TNGT.S TPG..L V..Y AIV..D...K N 9536 E3R1 E..H --. -T Q N..D...AVTTG.G 11481 E3R2 S..Q.NYT...NQG.Y.I. -AVG V Y N.R AW D...V.. 11940 E3R3 --..T PAQ C.S.F.N..D V.A 12405 Con TDSYRTDSAGDSFNDLITDFDASEDRIDLSALGFTGLGDGYNGTLLLQLNSAGTRTYLKS E4R1 F V DH. -D.S.A.V...A.G 1891 E1R1 Q FNDN.LL.DAA. 3834 E1R2 .DT..N.YS..LS.A..YQ.Q.S..Q SQ 4293 E1R3 F N.Q D.S.Q.DSG.VI.EPA- 4761 E1R4 ..E.L V DH..D.S.A.V...DDG 6351 E2R.1 L * .».............-•-&.•..• .G.XJL.jεR..A 8309 E2R2 ..NG.D.Y...FS YLE...N.D.I...PS 8768 E2R3 F.TN.A.E DLSA.CNLILD.R.V 9176 E2R4 R.S NQ E..HADQ.D.SD.I...AA 9635 E3R1 ..V..Q V.QFNDG.LL.DAA. 11580 E3R2 .DT..N.YN..LS.A..YQ Q P. 12039 E3R3 L D.Q D.S.Q.DSG.VI.EAG- 12502 Con YEADAEGRRFEIALDGNFTGLLGAENFVFATTP SEQUENCE LISTING NO 7 1 GCCAGTCTCA GGCACAGCAG CGCGCGAGCC GCTTCGCTTT 1 GTCCGCCCCC CGCTTTTCTC GCTGAACGCG ACGATCGCCG 1 GGCGCCGGGG AAGGGTTCGC GCATGCCGAG CCGGGGACGG 1 GAAAAGCCTG TTCGACCAGT CGACTCTTCC TCCCTTCACT 1 TTCCAGGCAG CCTGCGGGCT GCGCAGTAAC GGAACAGGAA ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ M D Y N V K D F G A L G 1 GCAGCATGGA TTACAACGTC AAAGATTTCG GGGCGCTGGG ★ ★ ★ ★ ★ ★ ★ D G V S D D T A A I Q A A 1 CGATGGCGTC AGCGACGATA CGGCCGCCAT CCAGGCGGCG I D A A Y A A G G G T V Y L 1 ATCGATGCCG CCTACGCGGC CGGCGGCGGC ACCGTCTACC P A G E Y R V S G G E E P 1 TGCCGGCCGG CGAATACCGG GTCAGCGGCG GCGAGGAGCC S D G C T I K S N V H I 1 TTCCGATGGT TGCCTGACCA TCAAGAGCAA CGTCCATATC V G A G M G E T V I K L V D 1 GTCGGCGCGG GGATGGGCGA GACGGTCATC AAGCTGGTCG G W D Q D V T G I V R S A 1 ACGGCTGGGA TCAGGACGTC ACCGGCATCG TCCGCTCGGC Y G E E T S N F G M S D L 1 CTACGGCGAG GAGACCAGCA ACTTCGGCAT GAGCGACCTG T L D G N R D N T S G K V D 1 ACCCTCGACG GCAACCGCGA CAACACCAGC GGCAAGGTCG G F N G Y I P G E D G A 1 ACGGCTGGTT CAACGGCTAC ATTCCCGGCG AGGACGGCGC D R D V T L E R V E I R E 1 CGACCGCGAC GTGACCCTGG AGCGGGTGGA AATCCGTGAA M S G Y G F D P H E Q T I N 1 ATGTCCGGTT ACGGTTTCGA TCCGCACGAG CAGACCATCA L T I R D S V A H D N G L 1 ACCTGACGAT CCGCGACAGC GTGGCCCACG ACAACGGCCT D G F V A D F Q I G G V F 721 CGACGGCTTC GTCGCCGATT TCCAGATCGG CGGGGTGTTC E N N V S Y N N D R H G F N 761 GAGAACAACG TCTCGTACAA CAACGACCGC CACGGCTTCA I V T S T N D F V L S N N 801 ACATCGTCAC CAGCACCAAC GACTTCGTCC TGAGCAACAA V A Y G N G G A G L V V Q 841 CGTCGCCTAC GGCAACGGCG GCGCCGGCCT GGTGGTGCAG R G S S D V A H P Y D I L I 881 CGCGGCTCGT CCGACGTGGC GCACCCCTAC GACATCCTGA D G G A Y Y D N G L E G V 921 TCGACGGCGG CGCCTACTAC GACAACGGCC TGGAAGGCGT Q I K M A H D V T L Q N A 961 GCAGATCAAG ATGGCCCACG ACGTCACCCT GCAGAACGCC E I Y G N G L Y G V R V Y G 1001 GAGATCTACG GCAACGGCCT ATACGGGGTG CGCGTCTACG A E D V Q I L D N Y I H D 1041 GCGCCGAGGA TGTGCAGATC CTCGACAACT ACATCCACGA N S Q N G S Y A E I L L Q 1081 CAATTCGCAG AACGGTTCCT ACGCGGAAAT CCTCCTGCAG S Y D D T A G V S G N F Y T 1121 TCCTACGACG ATACCGCCGG GGTGTCCGGC AATTTCTACA T T G T W I E G N T I V G 1161 CCACCACCGG CACCTGGATC GAAGGCAACA CCATCGTCGG S A N S T Y G I Q E R D D 1201 CTCGGCCAAC TCCACCTATG GCATCCAGGA GCGCGACGAC G T D Y S S L Y A N S V S N 1241 GGCACCGACT ACAGCAGCCT CTACGCCAAC AGCGTCAGCA V Q N G S V R L Y G A N S 1281 ATGTGCAGAA CGGCTCGGTG CGCCTCTACG GCGCCAACTC H D N D R 1401 ACGCCCACGA GACGCTGCTC GGGCTGGACG GCAACGACCG N G N D L D 1441 CCTGAACGGC GGCGCCGGCA ACGACATCCT CGACGGCGGC A R D N A G D 1481 GCCGGGCGCG ACAACCTGAC CGGCGGCGCG GGCGCCGACC F R V S A R T D S Y R T D 1521 TGTTCCGCGT CTCCGCGCGC ACCGACAGCT ACCGCACCGA S A S F N D L I T D F D A 1561 CAGCGCCAGC TTCAACGACC TGATCACCGA CTTCGACGCC S Q D R I D L S A L G F T G 1601 AGCCAGGACC GCATCGACCT GTCCGCGCTG GGCTTCACCG L G D G Y N G T L L L Q V 1641 GGCTGGGCGA CGGCTATAAC GGCACCCTGC TGCTGCAGGT S A D G S R T Y L K S L E 1681 CAGCGCCGAC GGCAGCCGCA CCTATCTGAA GAGCCTGGAG A D A E G R R F E I A L D G 1721 GCGGATGCCG AGGGGCGGCG TTTCGAGATC GCCCTGGACG N F A G L L G A G N L L F 1761 GCAACTTCGC CGGCCTGCTC GGTGCCGGCA ACCTGCTCTT π REP2 I E G D A G D N A 1801 CGAGCGCACC GCCATCGAGG GGGATGCCGG CGACAACGCC A E T L L G H 1841 CTGCTCGGTA CCTCGGCCGC CGAGACATTG CTCGGCCACG G N D T L D G G A G D P I 1881 CCGGCAACGA CACGCTCGAC GGCGGGGCCG GCGACGACAT V G G A G R D S L T G G 1921 CCTGGTCGGC GGCGCCGGGC GCGACAGCCT CACCGGCGGC A G A D V F R F D A L S D S 1961 GCCGGAGCGG ACGTGTTCCG CTTCGACGCG CTGTCCGACA Q R N Y D I G D N Q G D R 2001 GCCAGCGCAA CTACGACATC GGCGACAACC AGGGCGACCG I A D F A V G E D K L D V 2041 CATCGCCGAC TTCGCGGTGG GCGAAGACAA GCTCGACGTA S A L G F T G L G D G Y N G 2081 TCGGCGCTGG GCTTCACCGG GCTGGGCGAC GGCTACAACG T L A L V L N S A G D R T 2121 GCACCCTCGC CCTGGTGCTC AACAGCGCCG GCGACCGCAC Y V K S Y E N G A D G Y R 2161 CTACGTGAAA AGCTACGAGA ACGGCGCCGA CGGCTACCGC F E F S L D G N Y L E L L G 2201 TTCGAGTTTT CCCTCGACGG CAACTATCTG GAGCTACTCG N E D F I F A T P S| G Q Q 2241 GCAACGAGGA TTTCATCTTC GCCACGCCCA GCGGCCAGCA IEP3 L IL E G S A G N P S L 0 2281 ACTCCTCGAA GGCAGCGCCG GCAACGACAG CCTGCAGGGC T A A D E V I H G G G G R D 2321 ACGGCCGCCG ACGAGGTGAT CCACGGCGGC GGCGGGCGCG G A A D V R 2361 ACACGCTGGC CGGAGGGGCC GGGGCCGACG TGTTCCGCTT S E L T D S Y R D S A S Y 2401 TAGCGAACTG ACCGACAGCT ACCGAGACAG TGCCAGCTAT A D L I T D F D A S E D R I 2441 GCCGATCTGA TCACTGACTT CGATGCCAGC GAGGATCGTA D L S G L G F S G L G N G 2481 TCGACCTGTC CGGCCTCGGC TTCAGCGGTC TGGGCAACGG Y G G T L A L Q V N S A G 2521 CTACGGCGGT ACCCTGGCGC TGCAGGTGAA CAGCGCCGGT T R T Y L K S F E T N A A G 2561 ACCCGCACCT ACCTGAAGAG CTTCGAGACC AACGCCGCCG E R F E I A L D G D L S A 2601 GCGAGCGTTT CGAGATCGCC CTGGACGGCG ACCTGTCCGC L G G A N L I L D A R T V 2641 GCTCGGCGGG GCCAACCTGA TCCTCGACGC GCGTACCGTA J.EP4 A G G D G N D T L S 2681 CTGGCGGGCG GCGACGGCAA CGACACGCTT TCCGGCAGCA A A E E L L G G V G N P S 2721 GCGCGGCCGA GGAACTGCTC GGCGGGGTCG GCAACGACAG D G N P P 2761 CCTGGACGGC GGCGCCGGCA ACGACATCCT CGACGGCGGG A G R P T L S G G S G S P 2801 GCGGGGCGCG ACACCCTGAG TGGCGGCAGC GGCAGCGACA F R F G G A L P S F R N Y 2841 TCTTCCGCTT CGGCGGCGCG CTCGACAGCT TCCGCAACTA A S G T N G T P S I T P F 2881 CGCCAGCGGG ACGAACGGCA CCGACAGCAT CACCGACTTC T P G E P L I P L S V L G Y 2921 ACCCCCGGCG AGGATCTGAT CGACCTCTCC GTGCTCGGCT T G L G P G Y N G T L A I 2961 ACACCGGGCT GGGCGACGGC TACAACGGTA CCCTGGCGAT V L N D A G T K T Y L K N 3001 AGTGCTGAAC GACGCCGGCA CCAAGACCTA CCTGAAAAAC R E S D A E G N Q F E I A L 3041 CGCGAGAGCG ACGCCGAAGG CAACCAGTTC GAGATCGCCC E G N H A D Q L D A S D F 3081 TGGAGGGCAA CCACGCCGAC CAGCTCGATG CGAGCGACTT I F A T A A| A T T G I E V 3121 CATCTTCGCC ACGGCGGCCG CGACCACCGG AATCGAGGTG V G G S G T Q T D Q L A < 3161 GTCGGCGGCA GCGGCACCCA GACCGATCAG CTCGCCTGAT 3201 CCGACCCCGC CCGCACCCGC CCGGCCATTC CGGCCGGGCG 3241 AACCAATGGC CTTTTGATCA

Claims: Claims : 1. A DNA compound comprising a sequence encoding mannuronan C-5-epimerase, said sequence being isolated from a natural source or being derived synthetically. 2. An isolated DNA compound encoding mannuronan C-S- epimerase that comprises a DNA block A and/or a DNA block S and/or a DNA block R. 3. An isolated DNA compound according to claim 2 encoding mannuronan C-5-epimerase that comprises at least one DNA block A, one DNA block S and a DNA block R in a number of 0 to at least 5. 4. An isolated DNA compound according to claim 1 comprising at least a portion of the DNA sequence as set out in Figure 6. 5. An isolated DNA compound according to claim 1 that encodes at least a portion of the protein amino acid sequence as set out in Figure 6. 6. A recombinant DNA vector that comprises the DNA sequence of claim 1. 7. Use of a DNA sequence according to claims 1-6 for constructing a recombinant host cell for production of enzymes having mannuronan C-5-epimerase activity and/or the micro-biological production of alginates. 8. Use according to claim 7 of a DNA sequence for the micro-biological production of alginates having a high G block content of 75-98%. 9. Use according to Claim 7 of a DNA sequence for the micro-biological production of alginates wherein the DNA sequence is selected to produce an alginate having a desired M/G block content. 10. Process for the production of enzymes having mannuronan C-5-epimerase by constructing a recombinant host cell capable of expressing epimerase activity, characterized by transforming said host cell with a recbmbinant DNA expression vector that comprises: (a) a promoter and translational activating sequence that function in said host cell; and (b) a DNA sequence encoding mannuronan C-5-epimerase comprising at a DNA block A and/or a DNA block S and/or a DNA block R, positioned for expression from said promoter and translational activity sequence. 11. Process for the bacterial production of alginates characterized by selectively inactivating one, several or all DNA sequences encoding for mannuronan C-5-epimerases in a natural host cell. 12. Process according to claim 11 for the production of pure poly-M alginate or alginates having a G block content from 0-25%. 13. Process according to claim 11 for the production of alginates wherein said DNA sequences are selectively inactivated to produce alginates having a desired M/G block content and G block distribution. 14. Amino acid sequence for an enzyme having mannuronan C-5-epimerase activity encompassing at least a portion of the sequence as disclosed in Figure 6. 15. A DNA compound according to claim 1 wherein said compound is not naturally occurring in an alginate producing host and comprises at least one A block and at least one R block isolated from a natural source or being derived synthetically. 16. A DNA compound according to claim 15 wherein said compound further comprises at least one S block. 17. A polypeptide having mannuronan C-5-epimerase activity wherein said polypeptide is not naturally occurring in an alginate producing host and comprises at least one A block and at least one R block isolated from a natural source or being derived synthetically. 18. A polypeptide according to claim 17 wherein said polypeptide further comprises at least one S block. 1/12 FIG. NO 1 is identical with sequence listing no. 2

Inventors: ERTESVAAG HELGA, LARSEN BJORN, SKJAAK-BRAEK GUDMUND, VALLA SVEIN, ERTESVAAG, HELGA, LARSEN, BJOERN, SKJAAK-BRAEK, GUDMUND, VALLA, SVEIN
Applicants: ERTESVAAG HELGA, LARSEN BJORN, NOBIPOL, PRONOVA BIOPOLYMER AS, SKJAAK BRAEK GUDMUND, VALLA SVEIN, PRONOVA BIOPOLYMER A.S
Application year: 1993
Main classification: C12N9
IPC codes: C07K14, C12N1, C12N9, C12N15, C12P19, C12R1