08/835,292, filed April 7,1997, now abandoned, the contents of which are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION The microbial desulfurization of fossil fuels has been an area of active investigation for over fifty years. The object of these investigations has been to develop biotechnology based methods for the pre- combustion removal of sulfur from fossil fuels, such as coal, crude oil and petroleum distillates. The driving forces for the development of desulfurization methods are the increasing levels of sulfur in fossil fuel and the increasingly stringent regulation of sulfur emissions. Monticello et al.,"Practical Considerations in Biodesulfurization of Petroleum,"IGT's 3d Intl.

Symp. on Gas, Oil, Coal and Env. Biotech., (Dec. 3-5, 1990) New Orleans, LA.

Many biocatalysts and processes have been developed to desulfurize fossil fuels, including those described in U. S. Patent Nos. 5,356,801,5,358,870,5,358,813, 5,198,341,5,132,219,5,344,778,5,104,801 and 5,002,888, incorporated herein by reference. Economic

analyses indicate that one limitation in the commercialization of the technology is improving the reaction rates and specific activities of the biocatalysts, such as the bacteria and enzymes that are involved in the desulfurization reactions. The reaction rates and specific activities (sulfur removed/hour/gram of biocatalyst) that have been reported in the literature are much lower than those necessary for optimal commercial technology. Therefore, improvements in the longevity and specific activity of the biocatalyst are desirable.

SUMMARY OF THE INVENTION The invention relates to a novel microorganism, designated Sphingomonas sp. strain AD109, as well as isolated proteins and nucleic acid sequences obtained from this microorganism. This microorganism was obtained using a soil enrichment process using 2- (2- hydroxyphenyl) benzenesulfinate as the sole sulfur source. A biologically pure sample of this microorganism has been isolated and characterized.

The invention also relates to a collection of desulfurization enzymes isolated from Sphingomonas sp. strain AD109 which, together, catalyze the oxidative desulfurization of dibenzothiophene (DBT).

In another embodiment, the invention includes an isolated nucleic acid molecule, such as a DNA or RNA nucleotide sequence or molecule, which encodes one or more of the Sphingomonas desulfurization enzymes, or a homologue or active fragment thereof. The invention also includes a recombinant microorganism containing one or more heterologous nucleic acid molecules which encode

one or more of the Sphingomonas desulfurization enzymes or homologues or active fragments thereof.

In a further embodiment, the invention provides a method of using the Sphingomonas microorganism or an enzyme preparation derived therefrom as a biocatalyst in the biocatalytic desulfurization of a fossil fuel containing organosulfur compounds. The method comprises the steps of (1) contacting the fossil fuel with an aqueous phase containing a Sphingomonas biocatalyst which is capable of biocatalytic desulfurization and, optionally, a flavoprotein, thereby forming a fossil fuel and aqueous phase mixture; (2) maintaining the mixture under conditions sufficient for sulfur oxidation and/or cleavage of the carbon-sulfur bonds of the organosulfur molecules by the biocatalyst, and (3) separating the fossil fuel having a reduced organic sulfur content from the resulting aqueous phase.

The invention also provides a method of oxidizing an organic compound. The method comprises the steps of: (1) contacting the organic compound with an aqueous phase containing a Sphingomonas biocatalyst comprising at least one enzyme capable of catalyzing at least one step in the oxidative cleavage of carbon-sulfur bonds, thereby forming an organic compound and aqueous phase mixture; (2) maintaining the mixture of step (1) under conditions sufficient for oxidation of the organic compound by the biocatalyst, thereby resulting in an oxidized organic compound, and, optionally, separating the oxidized organic compound from the aqueous phase.

BRIEF DESCRIPTION OF THE DRAWTNGS

Figures 1A, 1B, 1C and 1D together set forth the DNA sequence and the corresponding amino acid sequence of open reading frame 1 (ORF-1, dszA) of the nucleotide sequence required for desulfurization activity in Sphingomonas sp. strain AD109.

Figures 2A, 2B and 2C together set forth the DNA sequence and the corresponding amino acid sequence of open reading frame 2 (ORF-2, dszB) of the nucleotide sequence required for desulfurization activity in Sphingomonas sp. strain AD109.

Figures 3A, 3B and 3C together set forth the DNA sequence and the corresponding amino acid sequence of open reading frame 3 (ORF-3, dszC) of the nucleotide sequence required for desulfurization activity in Sphingomonas sp. strain AD109.

Figure 4 is a graph showing the disappearance of 2- (2-phenyl) benzenesulfinate (HPBS) and the appearance of 2-hydroxybiphenyl (2-HBP) in the presence of Sphingomonas AD109 cell-free lysates.

Figure 5 shows a physical map of the Sphingomonas dsz gene cluster.

Figures 6A, 6B, 6C, 6D, 6E, 6F and 6G together set forth the nucleotide sequence of the Sphingomonas dsz gene cluster.

Figure 7 is a physical map of the plasmid pDA296.

Figure 8 presents the results of a GAP analysis of the DszA proteins from Sphingomonas sp. strain AD109 and Rhodococcus IGTS8.

Figure 9 presents the results of a GAP analysis of the DszB proteins from Sphingomonas sp. strain AD109 and Rhodococcus IGTS8.

Figure 10 presents the results of a GAP analysis of the sequences of the DszC proteins from Sphingomonas sp. strain AD109 and Rhodococcus IGTS8.

Figure 11 is a physical map of the plasmid pEBCtac.

Figure 12 is a graph of substrate concentration versus time for the desulfurization of DBT, 2,8- dimethyl-DBT and 4,6-dimethyl-DBT by a cell-free Sphingomonas AD109 lysate.

Figure 13 is a graph of substrate a dprodcut concentrations versus time for the desulfurization of DBT by a cell-free Sphingomonas AD109 lysate.

Figure 14 is a graph of product concentration versus time for the desulfurization of 2,8-dimethyl-DBT and 4,6-dimethyl-DBT by a cell-free Sphingomonas AD109 lysate.

Figure 15 is a graph of substrate concentration versus time for the desulfurization of DBT, 2,8- dimethyl-DBT and 4,6-dimethyl-DBT by a cell-free Rhodococcus lysate.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery and isolation of a novel microorganism which is capable of selectively desulfurizing dibenzothiophene ("DBT"). As described in Example 1, this microorganism was obtained from soil samples obtained at sites contaminated with petroleum and petroleum by-products by a soil enrichment procedure using 2- (2-hydroxyphenyl) benzenesulfinate as the sole sulfur source. A biologically pure sample of the novel microorganism has been isolated and characterized. The microorganism is a motile, gram- negative rod. Based on a fatty acid analysis, as

described in Example 2, this microorganism has been identified as a Sphingomonas species, and designated strain AD-109. This microorganism has been deposited at the American Type Culture Collection (ATCC), 12301 Park Lawn Drive, Rockville, Maryland, U. S. A. 20852 under the terms of the Budapest Treaty and has been designated as ATCC Deposit No. 55954 on April 21,1997.

The novel microorganism of the invention can be grown by fermentation under aerobic conditions in the presence of a sulfur-free mineral salts medium (e. g., 4 g/L KHPO,, 4 g/L Na2HPO4, 2 g/L NH4Cl, 0.2 g/L MgCl2. 6H20, 0.001 g/L CaCl2. 2H20, and 0.001 g/L FeCl3. 6H20), containing a sulfur-free source of assimilable carbon such as glucose. The sole source of sulfur provided can be a heterocyclic organosulfur compound, such as dibenzothiophene or a derivative thereof.

Sphingomonas sp. strain AD109 expresses a collection of enzymes which together catalyze the conversion of DBT to 2-hydroxybiphenyl (also referred to as"2-HBP") and inorganic sulfur. An enzyme which catalyzes one or more steps in this overall process is referred to herein as a"desulfurization enzyme". The nucleic acid sequence required for this overall process has been identified and cloned using the general method described in U. S. Patent No. 5,356,801, the contents of which are incorporated herein by reference, and is set forth in Figure 6 (SEQ ID NO.: 12). This nucleic acid sequence (also referred to as the"Sphingomonas dsz sequence") comprises three open reading frames, designated ORF-1 (base pairs 442-1800, also set forth in Figures 1A-lD and SEQ ID NO.: 1), ORF-2 (base pairs 1800-2909, also set forth in Figures 2A-2C and SEQ ID

NO.: 3) and ORF-3 (base pairs 2906-4141, sequence also set forth in Figures 3A-3C and SEQ ID NO.: 5). The predicted amino acid sequences encoded by these open reading frames are set forth in Figures 1A-lD (ORF-1, SEQ ID NO: 2), Figures 2A-2C (ORF-2, SEQ ID NO.: 4) and Figures 3A-3C (ORF-3, SEQ ID NO.: 6). Each of these open reading frames is homologous to the corresponding open reading frame of Rhodococcus sp. IGTS8; the sequences of the Rhodococcus open reading frames are disclosed in U. S. Patent No. 5,356,801.

In one embodiment, the present invention provides an isolated nucleic acid molecule comprising one or more nucleotide sequences which encode one or more of the biodesulfurization enzymes of Sphingomonas sp. strain AD109. The isolated nucleic acid molecule can be, for example, a nucleotide sequence, such as a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence. Such a nucleic acid molecule comprises one or more nucleotide sequences which encode one or more of the amino acid sequences set forth in SEQ ID NO.: 2, SEQ ID NO.: 4, and SEQ ID NO.: 6. For example, the isolated nucleic acid molecule can comprise one or more of the nucleotide sequences of SEQ ID NO.: 1, SEQ ID NO.: 3, and SEQ ID NO.: 5, or a complement of any of these sequences. The isolated nucleic acid molecule can also comprise a nucleotide sequence which results from a silent mutation of one or more of the sequences set forth in SEQ ID NO.: 1, SEQ ID NO.: 3, and SEQ ID NO.: 5. Such a nucleotide sequence can result, for example, from a mutation of the native sequence in which one or more codons have been replaced with a degenerate codon, i. e., a codon which encodes the same

amino acid. Such mutant nucleotide sequences can be constructed using methods which are well known in the art, for example the methods discussed by Ausubel et al., Current Protocols in Molecular Biology, Wiley- Interscience, New York (1997) (hereinafter"Ausubel et al.") and by Sambrook et al., Molecular Cloning : A Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press (1992) (hereinafter"Sambrook et al."), each of which are incorporated herein by reference.

In another embodiment, the invention includes an isolated nucleic acid molecule comprising a nucleotide sequence which is homologous to one or more of the sequences of SEQ ID NO.: 1, SEQ ID NO.: 3, and SEQ ID NO.: 5, or complements thereof. Such a nucleotide sequence exhibits at least about 80% homology, or sequence identity, with one of these Sphingomonas nucleotide sequences, preferably at least about 90% homology or sequence identity. Particularly preferred sequences have at least about 95% homology or have essentially the same sequence. Preparation of mutant nucleotide sequences can be accomplished by methods known in the art as are described in Old, et al., Principles of Gene Manipulation, Fourth Edition, Blackwell Scientific Publications (1989), in Sambrook et al., and in Ausubel et al.

The invention further includes nucleic acid molecules which are useful as hybridization probes, for example, for the isolation of the Sphingomonas genes encoding desulfurization enzymes or identical or homologous genes from other organisms. Such molecules comprise nucleotide sequences which hybridize to all or a portion of the nucleotide sequence of SEQ ID NO.: 1,

SEQ ID NO.: 3 or SEQ ID NO.: 5 or to non-coding regions immediately (within about 1000 nucleotides) 5'or 3'of each open reading frame. The invention also includes an isolated nucleic acid molecule which comprises a fragment of one or more of the nucleotide sequences set forth in SEQ ID NO.: 1, SEQ ID NO.: 3 or SEQ ID NO.: 5 or complements of any of these sequences. Such a fragment will generally comprise at least about 20 or at least about 40 contiguous nucleotides and, preferably, at least about 50 contiguous nucleotides of one of the disclosed sequences. Preferably, the hybridization probe of the invention hybridizes to one of these sequences under stringent conditions, such as those set forth by Sambrook et al. and Ausubel et al. For example, under conditions of high stringency, such as high temperatures and low salt concentrations, only DNA molecules which are essentially exact matches, or complements, will hybridize, particularly if the probe is relatively short. Hybridization under conditions of lower stringency, such as low temperatures, low formamide concentrations and high salt concentrations, allows greater mismatch between the probe and the target DNA molecule. It is particularly preferred that the nucleic acid molecule hybridizes selectively to the disclosed sequence (s).

The nucleic acid molecules can be synthesized chemically from the disclosed sequences. Alternatively, the nucleic acid molecules can be isolated from a suitable nucleic acid library (such as a DNA library) obtained from a microorganism which is believed to possess the nucleic acid molecule (such as, Sphingomonas sp. strain AD109), employing hybridizing primers and/or

probes designed from the disclosed sequences. Such a method can result in isolating the disclosed molecules (or spontaneous mutants thereof) for use in preparing recombinant enzymes, confirming the disclosed sequences, or for use in mutagenizing the native sequences.

In yet another embodiment, the nucleic acid molecule of the present invention can be a nucleic acid molecule, such as a recombinant DNA molecule, resulting from the insertion into its chain by chemical or biological means, of one or more of the nucleotide sequences described above. Recombinant DNA includes any DNA synthesized by procedures using restriction nucleases, nucleic acid hybridization, DNA cloning, DNA synthesis or any combination of the preceding. Methods of construction can be found in Sambrook et al. and Ausubel et al., and additional methods are known by those skilled in the art.

The isolated nucleic acid molecule of the invention can further comprise a nucleotide sequence which encodes an oxidoreductase, such as a flavoprotein, such as a flavin reductase. For example, the nucleic acid molecule can encode an oxidoreductase which is native to Sphingomonas sp. strain AD109. The nucleic acid molecule can also encode the oxidoreductase denoted DszD described in copending U. S. Patent Application Serial No. 08/583,118; the flavin reductase from Vibrio harveyii described in copending U. S. Patent Application Serial No. 08/351,754; or the flavin reductase from Rhodococcus sp. IGTS8, described in copending U. S.

Patent Application Serial No. 08/735,963. The contents of each of these applications are incorporated herein by reference.

The invention also includes a plasmid or vector comprising a recombinant DNA sequence or molecule which comprises one or more of the nucleic acid molecules, e. g. nucleotide sequences, of the invention, as described above. The terms"plasmid"and"vector"are intended to encompass any replication competent or replication incompetent plasmid or vector capable of having foreign or exogenous DNA inserted into it by chemical or biological means and subsequently, when transformed into an appropriate non-human host organism, of expressing the product of the foreign or exogenous DNA insert (e. g., of expressing the biocatalyst and flavoprotein of the present invention). In addition, the plasmid or vector is receptive to the insertion of a DNA molecule or fragment thereof containing the gene or genes of the present invention, said gene or genes encoding a biocatalyst as described herein. Procedures for the construction of DNA plasmid vectors include those described in Sambrook et al. and Ausubel et al. and others known by those skilled in the art.

The plasmids of the present invention include any DNA fragment containing a nucleotide sequence as described above. The DNA fragment should be transmittable, for example, to a host microorganism by transformation or conjugation. Procedures for the construction or extraction of DNA plasmids include those described in Sambrook et al. and Ausubel et al., and others known by those skilled in the art. In one embodiment, the plasmid comprises a nucleotide sequence of the invention operatively linked to a competent or functional regulatory sequence. Examples of suitable regulatory sequences include promoters, enhancers,

transcription binding sites, ribosomal binding sites, transcription termination sequences, etc.

In one preferred embodiment, the regulatory or promoter sequences are those native to the Sphingomonas operon containing the genes disclosed herein. In yet another embodiment, one or more regulatory sequences (e. g. the promoter) is native to the selected host cell for expression. The promoter can be selected so that the gene or genes are inducible or constitutively expressed. Furthermore, the sequences can be regulated individually or together, as an operon. Examples of suitable promoters include the E. coli lac and tac promoters and the Pseudomonas PG promoter (Yen, J.

Bacteriol. 173 : 5328-5335 (1991)). An example of such a plasmid and its construction are described in Example 8.

In another embodiment, the invention relates to a recombinant or transformed non-human host organism which contains a heterologous DNA molecule of the invention as described above. The recombinant non-human host organisms of the present invention can be created by various methods by those skilled in the art. Any method for introducing a recombinant plasmid, such as a plasmid of the invention described above, into the organism of choice can be used, and a variety of such methods are described by Sambrook et al. and Ausubel et al. For example, the recombinant plasmid can be introduced via a suitable vector by transformation, conjugation, transduction or electroporation. By the term'non-human host organism"is intended any non-human organism capable of the uptake and expression of foreign, exogenous or recombinant DNA.

The recombinant microorganism can be derived from a host organism which does not express a native desulfurization biocatalyst. Such microorganisms include bacteria and yeasts, e. g., E. coli, Bacillus, and non-desulfurizing pseudomonads (as described in U.

S. Patent Application Serial Number 08/851,088). In another embodiment, the recombinant microorganism is derived from a host organism which expresses a native biodesulfurization catalyst. Preferred microorganisms of this type are Rhodococcus sp. IGTS8 (ATCC 53968), recombinant microorganisms comprising one or more of the IGTS8 desulfurizing genes and Sphingomonas sp. strain AD109. Other desulfurizing microorganisms which are suitable host organisms include Corynebacterium sp. strain SY1, as disclosed by Omori et al., Appl. Env.

Microbiol., 58 : 911-915 (1992); Rhodococcus erythropolis D-1, as disclosed by Izumi et al., Appl.

Env. Microbiol., 60: 223-226 (1994); the Arthrobacter strain described by Lee et al., Appl. Environ.

Microbiol. 61 : 4362-4366 (1995); the Agrobacterium strain disclosed by Constanti et al., Enzyme Microb.

Tech. 19 : 214-219 (1996) and the Rhodococcus strains (ATCC 55309 and ATCC 55310) disclosed by Grossman et al., U. S. Patent No. 5,607,857, each of which is incorporated herein by reference in its entirety. Each of these microorganisms produces one or more enzymes (protein biocatalysts) that catalyze one or more reactions in the desulfurization of DBT.

The invention also relates to desulfurization enzymes which can be isolated from Sphingomonas sp. strain AD109. These include desulfurization enzymes which catalyze one or more steps in the oxidative

desulfurization of DBT. The enzyme encoded by ORF-2 has been partially purified and exhibits 2- (2- hydroxyphenyl) benzenesulfinate (HPBS) desulfinase activity and has an apparent molecular weight by denaturing gel electrophoresis of about 40,000 daltons.

In one embodiment, the invention includes an isolated desulfurization enzyme from Sphingomonas sp. strain AD109 using methods and assays which are known the art, for example, the methods used by Gray et al. to isolate and characterize desulfurization enzymes from Rhodococcus IGTS8 (Gray et al., Nature Biotech. 14 : 1705-1709 (1996)). These enzymes can be isolated or purified from the cell by lysing the cell and subjecting the cell lysate to known protein purification methods, and testing the fractions obtained thereby for the desired enzymatic activity. Examples of suitable protein purification methods include ammonium sulfate precipitation, ultrafiltration, diafiltration, immunoabsorption, anion exchange chromatography, gel filtration chromatography and hydrophobic interaction chromatography. The enzymes of the invention can also be recombinant proteins produced by heterologous expression of a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO.: 1, SEQ ID NO.: 3 or SEQ ID NO.: 5; or a mutation or fragment thereof, as discussed above. When the recombinant organism is derived from a non-Sphingomonas host, the recombinant proteins can be prepared in a form which is substantially free of other Sphingomonas proteins.

The invention also includes an isolated enzyme having an amino acid sequence which is homologous to the amino acid sequence of SEQ ID NO.: 2, SEQ ID NO.: 4 or

SEQ ID NO.: 6, or fragments thereof. The term "homologous"or"homologue", as used herein, describes a protein (which is not obtained from Rhodococcus or Rhodococcus sp IGTS8) having at least about 80% sequence identity or homology with the reference protein, and preferably about 90% sequence homology, in an amino acid alignment. Most preferably, the protein exhibits at least about 95% homology or essentially the same sequence as the disclosed sequence. An amino acid alignment of two or more proteins can be produced by methods known in the art, for example, using a suitable computer program, such as BLAST (Altschul et al., J.

Mol. Biol. 215 : 403-410 (1990)). A homologous protein can also have one or more additional amino acids appended at the carboxyl terminus or amino terminus, such as a fusion protein.

The homologous enzymes described herein can be native to an organism, such as a desulfurizing microorganism, including Sphingomonas sp. strain AD109 and mutants thereof. Such enzymes can be isolated from such sources using standard techniques and assays, as are described in the Exemplification and others known in the art. For example, the Sphingomonas desulfurization enzymes can be used to induce the formation of antibodies, such as monoclonal antibodies, according to known methods. The antibodies can then be used to purify the desulfurization enzymes from a desulfurizing organism via affinity chromatography, as is well known in the art.

The homologous enzymes of the invention can also be non-naturally occurring. For example, a homologous enzyme can be a mutant desulfurization enzyme which has

a modified amino'acid sequence resulting from the deletion, insertion or substitution of one or more amino acid residues in the amino acid sequence of a Sphingomonas desulfurization enzyme. Such amino acid sequence variants can be prepared by methods known in the art. For example, the desired polypeptide can be synthesized in vitro using known methods of peptide synthesis. The amino acid sequence variants are preferably made by introducing appropriate nucleotide changes into a DNA molecule encoding the native enzyme, followed by expression of the mutant enzyme in an appropriate vector, such as E. coli. These methods include site-directed mutagenesis or random mutagenesis, for example.

Particularly preferred mutants include those having amino acid sequences which include the amino acid residues which are encoded by both SEQ ID NO.: 1, SEQ ID NO.: 3 or SEQ ID NO.: 5 and the corresponding open reading frame of Rhodococcus sp. IGTS8, as disclosed in U. S. Patent No. 5,356,801. That is, these mutants include the amino acid residues which are conserved in these two organisms in an amino acid alignment. Mutants which result from conservative substitution of one or more of these conserved residues, as well as non- conserved residues, are also included. Conservative and non-conservative substitutions (including deletions and insertions) can be made in non-conserved regions of the amino acid sequence and mutants resulting from both conservative and non-conservative substitutions of these residues are included herein.

Conservative substitutions are those in which a first amino acid residue is substituted by a second

residue having similar side chain properties. An example of such a conservative substitution is replacement of one hydrophobic residue, such as valine, with another hydrophobic residue, such as leucine. A non-conservative substitution involves replacing a first residue with a second residue having different side chain properties. An example of this type of substitution is the replacement of a hydrophobic residue, such as valine, with an acidic residue, such as glutamic acid.

The two primary variables in the construction of amino acid sequence variants are (1) the location of the mutation site and (2) the nature of the mutation. These variables can be manipulated to identify amino acid residues at the active site of the enzyme. For example, an amino acid substitution which yields a mutant enzyme having significantly different activity than the native enzyme suggests that the substituted amino acid residue is at the active site. Such mutants can have the same or similar, increased or decreased activity relative to that of the native enzyme.

Amino acids can be modified, for example, by substituting first with a conservative choice, followed by non-conservative choices depending upon the results achieved, by deleting the target residue (s) or by inserting residues adjacent to a particular site.

Variants can also be constructed using a combination of these approaches.

The proteins of the present invention can be produced using techniques to overexpress the gene, as are described by Sambrook et al. and Ausubel et al.

Improved expression, activity or overexpression of the

Sphingomonas desulfurization enzymes (in Sphingomonas sp AD 109 or in recombinant host cells harboring the disclosed nucleic acid molecules) can also be accomplished by mutagenesis. Suitable mutagens include radiation, e. g., ultraviolet radiation, and chemical mutagens, such as N-methyl-N'-nitroso-guanidine, hydroxylamine, ethylmethanesulfonate and nitrous acid.

Furthermore, spontaneous mutants can be selected where the microorganism is subjected to an enrichment culture, as exemplified herein. The mutagenesis and subsequent screening for mutants harboring increased enzymatic activity can be conducted according to methods generally known in the art.

The present invention also provides a method of desulfurizing a carbonaceous material containing organosulfur molecules. The carbonaceous material can be, for example, a DBT-containing material or a fossil fuel, such as petroleum, a petroleum distillate fraction or coal. The method comprises the steps of (1) contacting the carbonaceous material with an aqueous phase containing a Sphingomonas-derived biocatalyst comprising at least one enzyme capable of catalyzing at least one step in the oxidative cleavage of carbon- sulfur bonds, thereby forming a carbonaceous material and aqueous phase mixture; (2) maintaining the mixture of step (1) under conditions sufficient for biocatalysis; and (3) separating the carbonaceous material having a reduced organic sulfur content from the resulting aqueous phase.

The term"Sphingomonas-derived biocatalyst", as used herein, is a biocatalyst which includes one or more desulfurization enzymes encoded by SEQ ID NO. : 1, SEQ ID

NO.: 3 and SEQ ID NO.: 5; or a mutant or homologue thereof. In one embodiment, the biocatalyst is a microorganism, such as Sphingomonas sp. strain AD109.

The biocatalyst can also be a recombinant organism which contains one or more heterologous nucleotide sequences or nucleic acid molecules as described above.

Although living microorganisms (e. g., a culture) can be used as the biocatalyst herein, this is not required. Biocatalytic enzyme preparations that are useful in the present invention include microbial lysates, extracts, fractions, subfractions, or purified products obtained by conventional means and capable of carrying out the desired biocatalytic function.

Generally, such enzyme preparations are substantially free of intact microbial cells. In a particularly preferred embodiment, the biocatalyst is overexpressed in the recombinant host cell (such as a cell which contains more than one copy of the gene or genes).

Enzyme biocatalyst preparations suitable for use herein can optionally be affixed to a solid support, e. g., a membrane, filter, polymeric resin, glass particles or beads, or ceramic particles or beads. The use of immobilized enzyme preparations facilitates the separation of the biocatalyst from the treated fossil fuel which has been depleted of refractory organosulfur compounds.

A fossil fuel that is suitable for desulfurization treatment according to the present invention is one that contains organic sulfur. Such a fossil fuel is referred to as a"substrate fossil fuel". Substrate fossil fuels that are rich in thiophenic sulfur are particularly suitable for desulfurization according to the method

described herein. Examples of such substrate fossil fuels include Cerro Negro or Orinoco heavy crude oils; Athabascan tar and other types of bitumen; petroleum refining fractions such as gasoline, kerosene, diesel, fuel oil, residual oils and miscellaneous refinery by- products; shale oil and shale oil fractions; and coal- derived liquids manufactured from sources such as Pocahontas #3, Lewis-Stock, Australian Glencoe or Wyodak coal.

In the petroleum extraction and refining arts, the term"organic sulfur"is generally understood as referring to organic molecules having a hydrocarbon framework to which one or more sulfur atoms are covalently joined. These sulfur atoms can be directly bonded to the hydrocarbon framework, e. g., by one or more carbon-sulfur bonds, or can be present in a substituent bonded to the hydrocarbon framework of the molecule, e. g., a sulfate group. The general class of organic molecules having one or more sulfur heteroatoms are sometimes referred to as"organosulfur compounds".

The hydrocarbon portion of these compounds can be aliphatic and/or aromatic.

Sulfur-bearing heterocycles, such as substituted and unsubstituted thiophene, benzothiophene, and dibenzothiophene, are known to be stable to conventional desulfurization treatments, such as hydrodesulfurization (HDS). Sulfur-bearing heterocycles can have relatively simple or relatively complex chemical structures. In complex heterocycles, multiple condensed aromatic rings, one or more of which can be heterocyciic, are present.

The difficulty of desulfurization generally increases with the structural complexity of the molecule. That

is, refractory behavior is particularly accentuated in complex sulfur-bearing heterocycles, such as dibenzothiophene (DBT, C12H8s).

Much of the residual post-HDS organic sulfur in fossil fuel refining intermediates and combustible products is thiophenic sulfur. The majority of this residual thiophenic sulfur is present in DBT and derivatives thereof having one or more alkyl or aryl groups attached to one or more carbon atoms present in one or both flanking benzo rings. DBT itself is accepted as a model compound illustrative of the behavior of the class of compounds encompassing DBT and derivatives thereof in reactions involving thiophenic sulfur (Monticello and Finnerty, Ann. Rev. Microbiol., 39 : 371-389 (1985)). DBT and derivatives thereof can account for a significant percentage of the total sulfur content of particular crude oils, coals and bitumen.

For example, these sulfur-bearing heterocycles have been reported to account for as much as 70 wt% of the total sulfur content of West Texas crude oil, and up to 40 wt% of the total sulfur content of some Middle East crude oils. Thus, DBT is considered to be particularly relevant as a model compound for the forms of thiophenic sulfur found in fossil fuels, such as crude oils, coals or bitumen of particular geographic origin, and various refining intermediates and fuel products manufactured therefrom (Monticello and Finnerty (1985), supra).

Another characteristic of DBT and derivatives thereof is that, following a release of fossil fuel into the environment, these sulfur-bearing heterocycles persist for long periods of time without significant biodegradation. Gundlach et al., Science 221 : 122-129

(1983). Thus, most prevalent naturally occurring microorganisms do not effectively metabolize and break down sulfur-bearing heterocycles.

Biocatalytic desulfurization (biocatalysis or BDS) is the excision (liberation or removal) of sulfur from organosulfur compounds, including refractory organosulfur compounds such as sulfur-bearing heterocycles, as a result of the oxidative, preferably selective, cleavage of carbon-sulfur bonds in said compounds by a biocatalyst. BDS treatment yields the desulfurized combustible hydrocarbon framework of the former refractory organosulfur compound, along with inorganic sulfur substances which can be readily separated from each other by known techniques such as fractional distillation or water extraction. For example, DBT is converted into 2-hydroxybiphenyl when subjected to BDS treatment. A suitable biocatalyst for BDS comprises Sphingomonas sp. strain AD109 or an enzyme preparation derived therefrom, optionally, in combination with one or more additional non-human desulfurizing organisms (e. g., microorganisms); or an enzyme preparation derived from such an organism.

Suitable additional desulfurizing organisms include those described above.

The specific activity of a given biocatalyst is a measure of its biocatalytic activity per unit mass.

Thus, the specific activity of a particular biocatalyst depends on the nature or identity of the microorganism used or used as a source of biocatalytic enzymes, as well as the procedures used for preparing and/or storing the biocatalyst preparation. The concentration of a particular biocatalyst can be adjusted as desired for

use in particular circumstances. For example, where a culture of living microorganisms, such as Sphingomonas sp. strain AD109, is used as the biocatalyst preparation, a suitable culture medium lacking a sulfur source other than sulfur-bearing heterocycles can be inoculated with suitable microorganisms and grown until a desired culture density is reached. The resulting culture can be diluted with additional medium or another suitable buffer, or microbial cells present in the culture can be retrieved e. g., by centrifugation, and resuspended at a greater concentration than that of the original culture. The concentrations of microorganism and enzyme biocatalyst can be adjusted similarly. In this manner, appropriate volumes of biocatalyst preparations having predetermined specific activities and/or concentrations can be obtained.

In the biocatalytic desulfurization stage, the liquid fossil fuel containing sulfur-bearing heterocycles is combined with the biocatalyst. The relative amounts of biocatalyst and liquid fossil fuel can be adjusted to suit particular conditions, or to produce a particular level of residual sulfur in the treated, deeply desulfurized fossil fuel. The amount of biocatalyst preparation to be combined with a given quantity of liquid fossil fuel will reflect the nature, concentration and specific activity of the particular biocatalyst used, as well as the nature and relative abundance of inorganic and organic sulfur compounds present in the substrate fossil fuel and the degree of deep desulfurization sought or considered acceptable.

The method of desulfurizing a fossil fuel of the present invention involves two aspects. First, a host

organism or biocatalytic preparation obtained therefrom is contacted with a fossil fuel to be desulfurized.

This can be done in any appropriate container, optionally fitted with an agitation or mixing device.

The mixture is combined thoroughly and maintained or allowed to incubate for a sufficient time to allow for biocatalysis. In one embodiment, an aqueous emulsion or microemulsion is produced with an aqueous culture of the organism or enzyme fraction and the fossil fuel, allowing the organism to propagate in the emulsion while the expressed biocatalyst cleaves carbon-sulfur bonds.

Variables such as temperature, pH, oxidation levels, mixing rate and rate of desulfurization will vary according to the nature of the biocatalyst used.

Optimal parameters can generally be determined through no more than routine experimentation.

When the fossil fuel is a liquid hydrocarbon, such as petroleum, the desulfurized fossil fuel and the aqueous phase can form an emulsion. The components of such emulsions can be separated by a variety of methods, such as those described in U. S. Patent No. 5,358,870 and U. S. Patent Application Serial No. 08/640,129, which are incorporated herein by reference. For example, some emulsions reverse spontaneously when maintained under stationary conditions for a suitable period of time.

Other emulsions can be reversed by adding an additional amount of an aqueous phase. Still other emulsions can be separated by the addition of a suitable chemical agent, such as a demulsifying agent or by employing suitable physical conditions, such as a particular temperature range.

The biocatalyst can be recovered from the aqueous phase, for example, by centrifugation, filtration or lyophilization. When the biocatalyst is a microorganism, the biocatalyst can be resuspended in fresh sulfur-free nutrient medium and/or any fresh microorganism culture as necessary to reconstitute or replenish to the desired level of biocatalytic activity.

The biocatalyst can then be reintroduced into the reaction system.

Several suitable techniques for monitoring the rate and extent of desulfurization are well-known and readily available to those skilled in the art. Baseline and time course samples can be collected from the incubation mixture, and prepared for a determination of the residual organic sulfur in the fossil fuel. The disappearance of sulfur from organosulfur compounds, such as DBT, in the sample being subjected to biocatalytic treatment can be monitored using, e. g., X- ray fluorescence (XRF) or atomic emission spectrometry (flame spectrometry). Preferably, the molecular components of the sample are first separated, e. g., by gas chromatography.

Without being limited to any particular mechanism or theory, it is believed that the pathway of the desulfurization reaction in Sphingomonas sp. strain AD109 and other desulfurizing organisms, such as Rhodococcus sp. IGTS8, is set forth below:

Here the flavin reductase provides an electron transport chain which delivers, via FMNH2, the reducing equivalents from NADH (or other electron donor) to the enzymes DszC and/or DszA. The enzyme DszC is responsible for the biocatalysis of the oxidation reaction of DBT to DBTO2.

The enzyme DszA is responsible for the reaction of DBTO2 to 2- (2-hydroxyphenyl) benzenesulfinate (HPBS). The enzyme DszB catalyzes the conversion of HPBS to 2- hydroxybiphenyl and inorganic sulfur.

Another method of use of the Sphingomonas desulfurization enzymes, or mutants, homologues or active fragments thereof, is as a biocatalyst for the oxidation of organic compounds, such as substituted or unsubstituted dibenzothiophenes. The method comprises the steps of (1) contacting the organic compound with an aqueous phase containing a Sphingomonas-derived biocatalyst comprising at least one enzyme capable of

catalyzing at least one step in the oxidative cleavage of carbon-sulfur bonds, thereby forming an organic compound and aqueous phase mixture; (2) maintaining the mixture of step (1) under conditions sufficient for oxidation of the organic compound by the biocatalyst, thereby resulting in an oxidized organic compound, and, optionally, separating the oxidized organic compound from the aqueous phase. In one embodiment, the organic compound is a heteroorganic compound, such as an organonitrogen compound or an organosulfur compound. In one embodiment, the organic compound is an organosulfur compound which is a component of a fossil fuel, such as petroleum or a petroleum distillate fraction. In a second embodiment, the organic compound is a substituted or unsubstituted indole, as described in U. S.

Provisional Patent Application Serial Number 60/020563, filed July 2,1996, which is incorporated herein by reference.

The enzyme encoded by the nucleotide sequence of ORF-3 catalyzes the oxidation of dibenzothiophene to dibenzothiophene-5,5-dioxide (dibenzothiophene sulfone), and the enzyme encoded by the nucleotide sequence of ORF-1 catalyzes the oxidation of dibenzothiophene-5,5- dioxide to 2- (2-hydroxyphenyl) benzenesulfinate (also referred to as"HPBS"). In one embodiment the biocatalyst comprises the enzyme encoded by ORF-3, or a mutant, homologue or active fragment thereof; the organosulfur compound is substituted or unsubstituted dibenzothiophene; and the oxidized organosulfur is a substituted or unsubstituted dibenzothiophene-5,5- dioxide or dibenzothiophene-5-oxide (dibenzothiophene sulfoxide). In another embodiment the biocatalyst

comprises the enzymes encoded by ORF-1 and ORF-3, or a mutant, homologue or active fragment thereof; the organosulfur compound is a substituted or unsubstituted dibenzothiophene; and the oxidized organosulfur compound is a substituted or unsubstituted 2- (2- hydroxyphenyl) benzenesulfinate. In yet another embodiment, the biocatalyst comprises the enzyme encoded by ORF-1 or a mutant, homologue or active fragment thereof; the organosulfur compound is a substituted or unsubstituted dibenzothiophene-5,5-dioxide; and the oxidized organosulfur compound is a substituted or unsubstituted 2- (2-hydroxyphenyl) benzenesulfinate.

The oxidized organosulfur compound can, optionally, be further processed, for example, via a non-biological process or an enzyme-catalyzed reaction. In one embodiment, the oxidized organosulfur compound is desulfurized in a process employing suitable desulfurization enzymes from an organism other than a Sphingomonas.

The biocatalyst can be an organism, such as Sphingomonas sp. strain AD109, a desulfurizing mutant thereof, or a recombinant organism or enzyme preparation, as discussed above. When the organosulfur compound is a component of a fossil fuel, suitable reaction conditions and fossil fuel sources can be determined as described above.

The invention will now be further illustrated by the way of the following examples.

EXAMPLES General Methods and Materials

Bacterial strains and plasmids E. coli DH106 (F-mcrA A(mrr-hsdRMS-mcrBC) phi80dlacZAM15 AlacX74 deoR recAl endA1 araAl39 A (ara, leu) 7697 galU galK lambda-rpsL nupG ; Gibco-BRL, Gaithersburg, MD) was used as the cloning host. Plasmids pUC18 (ApR ; Vieria and Messing, Gene 19 : 259-268, (1982)), pOK12 (KmR ; Vieria and Messing, Gene 100 : 189-194 (1991)) and pSL1180 (ApR ; Brosius, DNA 8 : 759, (1989)) were used as cloning vectors. Plasmid pEBCtac (ApR TcR lacIq tac, shown in Figure 11, was used to overexpress the Sphingomonas dszB in E. coli.

Media and Reagents Luria broth (LB) medium was routinely used to propagate E. coli. LB medium is 1% tryptone (Difco), 0.5% yeast extract (Difco) and 0.5% NaCl. Rich medium (RM) was used to propagate Sphingomonas strain AD109.

RM medium is 0.8% nutrient broth, 0.05% yeast extract and 1% glucose. 2YT medium, used in gene expression studies, is 1.6% tryptone, 1% yeast extract and 0.5% NaCl. Basal salts medium (BSM-glucose) contained the following (per liter): phosphate buffer 100 mmol (pH 7.2); glucose, 20 g : NH4Cl, 2 g ; MgCl2 6H20, 644 mg; MnCl24H2O, 1 mg; nitriloacetic acid, 0.1 g ; FeCl2 4H20, 2.6 mg; Na2B407 10H20, 0.1 mg; CuCl2 2H20, 0.15 mg; Co (NO3) 2 6H20, 0.125 mg; ZnCl2, 2.6 mg; CaCl2 2H20, 33 mg; (NH4) 6Mo7024 4H20, 0.09 mg; and EDTA, 1.25 mg. When required the sulfur source was either 2 mM MgSO4, 300 ßM Dibenzothiophene (DBT), 300, uM Dibenzothiophene sulfone (DBTO2) or 300, uM 2-(2-hydroxyphenyl) benzenesulfinate

(HPBS). For solid media, agar or agarose was added at a concentration of 1.5% (wt/wt). The antibiotic concentrations for E. coli were as follows: ampicillin, 100 ßg/ml ; kanamycin, 30 Hg/ml ; tetracycline, 10 Fg/ml.

DNA Methods Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs, Inc. (Beverly, MA) and used as recommended by the supplier. Chromosomal DNA was isolated by the method described by Woo et al., BioTechniques 13: 696-698 (1992). Small scale plasmid preparations from E. coli were carried out as described by Birboim and Doly, Nuc. Acids Res. 7 : 1513-1523 (1979). Larger scale DNA preparations were carried out with Midi-prep columns from Qiagen (Chatsworth, CA).

DNA fragments were purified from agarose gels after electrophoretic separation by the method of Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA 76: 615-619 (1979). DNA fragments were cloned into vectors by using techniques described by Sambrook et al.

Degenerate oligonucleotide probes were end-labeled using standard digoxygenin protocols according to the Boehringer Mannheim DIG Oligonucleotide 3'-End Labeling Kit (Cat. No. 1362372). Hybridization was performed in 5X SSC with blocking solution containing 50% ultrapure deionized formamide at 42°C overnight (16 hr). Detection of hybrids was by enzyme immunoassay according to the Boehringer Mannheim Nonradioactive DIG DNA Labeling and Detection Kit (Cat. No. 1093657).

DNA samples were sequenced by SeqWright (Houston, TX) using a dye-terminator cycling sequencing kit from Perkin Elmer and the 373A and 377 ABI automatic DNA

sequencer. The sequence was extended by synthesizing overlapping oligonucleotides to previously read sequence. The synthesized oligonucleotides were used as primers for continuing sequence reactions. Sequencing reads were assembled and edited to 99.99% accuracy using Genecode's Sequencher, version 3.0 computer software.

DNA and protein sequence analysis was performed with the MacVector software program (Oxford Molecular Group, Campbell, CA). Nucleotide and amino acid sequences were compared to sequences in the available databases using BLAST. The Wisconsin Genetics Computer Group (GCG) software (Devereux et al., Nucl. Acids Res.

12 : 387-395 (1984)) program GAP was used to generate comparisons of the protein sequences.

Transformation of E. coli Plasmid DNA was introduced into E. coli DHlOß by electroporation. Competent ElectroMAX DH106 (Gibco-BRL, Gaithersburg, MD) were used according to the manufacturer's suggestions.

Preparation of cell-free extracts Cells grown in the appropriate medium were concentrated to an optical density at 600 nm of 50 by centrifugation and resuspended in 10 mM phosphate buffer (pH 7.0). Cells were disrupted in a French press and debris was removed by centrifugation at 32,000 x g for 20 min. Cell lysates were stored on ice at 4 C.

Desulfurization assays and analytical analysis HPBS desulfinase activity was assayed by the ability of cell-free lysates to convert HPBS (substrate)

to 2-HBP (product) in a one hour assay at 30 C. The amounts of product made and substrate consumed during the reaction were quantitated by high-pressure liquid chromatography (HPLC) analysis. HPBS desulfinase activity was also measured by fluorescence spectroscopy.

In a typical enzyme assay, enzyme activity is determined by the change in fluorescence at an excitation wavelength of 288 nm and an emission wavelength of 414 nm as HPBS is converted to 2-HBP. The assay is initiated by the addition of 20-100 Hg total protein to a 3 mL solution of 200 uM HPBS in 50 mM phosphate buffer pH 7.5 containing 0.1 M NaCl.

Expression studies E. coli DH106 harboring the Sphingomonas dszB overexpression plasmid pDA296 was inoculated into 100ml of 2medium containing ampicillin and allowed to grow with shaking at 30C. At an OD6oo of approximately 0.3, the culture was divided into two parts. One half of the culture was induced by the addition of isopropylthio-ß- galactoside (IPTG) (final conc. 1 mM) and the remaining culture was used as an uninduced control (no IPTG was added). Following incubation for an additional 3 hr, both cultures were harvested and cell-free lysates were prepared.

Protein purification and N-terminal sequencing Sphingomonas AD109 cell paste was resuspended in an approximately equal weight of 25 mM phosphate buffer pH 7.5 containing 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 10 Hg/mL DNAse and 1 mM phenylmethylsulfonyl fluoride and passed through a French press mini-cell at about

20,000 psi. Cell debris was removed by centrifugation and the cell lysate was fractionated over an Econo-Pac High Q cartridge manufactured by Bio-Rad. A linear 0-0.5 M NaCl gradient was used to elute the bound protein into fractions. The active fractions were identified by a 2-HBP fluorescence enzyme assay (excitation/emission wavelengths set at 288/414 nm).

The active fractions were pooled and desalted over a Bio-Rad P6 gel filtration cartridge, diluted to 1.7 M ammonium sulfate and fractionated over a Phenyl Superose HR 5/5 column manufactured by Pharmacia. A linear 1.7-0.0 M ammonium sulfate gradient was used to elute protein into fractions. Active fractions were identified and pooled as described above. Identity and purity of the AD109 HPBS desulfinase protein was also determined by SDS-PAGE and Western blots using antibodies generated against the DszB protein from Rhodococcus erythroplis strain IGTS8. N-terminal microsequencing of the HPBS desulfinase was carried out by Edman degradation after transfer of the purified protein to a polyvinylidene difluoride (PVDF) membrane.

SDS-PAGE and Western Blot Analysis Protein separations were done with Novex (San Diego, CA) precast 10% polyacrylamide gels with Tris-Glycine-sodium dodecyl sulfate (SDS) (Laemmli) running buffer. Western blot analysis was carried out by first transferring the proteins electrophoretically to nitrocellulose membranes as recommended by Biorad (Hercules, CA). Blots were treated with antisera raised against the purified IGTS8 DszB protein (primary antibody) and then with goat anti-rabbit antisera

conjugated to horseradish peroxidase as the secondary antibody. Finally, the proteins were detected with a horseradish peroxidase catalyzed chemiluminescent reaction.

Example 1 Soil enrichments and isolation of a microorganism that can use HPBS as a sole sulfur source Three independent soil samples from oil- contaminated sites were used to perform soil enrichments for microorganisms able to use HPBS as a sole sulfur source. Approximately 5 grams of each soil sample was placed into a sterile 250 ml flask along with 50 ml of BSM Glucose medium containing HPBS (300 AM) as the sole source of sulfur. Following incubation for 96 hrs at 30°C, a 3 ml sample of each enrichment was transferred to fresh BSM Glucose medium containing HPBS. After 72 hrs, one of the three flasks (flask #3) showed visible turbidity, while the two remaining flasks showed no visible increase in turbidity (even after more than a week of incubation). Microscopic analysis of the contents of flask #3 revealed the presence of a mixed population of bacterial cells (i. e., sessile and motile rods of varying shapes; large and small coccoid shaped bacteria). After repeated liquid subculture enrichments with HPBS as the sole sulfur source, the contents of the flask was plated onto several RM and LB agar plates.

Following incubation at 30°C, a variety of microorganisms with different colony morphologies was present. Analysis of individual colonies from these plates identified a pure isolate that efficiently used

HPBS as a sole sulfur source. This strain, designated AD109, was selected for further analysis.

Example 2 Characterization and identification of strain AD109 The HPBS utilizing strain AD109 is a Gram-negative, motile rod that forms distinctive yellow colonies on agar plates. It grows somewhat poorly on LB agar, but grows rather well on RM agar plates. Like Rhodococcus IGTS8, strain AD109 also has the ability to produce clearing zones on a BSM Glucose DBT-sulfone plate. The optimal growth temperature of AD109 was found to be between 30 and 37 C.

Based on fatty acid analysis (Acculab, Inc., Newark, DE), this strain was identified as a Sphingomonas species. Strain AD109 was a"good"match to S. paucimobilis (formerly Pseudomonas paucimobilis) based on its"similarity index". The similarity index is a mathematical expression of the extent to which the fatty acid profile of a given unknown matches the mean profile for an organism in the TSBA database. Strain AD109 had an index value of 0.426 which indicates that it is from a strain of a species that differs significantly from those represented in the database.

A similarity index of 0.5 or above is considered to be an"excellent"match (a value of 1.0 being the highest possible). On the other hand, an index below 0.3 indicates that the sample is from a species that is not likely to be in the database. Based on 16S rRNA sequence analysis and the presence of sphingoglycolipids, Yabuuchi et al. (Microbiol. Immunol.

34 : 99-119 (1990)) proposed that P. paucimobilis be reclassified and placed into the genus Sphingomonas.

Example 3 Growth characteristics of Sphingomonas species strain AD109 Evidence for the existence of an HPBS desulfinase activity was demonstrated by monitoring the supernatant of a AD109 culture growing in BSM Glucose HPBS (300 RM).

By the time the culture was well into stationary phase all of the HPBS had been converted with no apparent accumulation of identifiable intermediates. There was, however, a transient production of a small amount of 2-HBP, as determined by HPLC analysis, which also disappeared with time. This preliminary result suggested that AD109 may also be capable of metabolizing 2-HBP. Sphingomonas strain AD109 was also capable of utilizing DBT-sulfone (DBTO2) as a sole sulfur source.

The ability to utilize DBT-sulfone as a sole sulfur source suggests that strain AD109 may also contain a gene that encodes DBT-sulfone monooxygenase activity.

During the course of growth studies it was discovered that strain AD109 could utilize DBT as a sole sulfur source. While growing with DBT, however, the culture supernatant takes on a very characteristic orange/brown color with an absorption maximum of approximately 470 nm. Orange-colored oxidation products have been previously identified in a number of Pseudomonas species that are capable c-degrading DBT (Monticello et al., Appl. Environ. Microbiol. 49 : 756- 760 (1985)); Foght and Westlake, Can. J. Microbiol. 36 : 718-724 (1990)). No such color development was detected

in cultures growing with either HPBS or DBT-sulfone as sulfur sources.

Example 4 Demonstration of HPBS desulfinase activity in AD109 cell-free lysates A cell-free lysate prepared from a culture of Sphingomonas strain AD109 (grown in BSM Glucose medium containing HPBS) was used in a time course study to examine the rate at which HPBS is converted to 2-HBP.

As presented in Figure 4, at a protein concentration of 4 mg/ml there was a linear increase in 2-HBP production and a concomitant disappearance of HPBS.

The product of the in vitro reaction was confirmed to be 2-HBP by a spectral comparison to authentic 2-HBP.

The ultraviolet absorption spectrum of the suspected 2-HBP peak produced by the action of the AD109 lysate is virtually identical with that of the 2-HBP standard.

Furthermore, the molecular weight of the unknown compound was exactly that of authentic 2-HBP as determined by GC-MS analysis.

Example 5 Purification of the HPBS desulfinase from Sphingomonas AD109 HPBS desulfinase was purified from AD109 by a series of chromatographic steps using a Bio-Rad low pressure column chromatography Econo system and a Pharmacia FPLC (Gray et al., Nature Biotech. 14 : 1705-1709 (1996)). The steps included fractionation over an anion exchange resin followed by a hydrophobia interaction column chromatography step. These protein

purification steps are described above. A 15-20 fold purification was achieved in these two steps which is comparable to protein preparations from a Rhodococcus IGTS8 lysate.

The molecular weight of this protein by SDS-PAGE was estimated to be 40,000 daltons, which is approximately the same size as DszB purified from IGTS8.

Western analysis demonstrated that the purified protein shows some cross-reactivity with anti-DszB antisera.

Nonlinear regression analysis of an enzyme progress curve was performed according to the general method described by Duggleby, Methods Enzymol. 249 : 61-90 (1995). The analysis involves fitting the integrated Michealis-Menton rate equation Vmt = y-Kmln (1-y/[A] 0) to concentration vs. time data from the enzyme catalyzed reaction of 2- (2-phenyl) benzenesulfinate to 2- hydroxybiphenyl monitored to completion by fluorescence.

The semi-pure protein sample was generated by fractionation of a crude lysate over Q Sepharose Fast Flow resin (Pharmacia) by a linear 0-0.5 M NaCl gradient, as discussed in more detail above. The purity of the active fraction was determined by SDS-PAGE. Pure enzyme is not necessary for the application of enzyme progress curve analysis, however, the calculation of kzat (Vm = [E] tvkCat) was limited to a value range as only a crude estimate of the enzyme concentration was available. The reaction conditions were as follows. A 3 mL reaction solution containing 1 M HPBS and 0.1 M NaCl in 50 mM phosphate at pH 7.5 and 30C was initiated by the addition of 0.023 mg total protein and was monitored for 30 min by fluorescence at an excitation wavelength of 288 nm and an emission wavelength of 414

nm. The data were fit to the equation using the Kaleidagraph data analysis/graphics application (Abelleck Software).

Based on the kinetic parameters calculated from the enzyme progress assay (K=0. 3 UM and Vm=O. 1 HM/min), the minimum kyat= 0.5 min-1. However, a more realistic value would be on the order of 2 min-'in view of the fact that the preparation is estimated to be about 25% pure.

Therefore, the HPBS desulfinase from Sphingomonas AD109 appears to be comparable to that from Rhodococcus IGTS8 with the possibility of a higher catalytic efficiency (kat/").

The N-terminal amino acid sequence of the purified Sphingomonas HPBS desulfinase was also determined.

Protein microsequencing using standard methods of analysis resulted in the following amino acid sequence: 1 10 20 TTDIHPASAA SSPAARATIT YS (SEQ ID NO.: 7) A comparison of the putative AD109 HPBS desulfinase N-terminal sequence with that of the N-terminus of the IGTS8 DszB protein revealed that 9 out the 22 amino acid residues were identical (41%). In order to determine whether the purified protein is, in fact, the Sphingomonas desulfinase protein, a degenerate (192 permutations) 17-mer oligonucleotide probe with the following sequence: 5'ACN GAY ATH CAY CCN GC 3' (SEQ ID NO.: 8), was designed based on the determined N-terminal sequence. Following labeling with a

non-isotopic label this probe was used in hybridization studies using the cloned Sphingomonas AD109 HPBS desulfinase gene (see below) and the dszB gene from IGTS8 (Denome et al., J. Bacteriol. 176 : 6707-6716 (1994); Piddington et al., App. Environ. Microbiol. 61 : 468-475 (1995). The labeled oligonucleotide probe hybridized to the cloned Sphingomonas HPBS desulfinase gene which indicated that the correct protein had been purified. However, no signal was detected in the lane containing a fragment harboring the Rhodococcus dsz B gene.

Example 6 Cloning of the Sphingomonas AD109 HPBS desulfinase gene Strain AD109 has been shown to be capable of using HPBS as a sole sulfur source and clearing a DBTO2 plate.

On the assumption that the gene (s) responsible for DBT02 clearing and HPBS desulfinase activity are genetically closely linked, as they are in Rhodococcus IGTS8, a cloning scheme was devised to isolate the HPBS desulfinase gene from Sphingomonas strain AD109. Total genomic DNA from strain AD109 was digested with either EcoRI, BamHI, and HindIII and the resulting fragments were ligated into pUC18 or pSL1180. Following transformation of E. coli DH10S, approximately 1000-2000 Lac-negative, ampicillin-resistant colonies of each library were screened for the ability to clear a DBTO2 plate. No clearing colonies were detected amongst transformants derived from either the EcoRI or BamHI libraries. However, two clearing colonies were detected utilizing the HindIII library and one clearing colony

was detected with the NotI library. Based on restriction endonuclease profiles, both colonies from the HindIII library contained the same large fragment (-20 kb). Furthermore, there was measurable HPBS desulfinase activity in cell-free lysates of these strains.

The single clearing colony from the NotI library contained a 6.5 kb fragment which, according to restriction endonuclease mapping, overlapped the 20 kb HindIII fragment. This clone also contained measurable HPBS desulfinase activity.

Subcloning analysis localized the genes responsible for DBT02 clearing and HPBS desulfinase activity to a 6 kb HindIII-NotI fragment. A smaller 2.7 kb HindIII-SmaI fragment was subsequently found to retain HPBS desulfinase activity, but lost the ability to clear a DBTO2 plate. It is likely, therefore, that the gene that confers the ability to produce clearing zones on a DBT-sulfone plate spans the SmaI site.

Example 7 DNA sequence analysis of the Sphingomonas sp. strain AD109 desulfurization gene cluster The nucleotide sequence of a 4144 bp region which encompasses the AD109 HPBS desulfinase gene was determined from both DNA strands and is present in Figure 6 (SEQ ID NO.: 12). The overall G+C content of the first 3837 base pairs of the AD109 sequence is 64.5%, a value which is consistent with the range of G+C values (61.7-67.2%) reported for various Sphingomonas species (Yabuuchi et al. (1990)). A

comparison of the AD109 nucleotide sequence with the IGTS8 dsz sequence by DNA matrix analysis revealed that a considerable amount of homology exists between the two sequences as evidenced by the presence of a near continuous diagonal line.

Open reading frame analysis of the AD109 sequence revealed the presence of a number of ORFs on both DNA strands, but of these, only three contained the codon-choice pattern characteristic of microorganisms with G-C rich genomes (West et al., Nucl. Acids Res.

16: 9323-9334 (1988)). All three identified ORFs were in the same transcriptional orientation. A strong preference for codons with either G or C occurred in positions 1 and 3. The first codon position of all three ORFs ranged from 67 to 72%, while the third codon position of all three ORFs ranged from 79-81%. In addition, the predicted translation initiation sites of all three ORFs are preceded by sequences that resemble a consensus ribosome binding site.

The entire nucleotide sequence of the AD109 region was used to conduct a BLAST search of the available DNA databases. The Rhodococcus IGTS8 dsz genes were the highest scoring sequences that demonstrated homology to the Sphingomonas sequences.

The only other nucleotide sequence that demonstrated any significant homology to the Sphingomonas DNA, was the Streptomyces pristinaespiralis snaA gene which encodes the large subunit of the PIIA synthase (Blanc et al., J. Bacteriol. 177 : 5206-5214 (1995)). The Sphingomonas dszA and S. pristinaespiralis snaA genes

demonstrate about 60% identity over a 800 bp region proximal to the 5'end of each gene.

The first ORF (bp 442-1800; Figures 1A-1D) is 71% identical (at the nucleotide level) to the Rhodococcus dszA gene. The primary translation product of ORF-1 would encode a protein (Sphingomonas DszA or Dsz (S)) that contains 453 amino acids with a predicted molecular weight of 50,200. More importantly, this protein demonstrates considerable homology to the amino acid sequence of Rhodococcus DszA (Dsz (R), SEQ ID NO.: 9) over the entire length of the polypeptide (76% identity and 87% similarity; Figure 8). The protein databases were also searched with the Sphingomonas DszA protein sequences. Aside from the DszA protein of Rhodococcus IGTS8, several other proteins demonstrated significant homology to the Sphingomonas DszA protein. These include a hypothetical 49.3 kD protein in the IDH-DEOR intergenic region of Bacillus subtilis which showed 45% identity over 382 residues, the PIIA synthase SnaA subunit of S. pristineaspiralis (Blanc et al., J.

Bacteriol. 177 : 5206-5214 (1995)) which was 49% identical over 358 residues and the nitrilotriacetate monooxygenase of Chelatobacter heintzii (Xu et al.

Abstracts of the 95th General Meeting of the American Society for Microbiology, Q-281) which was 50% identical over the 335 residues examined.

The stop site of the Sphingomonas ORF-1 shows a 4-bp overlap with the translation start site of the second ORF (bp 1800-2906; Figures 2A-2C), which shows a high degree of homology to the Rhodococcus IGTS8 dszB gene (67% identity). It was determined that the

primary translation product of ORF-2 would encode a 369-amino acid polypeptide with a predicted molecular weight of 40,000 (Sphingomonas DszB or Dsz (S)). At the amino acid level this putative protein is 66% identical (75% similarity) to the Rhodococcus HPBS desulfinase protein DszB (DszB (R), SEQ ID NO: 10), as shown in Figure 9. Except for the IGTS8 DszB protein, a BLAST search with the Sphingomonas DszB sequence did not identify any other significant homologous sequences in the available databases. The predicted N-terminus of the Sphingomonas DszB protein matches identically the N-terminus of the HPBS desulfinase purified from AD109 cell lysates, except that the amino-terminal methionine was absent. Removal of the methionine residue has been shown to occur when the second amino acid is Ala, Ser, Gly, Pro, Thr or Val (Hirel et al., Proc. Nat. Acad. Sci. USA 86 : 8247- 8251 (1989)).

The stop site of the Sphingomonas dszB gene also shows a 4-bp overlap with the translation start site of the third ORF. This ORF (bp 2906-4141; Figures 3A- 3C), shows significant homology to the Rhodococcus IGTS8 dszC gene. For example, over the first 931 bp, this ORF is 69% identical to the IGTS8 dszC gene and the N-terminus of the protein predicted by this sequence (Sphingomonas DszC, DszC (S)) is 67% identical to the N-terminus of Rhodococcus DszC (DszC (R), SEQ ID NO: 11), as shown in Figure 10. A BLAST search of the protein databases with the available Sphingomonas DszC sequence identified a number of proteins in addition to the IGTS8 DszC protein. The Sphingomonas DszC protein is 32% identical (over 199 residues) to

Isobutylamine N-Hydroxylase (IBAH) of Streptomyces viridifaciens. It has previously been shown that IBAH exhibits the greatest similarity to the IGTS8 DszC protein (Parry et al., J. Bacteriol., 179: 409-416 (1997)). In addition, the AD109 DszC protein showed variable homology to a number of acyl coenzyme A dehydrogenases. For example, the N-terminal 300 residues of the Sphingomonas DszC protein is 29% identical to the acyl CoA dehydrogenase of B. subtilis.

The sequences (400 bp) directly upstream of the dszA start site contain regulatory elements (i. e., promoter elements) that control transcription of the AD109 dsz gene cluster. A comparison of this potential promoter region with the IGTS8 dsz promoter region failed to reveal any significant homology. It has been shown that the IGTS8 dsz promoter region encompasses a region of potential diad symmetry that may contain an operator (Li et al., J. Bacteriol. 178 : 6409-6418 (1996)). An examination of the AD109 sequences directly upstream of dszA revealed no such palindromic sequence.

Example 8 Expression of the Sphingomonas dszB gene in E. coli The AD109 dszB gene was subcloned into the tac promoter expression vector, pEBCtac, in two steps.

The first step involved cloning a 1.2 kb PstI-BglII fragment that contained the entire coding region of the AD109 dszB gene (Figures 2A-2C) into the polylinker plasmid pOK12. The resulting plasmid,

designated pDA295, contained a unique XbaI site upstream of the dszB gene. In the second step, a 1.2 kb XbaI-BglII fragment from pDA295 that contained the entire dszB gene was cloned into the XbaI and BglII sites of pEBCtac, thus placing the AD109 dszB gene under the transcriptional control of the tac promoter. This plasmid, designated pDA296 and presented in Figure 7, was introduced into E. coli DH106 for expression studies.

HPBS desulfinase assays (2 mg/ml protein) using cell-free lysates prepared from induced and uninduced cultures of DHlOß/pDA296 were performed. In the absence of IPTG the cell-free lysate contained very little HPBS desulfinase activity. Only 22 nmoles of 2-HBP were produced during the 60 min. incubation period which corresponds to a specific activity of 0.2 (nmoles 2-HBP formed/min/mg protein). The lysate prepared from the IPTG-induced culture, however, had approximately 20 times more HPBS desulfinase activity (4.2 nmoles 2-HBP formed/min/mg protein) than the lysate prepared from the uninduced culture.

Example 9 Desulfurization of DBT and alkylated derivatives by AD109 cell-free lysates To a cell free Sphingomonas AD109 lysate having a total protein concentration of 10 mg/mL were added NADH (4 mM) and FMN (10 M). The lysate was then treated with either DBT, 2,8-diMeDBT or 4,6-diMeDBT at a concentration of approximately 90 uM and maintained at 37°C. Aliquots were removed from the reaction mixture at approximately 10 minute time intervals, and

the substrate and product concentrations of each aliquot were determined using high performance liquid chromatography. A similar set of experiments was conducted using a cell-free lysate of a Rhodococcus strain which expresses the Rhodococcus ATCC 53968 DszA, DszB and DszC enzymes.

The results of substrate consumption by the Sphingomonas AD109 lysate are presented in Figures 12- 14. The time dependence of substrate concentration for each of the three substrates is illustrated in Figure 12, which shows that 4,6-diMeDBT is more rapidly consumed than the other two substrates, which disappear at similar rates. Figure 13 indicates that the concentration of 2-HBP, the expected product of DBT desulfurization, increases as the DBT concentration decreases. Figure 14 shows the time dependence of product formation for both 4,6-diMeDBT (product: 2- (2-hydroxy-3-methylphenyl)-6- methylbenzenesulfinate (4,6-dimethyl HBP)) and 2,8- diMeDBT (product: 2- (2-hydroxy-6-methylphenyl)-3- methylbenzenesulfinate (2,8-dimethyl HBP)). The desulfurization product of 4,6-diMeDBT is formed more rapidly than the product resulting from 2,8-diMeDBT.

Figure 15 shows the time dependence of substrate disappearance in similar experiments with the Rhodococcus cell-free lysate. In this case, DBT and 2,8-diMeDBT are consumed at similar rates, while 4,6- diMeDBT is consumed at a much slower rate.

The results indicate that the Sphingomonas and Rhodococcus desulfurization enzymes have different substrate preferences. In particular, the Sphingomonas AD109 lysate desulfurizes 4,6-diMeDBT, in

which the sulfur atom is sterically hindered by the adjacent methyl groups, more rapidly than the unhindered 2,8-diMeDBT and DBT. Rhodococcus shows the opposite preference, desulfurizing the unhindered substrates significantly more rapidly than 4,6- diMeDBT. SEQUENCE LISTING (1) GENERAL INFORMATION: APPLICANT : (A) NAME: Energy BioSystems Corporation (B) STREET: 4200 Research Forest Drive (C) CITY: The Woodlands (D) STATE/PROVINCE: Texas (E) COUNTRY: USA (F) POSTAL CODE/ZIP: 77381 (G) TELEPHONE: (281) 364-6100 (i) TELEFAX: (281) 364-6112 (ii) TITLE OF INVENTION: A Sphingomonas Biodesulfurization Catalyst (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Hamilton, Brook, Smith & Reynolds, P. C. (B) STREET: Two Militia Drive (C) CITY: Lexington (D) STATE: Massachusetts (E) COUNTRY: USA (F) ZIP: 02173 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1. 0, Version #1. 30 (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/851,089 (B) FILING DATE: 05-MAY-1997 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/835,292 (B) FILING DATE: 07-APR-1997 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Elmore, Carolyn S. (B) REGISTRATION NUMBER : 37,567 (C) REFERENCE/DOCKET NUMBER: EBC97-06A2 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (781) 861-6240 (B) TELEFAX: (781) 861-9540 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1362 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix)FEATURE: (A) NAME/KEY : CDS (B) LOCATION: 1.. 1359 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : ATG ACC GAT CCA CGT CAG CTG CAC CTG GCC GGA TTC TTC TGT GCC GGC 48 Met Thr Asp Pro Arg Gln Leu His Leu Ala Gly Phe Phe Cys Ala Gly 1 5 10 15 AAC GTC ACG CAC GCC CAC GGA GCG TGG CGC CAC GCC GAC GAC TCC AAC 96 Asn Val Thr His Ala His Gly Ala Trp Arg His Ala Asp Asp Ser Asn 20 25 30 GGC TTC CTC ACC AAG GAG TAC TAC CAG CAG ATT GCC CGC ACG CTC GAG 144 Gly Phe Leu Thr Lys Glu Tyr Tyr Gln Gln Ile Ala Arg Thr Leu Glu 35 40 45 CGC GGC AAG TTC GAC CTG CTG TTC CTT CCC GAC GCG CTC GCC GTG TGG 192 Arg Gly Lys Phe Asp Leu Leu Phe Leu Pro Asp Ala Leu Ala Val Trp 50 55 60 GAC AGC TAC GGC GAC AAT CTG GAG ACC GGT CTG CGG TAT GGC GGG CAA 240 Asp Ser Tyr Gly Asp Asn Leu Glu Thr Gly Leu Arg Tyr Gly Gly Gln 65 70 75 80 GGC GCG GTG ATG CTG GAG CCC GGC GTA GTT ATC GCC GCG ATG GCC TCG 288 Gly Ala Val Met Leu Glu Pro Gly Val Val Ile Ala Ala Met Ala Ser 85 90 95 GTG ACC GAA CAT CTG GGG CTG GGC GCC ACC ATT TCC ACC ACC TAC TAC 336 Val Thr Glu His Leu Gly Leu Gly Ala Thr Ile Ser Thr Thr Tyr Tyr 100 105 110 CCG CCC TAC CAT GTA GCC CGG GTC GTC GCT TCG CTG GAC CAG CTG TCC 384 Pro Pro Tyr His Val Ala Arg Val Val Ala Ser Leu Asp Gln Leu Ser 115 120 125 TCC GGG CGA GTG TCG TGG AAC GTG GTC ACC TCG CTC AGC AAT GCA GAG 432 Ser Gly Arg Val Ser Trp Asn Val Val Thr Ser Leu Ser Asn Ala Glu 130 135 140 GCG CGC AAC TTC GGC TTC GAT GAA CAT CTC GAC CAC GAT GCC CGC TAC 480 Ala Arg Asn Phe Gly Phe Asp Glu His Leu Asp His Asp Ala Arg Tyr 145 150 155 160 GAT CGC GCC GAT GAA TTC CTC GAG GTC GTG CGC AAG CTC TGG AAC AGC 528 Asp Arg Ala Asp Glu Phe Leu Glu Val Val Arg Lys Leu Trp Asn Ser 165 170 175 TGG GAT CGC GAT GCG CTG ACA CTC GAC AAG GCA ACC GGC CAG TTC GCC 576 Trp Asp Arg Asp Ala Leu Thr Leu Asp Lys Ala Thr Gly Gln Phe Ala 180 185 190 GAT CCG GCT AAG GTG CGC TAC ATC GAC CAC CGC GGC GAA TGG CTC AAC 624 Asp Pro Ala Lys Val Arg Tyr Ile Asp His Arg Gly Glu Trp Leu Asn 195 200 205 GTA CGC GGG CCG CTT CAG GTG CCG CGC TCC CCC CAG GGC GAG CCT GTC 672 Val Arg Gly Pro Leu Gln Val Pro Arg Ser Pro Gln Gly Glu Pro Val 210 215 220 ATT CTG CAG GCC GGG CTT TCG GCG CGG GGC AAG CGC TTC GCC GGG CGC 720 Ile Leu Gln Ala Gly Leu Ser Ala Arg Gly Lys Arg Phe Ala Gly Arg 225 230 235 240 TGG GCG GAC GCG GTG TTC ACG ATT TCG CCC AAT CTG GAC ATC ATG CAG 768 Trp Ala Asp Ala Val Phe Thr Ile Ser Pro Asn Leu Asp Ile Met Gln 245 250 255 GCC ACG TAC CGC GAC ATA AAG GCG CAG GTC G GCC GCC GGA CGC GAT 816 Ala Thr Tyr Arg Asp Ile Lys Ala Gln Val Glu Ala Ala Gly Arg Asp 260 265 270 CCC GAG CAG GTC AAG GTG TTT GCC GCG GTG ATG CCG ATC CTC GGC GAG 864 Pro Glu Gln Val Lys Val Phe Ala Ala Val Met Pro Ile Leu Gly Glu 275 280 285 ACC GAG GCG ATC GCC AGG CAG CGT CTC GAA TAC ATA AAT TCG CTG GTG 912 Thr Glu Ala Ile Ala Arg Gln Arg Leu Glu Tyr Ile Asn Ser Leu Val 290 295 300 CAT CCC GAA GTC GGG CTT TCT ACG TTG TCC AGC CAT GTC GGG GTC AAC 960 His Pro Glu Val Gly Leu Ser Thr Leu Ser Ser His Val Gly Val Asn 305 310 315 320 CTT GCC GAC TAT TCG CTC GAT ACC CCG CTG ACC GAG GTC CTG GGC GAT 1008 Leu Ala Asp Tyr Ser Leu Asp Thr Pro Leu Thr Glu Val Leu Gly Asp 325 330 335 CTC GCC CAG CGC AAC GTG CCC ACC CAA CTG GGC ATG TTC GCC AGG ATG 1056 Leu Ala Gln Arg Asn Val Pro Thr Gln Leu Gly Met Phe Ala Arg Met 340 345 350 TTG CAG GCC GAG ACG CTG ACC GTG GGA GAA ATG GGC CGG CGT TAT GGC 1104 Leu Gln Ala Glu Thr Leu Thr Val Gly Glu Met Gly Arg Arg Tyr Gly 355 360 365 GCC AAC GTG GGC TTC GTC CCG CAG TGG GCG GGA ACC CGC GAG CAG ATC 1152 Ala Asn Val Gly Phe Val Pro Gln Trp Ala Gly Thr Arg Glu Gln Ile 370 375 380 GCG GAC CTG ATC GAG ATC CAT TTC AAG GCC GGC GGC GCC GAT GGC TTC 1200 Ala Asp Leu Ile Glu Ile His Phe Lys Ala Gly Gly Ala Asp Gly Phe 385 390 395 400 ATC ATC TCG CCG GCG TTC CTG CCC GGA TCT TAC GAG GAA TTC GTC GAT 1248 Ile Ile Ser Pro Ala Phe Leu Pro Gly Ser Tyr Glu Glu Phe Val Asp 405 410 415 CAG GTG GTG CCC ATC CTG CAG CAC CGC GGA CTG TTC CGC ACT GAT TAC 1296 Gln Val Val Pro Ile Leu Gln His Arg Gly Leu Phe Arg Thr Asp Tyr 420 425 430 GAA GGC CGC ACC CTG CGC AGC CAT CTG GGA CTG CGT GAA CCC GCA TAC 1344 Glu Gly Arg Thr Leu Arg Ser His Leu Gly Leu Arg Glu Pro Ala Tyr 435 440 445 CTG GGA GAG TAC GCA TGA 1362 Leu Gly Glu Tyr Ala 450 (2) INFORMATION FOR SEQ ID NO : 2: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 453 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: Met Thr Asp Pro Arg Gln Leu His Leu Ala Gly Phe Phe Cys Ala Gly 1 5 10 15 Asn Val Thr His Ala His Gly Ala Trp Arg His Ala Asp Asp Ser Asn 20 25 30 Gly Phe Leu Thr Lys Glu Tyr Tyr Gln Gln Ile Ala Arg Thr Leu Glu 35 40 45 Arg Gly Lys Phe Asp Leu Leu Phe Leu Pro Asp Ala Leu Ala Val Trp 50 55 60 Asp Ser Tyr Gly Asp Asn Leu Glu Thr Gly Leu Arg Tyr Gly Gly Gln 65 70 75 80 Gly Ala Val Met Leu Glu Pro Gly Val Val Ile Ala Ala Met Ala Ser 85 90 95 Val Thr Glu His Leu Gly Leu Gly Ala Thr Ile Ser Thr Thr Tyr Tyr 100 105 110 Pro Pro Tyr His Val Ala Arg Val Val Ala Ser Leu Asp Gln Leu Ser 115 120 125 Ser Gly Arg Val Ser Trp Asn Val Val Thr Ser Leu Ser Asn Ala Glu 130 135 140 Ala Arg Asn Phe Gly Phe Asp Glu His Leu Asp His Asp Ala Arg Tyr 145 150 155 160 Asp Arg Ala Asp Glu Phe Leu Glu Val Val Arg Lys Leu Trp Asn Ser 165 170 175 Trp Asp Arg Asp Ala Leu Thr Leu Asp Lys Ala Thr Gly Gln Phe Ala 180 185 190 Asp Pro Ala Lys Val Arg Tyr Ile Asp His Arg Gly Glu Trp Leu Asn 195 200 205 Val Arg Gly Pro Leu Gln Val Pro Arg Ser Pro Gln Gly Glu Pro Val 210 215 220 Ile Leu Gln Ala Gly Leu Ser Ala Arg Gly Lys Arg Phe Ala Gly Arg 225 230 235 240 Trp Ala Asp Ala Val Phe Thr Ile Ser Pro Asn Leu Asp Ile Met Gln 245 250 255 Ala Thr Tyr Arg Asp Ile Lys Ala Gln Val Glu Ala Ala Gly Arg Asp 260 265 270 Pro Glu Gln Val Lys Val Phe Ala Ala Val Met Pro Ile Leu Gly Glu 275 280 285 Thr Glu Ala Ile Ala Arg Gln Arg Leu Glu Tyr Ile Asn Ser Leu Val 290 295 300 His Pro Glu Val Gly Leu Ser Thr Leu Ser Ser His Val Gly Val Asn 305 310 315 320 Leu Ala Asp Tyr Ser Leu Asp Thr Pro Leu Thr Glu Val Leu Gly Asp 325 330 335 Leu Ala Gln Arg Asn Val Pro Thr Gln Leu Gly Met Phe Ala Arg Met 340 345 350 Leu Gln Ala Glu Thr Leu Thr Val Gly Glu Met Gly Arg Arg Tyr Gly 355 360 365 Ala Asn Val Gly Phe Val Pro Gln Trp Ala Gly Thr Arg Glu Gln Ile 370 375 380 Ala Asp Leu Ile Glu Ile His Phe Lys Ala Gly Gly Ala Asp Gly Phe 385 390 395 400 Ile Ile Ser Pro Ala Phe Leu Pro Gly Ser Tyr Glu Glu Phe Val Asp 405 410 415 Gln Val Val Pro Ile Leu Gln His Arg Gly Leu Phe Arg Thr Asp Tyr 420 425 430 Glu Gly Arg Thr Leu Arg Ser His Leu Gly Leu Arg Glu Pro Ala Tyr 435 440 445 Leu Gly Glu Tyr Ala 450 (2) INFORMATION FOR SEQ ID NO : 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1110 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1.. 1107 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3: ATG ACG ACA GAC ATC CAC CCG GCG AGC GCC GCA TCG TCG CCG GCG GCG 48 Met Thr Thr Asp Ile His Pro Ala Ser Ala Ala Ser Ser Pro Ala Ala 1 5 10 15 CGC GCG ACG ATC ACC TAC AGC AAC TGC CCC GTG CCT AAT GCC CTG CTC 96 Arg Ala Thr Ile Thr Tyr Ser Asn Cys Pro Val Pro Asn Ala Leu Leu 20 25 30 GCC GCG CTC GGC TCA GGT ATT CTG GAC AGT GCC GGG ATC ACA CTT GCC 144 Ala Ala Leu Gly Ser Gly Ile Leu Asp Ser Ala Gly Ile Thr Leu Ala 35 40 45 CTG CTG ACC GGA AAG CAG GGC GAG GTG CAC TTC ACC TAC GAC CGA GAT 192 Leu Leu Thr Gly Lys Gln Gly Glu Val His Phe Thr Tyr Asp Arg Asp 50 55 60 GAC TAC ACC CGC TTC GGC GGC GAG ATT CCG CCG CTG GTC AGC GAG GGA 240 Asp Tyr Thr Arg Phe Gly Gly Glu Ile Pro Pro Leu Val Ser Glu Gly 65 70 75 80 CTG CGT GCG CCG GGG CGG ACC CGC CTG CTG GGA CTG ACG CCG GTG CTG 288 Leu Arg Ala Pro Gly Arg Thr Arg Leu Leu Gly Leu Thr Pro Val Leu 85 90 95 GGC CGC TGG GGC TAC TTC GTC CGG GGC GAC AGC GCG ATC CGC ACC CCG 336 Gly Arg Trp Gly Tyr Phe Val Arg Gly Asp Ser Ala Ile Arg Thr Pro 100 105 110 GCC GAT CTT GCC GGC CGC CGC GTC GGA GTA TCC GAT TCG GCC AGG AGG 384 Ala Asp Leu Ala Gly Arg Arg Val Gly Val Ser Asp Ser Ala Arg Arg 115 120 125 ATA TTG ACC GGA AGG CTG GGC GAC TAC CGC GAA CTT GAT CCC TGG CGG 432 Ile Leu Thr Gly Arg Leu Gly Asp Tyr Arg Glu Leu Asp Pro Trp Arg 130 135 140 CAG ACC CTG GTC GCG CTG GGG ACA TGG GAG GCG CGT GCC TTG CTG AGC 480 Gln Thr Leu Val Ala Leu Gly Thr Trp Glu Ala Arg Ala Leu Leu Ser 145 150 155 160 ACG CTC GAG ACG GCG GGG CTT GGC GTC GGC GAC GTC GAG CTG ACG CGC 528 Thr Leu Glu Thr Ala Gly Leu Gly Val Gly Asp Val Glu Leu Thr Arg 165 170 175 ATC GAG AAC CCG TTC GTC GAC GTG CCG ACC GAA CGA CTG CAT GCC GCC 576 Ile Glu Asn Pro Phe Val Asp Val Pro Thr Glu Arg Leu His Ala Ala 180 185 190 GGC TCG CTC AAA GGA ACC GAC CTG TTC CCC GAC GTG ACC AGC CAG CAG 624 Gly Ser Leu Lys Gly Thr Asp Leu Phe Pro Asp Val Thr Ser Gln Gln 195 200 205 GCC GCA GTC CTT GAG GAT GAG CGC GCC GAC GCC CTG TTC GCG TGG CTT 672 Ala Ala Val Leu Glu Asp Glu Arg Ala Asp Ala Leu Phe Ala Trp Leu 210 215 220 CCC TGG GCG GCC GAG CTC GAG ACC CGC ATC GGT GCA CGG CCG GTC CTA 720 Pro Trp Ala Ala Glu Leu Glu Thr Arg Ile Gly Ala Arg Pro Val Leu 225 230 235 240 GAC CTC AGC GCA GAC GAC CGC AAT GCC TAT GCG AGC ACC TGG ACG GTG 768 Asp Leu Ser Ala Asp Asp Arg Asn Ala Tyr Ala Ser Thr Trp Thr Val 245 250 255 AGC GCC GAG CTG GTG GAC CGG CAG CCC GAA CTG GTG CAG CGG CTC GTC 816 Ser Ala Glu Leu Val Asp Arg Gln Pro Glu Leu Val Gln Arg Leu Val 260 265 270 GAT GCC GTG GTG GAT GCA GGG CGG TGG GCC GAG GCC AAT GGC GAT GTC 864 Asp Ala Val Val Asp Ala Gly Arg Trp Ala Glu Ala Asn Gly Asp Val 275 280 285 GTC TCC CGC CTG CAC GCC GAT AAC CTC GGT GTC AGT CCC GAA AGC GTC 912 Val Ser Arg Leu His Ala Asp Asn Leu Gly Val Ser Pro Glu Ser Val 290 295 300 CGC CAG GGA TTC GGA GCC GAT TTT CAC CGC CGC CTG ACG CCG CGG CTC 960 Arg Gln Gly Phe Gly Ala Asp Phe His Arg Arg Leu Thr Pro Arg Leu 305 310 315 320 GAC AGC GAT GCT ATC GCC ATC CTG GAG CGT ACT CAG CGG TTC CTG AAG 1008 Asp Ser Asp Ala Ile Ala Ile Leu Glu Arg Thr Gln Arg Phe Leu Lys 325 330 335 GAT GCG AAC CTG ATC GAT CGG TCG TTG GCG CTC GAT CGG TGG GCT GCA 1056 Asp Ala Asn Leu Ile Asp Arg Ser Leu Ala Leu Asp Arg Trp Ala Ala 340 345 350 CCT GAA TTC CTC GAA CAA AGT CTC TCA CGC CAG GTC GAA GGG CAG ATA 1104 Pro Glu Phe Leu Glu Gln Ser Leu Ser Arg Gln Val Glu Gly Gln Ile 355 360 365 GCA TGA 1110 Ala (2) INFORMATION FOR SEQ ID NO : 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 369 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4: Met Thr Thr Asp Ile His Pro Ala Ser Ala Ala Ser Ser Pro Ala Ala 1 5 10 15 Arg Ala Thr Ile Thr Tyr Ser Asn Cys Pro Val Pro Asn Ala Leu Leu 20 25 30 Ala Ala Leu Gly Ser Gly Ile Leu Asp Ser Ala Gly Ile Thr Leu Ala 35 40 45 Leu Leu Thr Gly Lys Gln Gly Glu Val His Phe Thr Tyr Asp Arg Asp 50 55 60 Asp Tyr Thr Arg Phe Gly Gly Glu Ile Pro Pro Leu Val Ser Glu Gly 65 70 75 80 Leu Arg Ala Pro Gly Arg Thr Arg Leu Leu Gly Leu Thr Pro Val Leu 85 90 95 Gly Arg Trp Gly Tyr Phe Val Arg Gly Asp Ser Ala Ile Arg Thr Pro 100 105 110 Ala Asp Leu Ala Gly Arg Arg Val Gly Val Ser Asp Ser Ala Arg Arg 115 120 125 Ile Leu Thr Gly Arg Leu Gly Asp Tyr Arg Glu Leu Asp Pro Trp Arg 130 135 140 Gln Thr Leu Val Ala Leu Gly Thr Trp Glu Ala Arg Ala Leu Leu Ser 145 150 155 160 Thr Leu Glu Thr Ala Gly Leu Gly Val Gly Asp Val Glu Leu Thr Arg 165 170 175 Ile Glu Asn Pro Phe Val Asp Val Pro Thr Glu Arg Leu His Ala Ala 180 185 190 Gly Ser Leu Lys Gly Thr Asp Leu Phe Pro Asp Val Thr Ser Gln Gln 195 200 205 Ala Ala Val Leu Glu Asp Glu Arg Ala Asp Ala Leu Phe Ala Trp Leu 210 215 220 Pro Trp Ala Ala Glu Leu Glu Thr Arg Ile Gly Ala Arg Pro Val Leu 225 230 235 240 Asp Leu Ser Ala Asp Asp Arg Asn Ala Tyr Ala Ser Thr Trp Thr Val 245 250 255 Ser Ala Glu Leu Val Asp Arg Gln Pro Glu Leu Val Gln Arg Leu Val 260 265 270 Asp Ala Val Val Asp Ala Gly Arg Trp Ala Glu Ala Asn Gly Asp Val 275 280 285 Val Ser Arg Leu His Ala Asp Asn Leu Gly Val Ser Pro Glu Ser Val 290 295 300 Arg Gln Gly Phe Gly Ala Asp Phe His Arg Arg Leu Thr Pro Arg Leu 305 310 315 320 Asp Ser Asp Ala Ile Ala Ile Leu Glu Arg Thr Gln Arg Phe Leu Lys 325 330 335 Asp Ala Asn Leu Ile Asp Arg Ser Leu Ala Leu Asp Arg Trp Ala Ala 340 345 350 Pro Glu Phe Leu Glu Gln Ser Leu Ser Arg Gln Val Glu Gly Gln Ile 355 360 365 Ala (2) INFORMATION FOR SEQ ID NO : 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1236 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1.. 1236 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5: ATG AAC GAA CTC GTC AAA GAT CTC GGC CTC AAT CGA TCC GAT CCG ATC 48 Met Asn Glu Leu Val Lys Asp Leu Gly Leu Asn Arg Ser Asp Pro Ile 1 5 10 15 GGC GCT GTG CGG CGA CTG GCC GCG CAG TGG GGG GCC ACC GCT GTT GAT 96 Gly Ala Val Arg Arg Leu Ala Ala Gln Trp Gly Ala Thr Ala Val Asp 20 25 30 CGG GAC CGG GCC GGC GGA TCG GCA ACC GCC GAA CTC GAT CAA CTG CGC 144 Arg Asp Arg Ala Gly Gly Ser Ala Thr Ala Glu Leu Asp Gln Leu Arg 35 40 45 GGC AGC GGC CTG CTC TCG CTG TCC ATT CCC GCC GCA TAT GGC GGC TGG 192 Gly Ser Gly Leu Leu Ser Leu Ser Ile Pro Ala Ala Tyr Gly Gly Trp 50 55 60 GGC GCC GAC TGG CCA ACG ACT CTG GAA GTT ATC CGC GAA GTC GCA ACG 240 Gly Ala Asp Trp Pro Thr Thr Leu Glu Val Ile Arg Glu Val Ala Thr 65 70 75 80 GTG GAC GGA TCG CTG GCG CAT CTA TTC GGC TAC CAC CTC GGC TGC GTA 288 Val Asp Gly Ser Leu Ala His Leu Phe Gly Tyr His Leu Gly Cys Val 85 90 95 CCG ATG ATC GAG CTG TTC GGC TCG GCG CCA CAA AAG GAA CGG CTG TAC 336 Pro Met Ile Glu Leu Phe Gly Ser Ala Pro Gln Lys Glu Arg Leu Tyr 100 105 110 CGC CAG ATC GCA AGC CAT GAT TGG CGG GTC GGG AAT GCG TCG AGC GAA 384 Arg Gln Ile Ala Ser His Asp Trp Arg Val Gly Asn Ala Ser Ser Glu 115 120 125 AAC AAC AGC CAC GTG CTC GAG TGG AAG CTT GCC GCC ACC GCC GTC GAT 432 Asn Asn Ser His Val Leu Glu Trp Lys Leu Ala Ala Thr Ala Val Asp 130 135 140 GAT GGC GGG TTC GTC CTC AAC GGC GCG AAG CAC TTC TGC AGC GGC GCC 480 Asp Gly Gly Phe Val Leu Asn Gly Ala Lys His Phe Cys Ser Gly Ala 145 150 155 160 AAA AGC TCC GAC CTG CTC ATC GTG TTC GGC GTG ATC CAG GAC GAA TCC 528 Lys Ser Ser Asp Leu Leu Ile Val Phe Gly Val Ile Gln Asp Glu Ser 165 170 175 CCC CTG CGC GGC GCG ATC ATC ACC GCG GTC ATT CCC ACC GAC CGG GCC 576 Pro Leu Arg Gly Ala Ile Ile Thr Ala Val Ile Pro Thr Asp Arg Ala 180 185 190 GGT GTT CAG ATC AAT GAC GAC TGG CGC GCA ATC GGG ATG CGC CAG ACC 624 Gly Val Gln Ile Asn Asp Asp Trp Arg Ala Ile Gly Met Arg Gln Thr 195 200 205 GAC AGC GGC AGC GCC GAA TTT CGC GAC GTC CGA GTC TAC CCA GAC GAG 672 Asp Ser Gly Ser Ala Glu Phe Arg Asp Val Arg Val Tyr Pro Asp Glu 210 215 220 ATC TTG GGG GCA CCA AAC TCA GTC GTT GAG GCG TTC GTG ACA AGC AAC 720 Ile Leu Gly Ala Pro Asn Ser Val Val Glu Ala Phe Val Thr Ser Asn 225 230 235 240 CGC GGC AGC CTG TGG ACG CCG GCG ATT CAG TCG ATC TTC TCG AAC GTT 768 Arg Gly Ser Leu Trp Thr Pro Ala Ile Gln Ser Ile Phe Ser Asn Val 245 250 255 TAT CTG GGG CTC GCG CGT GGC GCG CTC GAG GCG GCA GCG GAT TAC ACC 816 Tyr Leu Gly Leu Ala Arg Gly Ala Leu Glu Ala Ala Ala Asp Tyr Thr 260 265 270 CGG ACC CAG AGC CGC CCC TGG ACA CCC GCC GGC GTG GCG AAG GCG ACA 864 Arg Thr Gln Ser Arg Pro Trp Thr Pro Ala Gly Val Ala Lys Ala Thr 275 280 285 GAG GAT CCC CAC ATC ATC GCC ACC TAC GGT GAA CTG GCG ATC GCG CTC 912 Glu Asp Pro His Ile Ile Ala Thr Tyr Gly Glu Leu Ala Ile Ala Leu 290 295 300 CAG GGC GCC GAG GCG GCC GCG CGC GAG GTC GCG GCC CTG TTG CAA CAG 960 Gln Gly Ala Glu Ala Ala Ala Arg Glu Val Ala Ala Leu Leu Gln Gln 305 310 315 320 GCG TGG GAC AAG GGC GAT GCG GTG ACG CCC GAA GAG CGC GGC CAG CTG 1008 Ala Trp Asp Lys Gly Asp Ala Val Thr Pro Glu Glu Arg Gly Gln Leu 325 330 335 ATG GTG AAG GTT TCG GGT GTG AAG GCC CTC TCG ACG AAG GCC GCC CTC 1056 Met Val Lys Val Ser Gly Val Lys Ala Leu Ser Thr Lys Ala Ala Leu 340 345 350 GAC ATC ACC AGC CGT ATT TTC GAG ACA ACG GGC TCG CGA TCG ACG CAT 1104 Asp Ile Thr Ser Arg Ile Phe Glu Thr Thr Gly Ser Arg Ser Thr His 355 360 365 CCC AGA TAC GGA TTC GAT CGG TTC TGG CGT AAC ATC CGG ACT CAT ACG 1152 Pro Arg Tyr Gly Phe Asp Arg Phe Trp Arg Asn Ile Arg Thr His Thr 370 375 380 CTG CAC GAT CCG GTA TCG TAT AAA ATC GTC GAT GTG GGG AAC TAC ACG 1200 Leu His Asp Pro Val Ser Tyr Lys Ile Val Asp Val Gly Asn Tyr Thr 385 390 395 400 CTC AAC GGG ACA TTC CCG GTT CCC GGA TTT ACG TCA 1236 Leu Asn Gly Thr Phe Pro Val Pro Gly Phe Thr Ser 405 410 (2) INFORMATION FOR SEQ ID NO : 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 412 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6: Met Asn Glu Leu Val Lys Asp Leu Gly Leu Asn Arg Ser Asp Pro Ile 1 5 10 15 Gly Ala Val Arg Arg Leu Ala Ala Gln Trp Gly Ala Thr Ala Val Asp 20 25 30 Arg Asp Arg Ala Gly Gly Ser Ala Thr Ala Glu Leu Asp Gln Leu Arg 35 40 45 Gly Ser Gly Leu Leu Ser Leu Ser Ile Pro Ala Ala Tyr Gly Gly Trp 50 55 60 Gly Ala Asp Trp Pro Thr Thr Leu Glu Val Ile Arg Glu Val Ala Thr 65 70 75 80 Val Asp Gly Ser Leu Ala His Leu Phe Gly Tyr His Leu Gly Cys Val 85 90 95 Pro Met Ile Glu Leu Phe Gly Ser Ala Pro Gln Lys Glu Arg Leu Tyr 100 105 110 Arg Gln Ile Ala Ser His Asp Trp Arg Val Gly Asn Ala Ser Ser Glu 115 120 125 Asn Asn Ser His Val Leu Glu Trp Lys Leu Ala Ala Thr Ala Val Asp 130 135 140 Asp Gly Gly Phe Val Leu Asn Gly Ala Lys His Phe Cys Ser Gly Ala 145 150 155 160 Lys Ser Ser Asp Leu Leu Ile Val Phe Gly Val Ile Gln Asp Glu Ser 165 170 175 Pro Leu Arg Gly Ala Ile Ile Thr Ala Val Ile Pro Thr Asp Arg Ala 180 185 190 Gly Val Gln Ile Asn Asp Asp Trp Arg Ala Ile Gly Met Arg Gln Thr 195 200 205 Asp Ser Gly Ser Ala Glu Phe Arg Asp Val Arg Val Tyr Pro Asp Glu 210 215 220 Ile Leu Gly Ala Pro Asn Ser Val Val Glu Ala Phe Val Thr Ser Asn 225 230 235 240 Arg Gly Ser Leu Trp Thr Pro Ala Ile Gln Ser Ile Phe Ser Asn Val 245 250 255 Tyr Leu Gly Leu Ala Arg Gly Ala Leu Glu Ala Ala Ala Asp Tyr Thr 260 265 270 Arg Thr Gln Ser Arg Pro Trp Thr Pro Ala Gly Val Ala Lys Ala Thr 275 280 285 Glu Asp Pro His Ile Ile Ala Thr Tyr Gly Glu Leu Ala Ile Ala Leu 290 295 300 Gln Gly Ala Glu Ala Ala Ala Arg Glu Val Ala Ala Leu Leu Gln Gln 305 310 315 320 Ala Trp Asp Lys Gly Asp Ala Val Thr Pro Glu Glu Arg Gly Gln Leu 325 330 335 Met Val Lys Val Ser Gly Val Lys Ala Leu Ser Thr Lys Ala Ala Leu 340 345 350 Asp Ile Thr Ser Arg Ile Phe Glu Thr Thr Gly Ser Arg Ser Thr His 355 360 365 Pro Arg Tyr Gly Phe Asp Arg Phe Trp Arg Asn Ile Arg Thr His Thr 370 375 380 Leu His Asp Pro Val Ser Tyr Lys Ile Val Asp Val Gly Asn Tyr Thr 385 390 395 400 Leu Asn Gly Thr Phe Pro Val Pro Gly Phe Thr Ser 405 410 (2) INFORMATION FOR SEQ ID NO : 7: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7: Thr Thr Asp Ile His Pro Ala Ser Ala Ala Ser Ser Pro Ala Ala Arg 1 5 10 15 Ala Thr Ile Thr Tyr Ser 20 (2) INFORMATION FOR SEQ ID NO : 8 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : ACNGAYATHC AYCCNGC 17 (2) INFORMATION FOR SEQ ID NO : 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 453 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9: Met Thr Gln Gln Arg Gln Met His Leu Ala Gly Phe Phe Ser Ala Gly 1 5 10 15 Asn Val Thr His Ala His Gly Ala Trp Arg His Thr Asp Ala Ser Asn 20 25 30 Asp Phe Leu Ser Gly Lys Tyr Tyr Gln His Ile Ala Arg Thr Leu Glu 35 40 45 Arg Gly Lys Phe Asp Leu Leu Phe Leu Pro Asp Gly Leu Ala Val Glu 50 55 60 Asp Ser Tyr Gly Asp Asn Leu Asp Thr Gly Val Gly Leu Gly Gly Gln 65 70 75 80 Gly Ala Val Ala Leu Glu Pro Ala Ser Val Val Ala Thr Met Ala Ala 85 90 95 Val Thr Glu His Leu Gly Leu Gly Ala Thr Ile Ser Ala Thr Tyr Tyr 100 105 110 Pro Pro Tyr His Val Ala Arg Val Phe Ala Thr Leu Asp Gln Leu Ser 115 120 125 Gly Gly Arg Val Ser Trp Asn Val Val Thr Ser Leu Asn Asp Ala Glu 130 135 140 Ala Arg Asn Phe Gly Ile Asn Gln His Leu Glu His Asp Ala Arg Tyr 145 150 155 160 Asp Arg Ala Asp Glu Phe Leu Glu Ala Val Lys Lys Leu Trp Asn Ser 165 170 175 Trp Asp Glu Asp Ala Leu Val Leu Asp Lys Ala Ala Gly Val Phe Ala 180 185 190 Asp Pro Ala Lys Val His Tyr Val Asp His His Gly Glu Trp Leu Asn 195 200 205 Val Arg Gly Pro Leu Gln Val Pro Arg Ser Pro Gln Gly Glu Pro Val 210 215 220 Ile Leu Gln Ala Gly Leu Ser Pro Arg Gly Arg Arg Phe Ala Gly Lys 225 230 235 240 Trp Ala Glu Ala Val Phe Ser Leu Ala Pro Asn Leu Glu Val Met Gln 245 250 255 Ala Thr Tyr Gln Gly Ile Lys Ala Glu Val Asp Ala Ala Gly Arg Asp 260 265 270 Pro Asp Gln Thr Lys Ile Phe Thr Ala Val Met Pro Val Leu Gly Glu 275 280 285 Ser Gln Ala Val Ala Gln Glu Arg Leu Glu Tyr Leu Asn Ser Leu Val 290 295 300 His Pro Glu Val Gly Leu Ser Thr Leu Ser Ser His Thr Gly Ile Asn 305 310 315 320 Leu Ala Ala Tyr Pro Leu Asp Thr Pro Ile Lys Asp Ile Leu Arg Asp 325 330 335 Leu Gln Asp Arg Asn Val Pro Thr Gln Leu His Met Phe Ala Ala Ala 340 345 350 Thr His Ser Glu Glu Leu Thr Leu Ala Glu Met Gly Arg Arg Tyr Gly 355 360 365 Thr Asn Val Gly Phe Val Pro Gln Trp Ala Gly Thr Gly Glu Gln Ile 370 375 380 Ala Asp Glu Leu Ile Arg His Phe Glu Gly Gly Ala Ala Asp Gly Phe 385 390 395 400 Ile Ile Ser Pro Ala Phe Leu Pro Gly Ser Tyr Asp Glu Phe Val Asp 405 410 415 Gln Val Val Pro Val Leu Gln Asp Arg Gly Tyr Phe Arg Thr Glu Tyr 420 425 430 Gln Gly Asn Thr Leu Arg Asp His Leu Gly Leu Arg Val Pro Gln Leu 435 440 445 Gln Gly Gln Pro Ser 450 (2) INFORMATION FOR SEQ ID NO : 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 365 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 10: Met Thr Ser Arg Val Asp Pro Ala Asn Pro Gly Ser Glu Leu Asp Ser 1 5 10 15 Ala Ile Arg Asp Thr Leu Thr Tyr Ser Asn Cys Pro Val Pro Asn Ala 20 25 30 Leu Leu Thr Ala Ser Glu Ser Gly Phe Leu Asp Ala Ala Gly Ile Glu 35 40 45 Leu Asp Val Leu Ser Gly Gln Gln Gly Thr Val His Phe Thr Tyr Asp 50 55 60 Gln Pro Ala Tyr Thr Arg Phe Gly Gly Glu Ile Pro Pro Leu Leu Ser 65 70 75 80 Glu Gly Leu Arg Ala Pro Gly Arg Thr Arg Leu Leu Gly Ile Thr Pro 85 90 95 Leu Leu Gly Arg Gln Gly Phe Phe Val Arg Asp Asp Ser Pro Ile Thr 100105 110 Ala Ala Ala Asp Leu Ala Gly Arg Arg Ile Gly Val Ser Ala Ser Ala 115120 125 Ile Arg Ile Leu Arg Gly Gln Leu Gly Asp Tyr Leu Glu Leu Asp Pro 130135 140 Trp Arg Gln Thr Leu Val Ala Leu Gly Ser Trp Glu Ala Arg Ala Leu 145 150 155 160 Leu His Thr Leu Glu His Gly Glu Leu Gly Val Asp Asp Val Glu Leu 165 170 175 Val Pro Ile Ser Ser Pro Gly Val Asp Val Pro Ala Glu Gln Leu Glu 180185 190 Glu Ser Ala Thr Val Lys Gly Ala Asp Leu Phe Pro Asp Val Ala Arg 195200 205 Gly Gln Ala Ala Val Leu Ala Ser Gly Asp Val Asp Ala Leu Tyr Ser 210215 220 Trp Leu Pro Trp Ala Gly Glu Leu Gln Ala Thr Gly Ala Arg Pro Val 225 230 235 240 Val Asp Leu Gly Leu Asp Glu Arg Asn Ala Tyr Ala Ser Val Trp Thr 245 250 255 Val Ser Ser Gly Leu Val Arg Gln Arg Pro Gly Leu Val Gln Arg Leu 260265 270 Val Asp Ala Ala Val Asp Ala Gly Leu Trp Ala Arg Asp His Ser Asp 275280 285 Ala Val Thr Ser Leu His Ala Ala Asn Leu Gly Val Ser Thr Gly Ala 290295 300 Val Gly Gln Gly Phe Gly Ala Asp Phe Gln Gln Arg Leu Val Pro Arg 305 310 315 320 Leu Asp His Asp Ala Leu Ala Leu Leu Glu Arg Thr Gln Gln Phe Leu 325 330 335 Leu Thr Asn Asn Leu Leu Gln Glu Pro Val Ala Leu Asp Gln Trp Ala 340 345 350 Ala Pro Glu Phe Leu Asn Asn Ser Leu Asn Arg His Arg 355 360 365 (2) INFORMATION FOR SEQ ID NO : 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 11: Met Thr Leu Ser Pro Glu Lys Gln His Val Arg Pro Arg Asp Ala Ala 1 5 10 15 Asp Asn Asp Pro Val Ala Val Ala Arg Gly Leu Ala Glu Lys Trp Arg 20 25 30 Ala Thr Ala Val Glu Arg Asp Arg Ala Gly Gly Ser Ala Thr Ala Glu 35 40 45 Arg Glu Asp Leu Arg Ala Ser Ala Leu Leu Ser Leu Leu Val Pro Arg 50 55 60 Glu Tyr Gly Gly Trp Gly Ala Asp Trp Pro Thr Ala Ile Glu Val Val 65 70 75 80 Arg Glu Ile Ala Ala Ala Asp Gly Ser Leu Gly His Leu Phe Gly Tyr 85 90 95 His Leu Thr Asn Ala Pro Met Ile Glu Leu Ile Gly Ser Gln Glu Gln 100 105 110 Glu Glu His Leu Tyr Thr Gln Ile Ala Gln Asn Asn Trp Trp Thr Gly 115 120 125 Asn Ala Ser Ser Glu Asn Asn Ser His Val Leu Asp Trp Lys Val Ser 130135 140 Ala Thr Pro Thr Glu Asp Gly Gly Tyr Val Leu Asn Gly. Thr Lys His 145 150 155 160 Phe Cys Ser Gly Ala Lys Gly Ser Asp Leu Leu Phe Val Phe Gly Val 165 170 175 Val Gln Asp Asp Ser Pro Gln Gln Gly Ala Ile Ile Ala Ala Ala Ile 180 185 190 Pro Thr Ser Arg Ala Gly Val Thr Pro Asn Asp Asp Trp Ala Ala Ile 195 200 205 Gly Met Arg Gln Thr Asp Ser Gly Ser Thr Asp Phe His Asn Val Lys 210215 220 Val Glu Pro Asp Glu Val Leu Gly Ala Pro Asn Ala Phe Val Leu Ala 225 230 235 240 Phe Ile Gln Ser Glu Arg Gly Ser Leu Phe Ala Pro Ile Ala Gln Leu 245 250 255 Ile Phe Ala Asn Val Tyr Leu Gly Ile Ala His Gly Ala Leu Asp Ala 260 265 270 Ala Arg Glu Tyr Thr Arg Thr Gln Ala Arg Pro Trp Thr Pro Ala Gly 275 280 285 Ile Gln Gln Ala Thr Glu Asp Pro Tyr Thr Ile Arg Ser Tyr Gly Glu 290 295 300 Phe Thr Ile Ala Leu Gln Gly Ala Asp Ala Ala Ala Arg Glu Ala Ala 305 310 315 320 His Leu Leu Gln Thr Val Trp Asp Lys Gly Asp Ala Leu Thr Pro Glu 325 330 335 Asp Arg Gly Glu Leu Met Val Lys Val Ser Gly Val Lys Ala Leu Ala 340 345 350 Thr Asn Ala Ala Leu Asn Ile Ser Ser Gly Val Phe Glu Val Ile Gly 355 360 365 Ala Arg Gly Thr His Pro Arg Tyr Gly Phe Asp Arg Phe Trp Arg Asn 370 375 380 Val Arg Thr His Ser Leu His Asp Pro Val Ser Tyr Lys Ile Ala Asp 385 390 395 400 Val Gly Lys His Thr Leu Asn Gly Gln Tyr Pro Ile Pro Gly Phe Thr 405 410 415 Ser (2) INFORMATION FOR SEQ ID NO : 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4144 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12: GGTTCGAGAT CGATCTGACC GTCGAACCCG GCGCGGTTCA AACCATCCTC TGGGGCCTCT 60 TCTTGCACTT GACATAGGAA TCTCTACTAA ATAAATAGAT ATTTATTCGA CACTAAGTTC 120 GGTGATCAGG CCGACCGTGT GTCTCAAGTG CTCGCTCCGG GTTGCCACGA GCTAAAGCGC 180 GCGATGCTGG GGCGACAGCG CTAGGCATTG CGTTCCCTCA CACCAATGAT GAGATGATAC 240 GATGCGCATG ACCACTATCC GCACCTAGCA CGAAAGATCC GTGCATTTCG CGAATGCCAA 300 TGAAGAGGAC CGACGTACGG CAGCTTCCTA CGCTTTCGCG CCATCGTTCA TAGCCAAGGT 360 CTTTTCGACG CCGGTTCGCG TGGGCGACTG ACGGCGGTAG CGCCGCGACT ATTCGTTTCA 420 AACTCACGAG GATAAGAGCC TATGACCGAT CCACGTCAGC TGCACCTGGC CGGATTCTTC 480 TGTGCCGGCA ACGTCACGCA CGCCCACGGA GCGTGGCGCC ACGCCGACGA CTCCAACGGC 540 TTCCTCACCA AGGAGTACTA CCAGCAGATT GCCCGCACGC TCGAGCGCGG CAAGTTCGAC 600 CTGCTGTTCC TTCCCGACGC GCTCGCCGTG TGGGACAGCT ACGGCGACAA TCTGGAGACC 660 GGTCTGCGGT ATGGCGGGCA AGGCGCGGTG ATGCTGGAGC CCGGCGTAGT TATCGCCGCG 720 ATGGCCTCGG TGACCGAACA TCTGGGGCTG GGCGCCACCA TTTCCACCAC CTACTACCCG 780 CCCTACCATG TAGCCCGGGT CGTCGCTTCG CTGGACCAGC TGTCCTCCGG GCGAGTGTCG 840 TGGAACGTGG TCACCTCGCT CAGCAATGCA GAGGCGCGCA ACTTCGGCTT CGATGAACAT 900 CTCGACCACG ATGCCCGCTA CGATCGCGCC GATGAATTCC TCGAGGTCGT GCGCAAGCTC 960 TGGAACAGCT GGGATCGCGA TGCGCTGACA CTCGACAAGG CAACCGGCCA GTTCGCCGAT 1020 CCGGCTAAGG TGCGCTACAT CGACCACCGC GGCGAATGGC TCAACGTACG CGGGCCGCTT 1080 CAGGTGCCGC GCTCCCCCCA GGGCGAGCCT GTCATTCTGC AGGCCGGGCT TTCGGCGCGG 1140 GGCAAGCGCT TCGCCGGGCG CTGGGCGGAC GCGGTGTTCA CGATTTCGCC CAATCTGGAC 1200 ATCATGCAGG CCACGTACCG CGACATAAAG GCGCAGGTCG AGGCCGCCGG ACGCGATCCC 1260 GAGCAGGTCA AGGTGTTTGC CGCGGTGATG CCGATCCTCG GCGAGACCGA GGCGATCGCC 1320 AGGCAGCGTC TCGAATACAT AAATTCGCTG GTGCATCCCG AAGTCGGGCT TTCTACGTTG 1380 TCCAGCCATG TCGGGGTCAA CCTTGCCGAC TATTCGCTCG ATACCCCGCT GACCGAGGTC 1440 CTGGGCGATC TCGCCCAGCG CAACGTGCCC ACCCAACTGG GCATGTTCGC CAGGATGTTG 1500 CAGGCCGAGA CGCTGACCGT GGGAGAAATG GGCCGGCGTT ATGGCGCCAA CGTGGGCTTC 1560 GTCCCGCAGT GGGCGGGAAC CCGCGAGCAG ATCGCGGACC TGATCGAGAT CCATTTCAAG 1620 GCCGGCGGCG CCGATGGCTT CATCATCTCG CCGGCGTTCC TGCCCGGATC TTACGAGGAA 1680 TTCGTCGATC AGGTGGTGCC CATCCTGCAG CACCGCGGAC TGTTCCGCAC TGATTACGAA 1740 GGCCGCACCC TGCGCAGCCA TCTGGGACTG CGTGAACCCG CATACCTGGG AGAGTACGCA 1800 TGACGACAGA CATCCACCCG GCGAGCGCCG CATCGTCGCC GGCGGCGCGC GCGACGATCA 1860 CCTACAGCAA CTGCCCCGTG CCTAATGCCC TGCTCGCCGC GCTCGGCTCA GGTATTCTGG 1920 ACAGTGCCGG GATCACACTT GCCCTGCTGA CCGGAAAGCA GGGCGAGGTG CACTTCACCT 1980 ACGACCGAGA TGACTACACC CGCTTCGGCG GCGAGATTCC GCCGCTGGTC AGCGAGGGAC 2040 TGCGTGCGCC GGGGCGGACC CGCCTGCTGG GACTGACGCC GGTGCTGGGC CGCTGGGGCT 2100 ACTTCGTCCG GGGCGACAGC GCGATCCGCA CCCCGGCCGA TCTTGCCGGC CGCCGCGTCG 2160 GAGTATCCGA TTCGGCCAGG AGGATATTGA CCGGAAGGCT GGGCGACTAC CGCGAACTTG 2220 ATCCCTGGCG GCAGACCCTG GTCGCGCTGG GGACATGGGA GGCGCGTGCC TTGCTGAGCA 2280 CGCTCGAGAC GGCGGGGCTT GGCGTCGGCG ACGTCGAGCT GACGCGCATC GAGAACCCGT 2340 TCGTCGACGT GCCGACCGAA CGACTGCATG CCGCCGGCTC GCTCAAAGGA ACCGACCTGT 2400 TCCCCGACGT GACCAGCCAG CAGGCCGCAG TCCTTGAGGA TGAGCGCGCC GACGCCCTGT 2460 TCGCGTGGCT TCCCTGGGCG GCCGAGCTCG AGACCCGCAT CGGTGCACGG CCGGTCCTAG 2520 ACCTCAGCGC AGACGACCGC AATGCCTATG CGAGCACCTG GACGGTGAGC GCCGAGCTGG 2580 TGGACCGGCA GCCCGAACTG GTGCAGCGGC TCGTCGATGC CGTGGTGGAT GCAGGGCGGT 2640 GGGCCGAGGC CAATGGCGAT GTCGTCTCCC GCCTGCACGC CGATAACCTC GGTGTCAGTC 2700 CCGAAAGCGT CCGCCAGGGA TTCGGAGCCG ATTTTCACCG CCGCCTGACG CCGCGGCTCG 2760 ACAGCGATGC TATCGCCATC CTGGAGCGTA CTCAGCGGTT CCTGAAGGAT GCGAACCTGA 2820 TCGATCGGTC GTTGGCGCTC GATCGGTGGG CTGCACCTGA ATTCCTCGAA CAAAGTCTCT 2880 CACGCCAGGT CGAAGGGCAG ATAGCATGAA CGAACTCGTC AAAGATCTCG GCCTCAATCG 2940 ATCCGATCCG ATCGGCGCTG TGCGGCGACT GGCCGCGCAG TGGGGGGCCA CCGCTGTTGA 3000 TCGGGACCGG GCCGGCGGAT CGGCAACCGC CGAACTCGAT CAACTGCGCG GCAGCGGCCT 3060 GCTCTCGCTG TCCATTCCCG CCGCATATGG CGGCTGGGGC GCCGACTGGC CAACGACTCT 3120 GGAAGTTATC CGCGAAGTCG CAACGGTGGA CGGATCGCTG GCGCATCTAT TCGGCTACCA 3180 CCTCGGCTGC GTACCGATGA TCGAGCTGTT CGGCTCGGCG CCACAAAAGG AACGGCTGTA 3240 CCGCCAGATC GCAAGCCATG ATTGGCGGGT CGGGAATGCG TCGAGCGAAA ACAACAGCCA 3300 CGTGCTCGAG TGGAAGCTTG CCGCCACCGC CGTCGATGAT GGCGGGTTCG TCCTCAACGG 3360 CGCGAAGCAC TTCTGCAGCG GCGCCAAAAG CTCCGACCTG CTCATCGTGT TCGGCGTGAT 3420 CCAGGACGAA TCCCCCCTGC GCGGCGCGAT CATCACCGCG GTCATTCCCA CCGACCGGGC 3480 CGGTGTTCAG ATCAATGACG ACTGGCGCGC AATCGGGATG CGCCAGACCG ACAGCGGCAG 3540 CGCCGAATTT CGCGACGTCC GAGTCTACCC AGACGAGATC TTGGGGGCAC CAAACTCAGT 3600 CGTTGAGGCG TTCGTGACAA GCAACCGCGG CAGCCTGTGG ACGCCGGCGA TTCAGTCGAT 3660 CTTCTCGAAC GTTTATCTGG GGCTCGCGCG TGGCGCGCTC GAGGCGGCAG CGGATTACAC 3720 CCGGACCCAG AGCCGCCCCT GGACACCCGC CGGCGTGGCG AAGGCGACAG AGGATCCCCA 3780 CATCATCGCC ACCTACGGTG AACTGGCGAT CGCGCTCCAG GGCGCCGAGG CGGCCGCGCG 3840 CGAGGTCGCG GCCCTGTTGC AACAGGCGTG GGACAAGGGC GATGCGGTGA CGCCCGAAGA 3900 GCGCGGCCAG CTGATGGTGA AGGTTTCGGG TGTGAAGGCC CTCTCGACGA AGGCCGCCCT 3960 CGACATCACC AGCCGTATTT TCGAGACAAC GGGCTCGCGA TCGACGCATC CCAGATACGG 4020 ATTCGATCGG TTCTGGCGTA ACATCCGGAC TCATACGCTG CACGATCCGG TATCGTATAA 4080 AATCGTCGAT GTGGGGAACT ACACGCTCAA CGGGACATTC CCGGTTCCCG GATTTACGTC 4140 ATGA 4144 (2) INFORMATION FOR SEQ ID NO : 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4144 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 13: TCATGACGTA AATCCGGGAA CCGGGAATGT CCCGTTGAGC GTGTAGTTCC CCACATCGAC 60 GATTTTATAC GATACCGGAT CGTGCAGCGT ATGAGTCCGG ATGTTACGCC AGAACCGATC 120 GAATCCGTAT CTGGGATGCG TCGATCGCGA GCCCGTTGTC TCGAAAATAC GGCTGGTGAT 180 GTCGAGGGCG GCCTTCGTCG AGAGGGCCTT CACACCCGAA ACCTTCACCA TCAGCTGGCC 240 GCGCTCTTCG GGCGTCACCG CATCGCCCTT GTCCCACGCC TGTTGCAACA GGGCCGCGAC 300 CTCGCGCGCG GCCGCCTCGG CGCCCTGGAG CGCGATCGCC AGTTCACCGT AGGTGGCGAT 360 GATGTGGGGA TCCTCTGTCG CCTTCGCCAC GCCGGCGGGT GTCCAGGGGC GGCTCTGGGT 420 CCGGGTGTAA TCCGCTGCCG CCTCGAGCGC GCCACGCGCG AGCCCCAGAT AAACGTTCGA 480 GAAGATCGAC TGAATCGCCG GCGTCCACAG GCTGCCGCGG TTGCTTGTCA CGAACGCCTC 540 AACGACTGAG TTTGGTGCCC CCAAGATCTC GTCTGGGTAG ACTCGGACGT CGCGAAATTC 600 GGCGCTGCCG CTGTCGGTCT GGCGCATCCC GATTGCGCGC CAGTCGTCAT TGATCTGAAC 660 ACCGGCCCGG TCGGTGGGAA TGACCGCGGT GATGATCGCG CCGCGCAGGG GGGATTCGTC 720 CTGGATCACG CCGAACACGA TGAGCAGGTC GGAGCTTTTG GCGCCGCTGC AGAAGTGCTT 780 CGCGCCGTTG AGGACGAACC CGCCATCATC GACGGCGGTG GCGGCAAGCT TCCACTCGAG 840 CACGTGGCTG TTGTTTTCGC TCGACGCATT CCCGACCCGC CAATCATGGC TTGCGATCTG 900 GCGGTACAGC CGTTCCTTTT GTGGCGCCGA GCCGAACAGC TCGATCATCG GTACGCAGCC 960 GAGGTGGTAG CCGAATAGAT GCGCCAGCGA TCCGTCCACC GTTGCGACTT CGCGGATAAC 1020 TTCCAGAGTC GTTGGCCAGT CGGCGCCCCA GCCGCCATAT GCGGCGGGAA TGGACAGCGA 1080 GAGCAGGCCG CTGCCGCGCA GTTGATCGAG TTCGGCGGTT GCCGATCCGC CGGCCCGGTC 1140 CCGATCAACA GCGGTGGCCC CCCACTGCGC GGCCAGTCGC CGCACAGCGC CGATCGGATC 1200 GGATCGATTG AGGCCGAGAT CTTTGACGAG TTCGTTCATG CTATCTGCCC TTCGACCTGG 1260 CGTGAGAGAC TTTGTTCGAG GAATTCAGGT GCAGCCCACC GATCGAGCGC CAACGACCGA 1320 TCGATCAGGT TCGCATCCTT CAGGAACCGC TGAGTACGCT CCAGGATGGC GATAGCATCG 1380 CTGTCGAGCC GCGGCGTCAG GCGGCGGTGA AAATCGGCTC CGAATCCCTG GCGGACGCTT 1440 TCGGGACTGA CACCGAGGTT ATCGGCGTGC AGGCGGGAGA CGACATCGCC ATTGGCCTCG 1500 GCCCACCGCC CTGCATCCAC CACGGCATCG ACGAGCCGCT GCACCAGTTC GGGCTGCCGG 1560 TCCACCAGCT CGGCGCTCAC CGTCCAGGTG CTCGCATAGG CATTGCGGTC GTCTGCGCTG 1620 AGGTCTAGGA CCGGCCGTGC ACCGATGCGG GTCTCGAGCT CGGCCGCCCA GGGAAGCCAC 1680 GCGAACAGGG CGTCGGCGCG CTCATCCTCA AGGACTGCGG CCTGCTGGCT GGTCACGTCG 1740 GGGAACAGGT CGGTTCCTTT GAGCGAGCCG GCGGCATGCA GTCGTTCGGT CGGCACGTCG 1800 ACGAACGGGT TCTCGATGCG CGTCAGCTCG ACGTCGCCG_. CGCCAAGCCC CGCCGTCTCG 1860 AGCGTGCTCA GCAAGGCACG CGCCTCCCAT GTCCCCAGCG CGACCAGGGT CTGCCGCCAG 1920 GGATCAAGTT CGCGGTAGTC GCCCAGCCTT CCGGTCAATA TCCTCCTGGC CGAATCGGAT 1980 ACTCCGACGC GGCGGCCGGC AAGATCGGCC GGGGTGCGGA TCGCGCTGTC GCCCCGGACG 2040 AAGTAGCCCC AGCGGCCCAG CACCGGCGTC AGTCCCAGCA GGCGGGTCCG CCCCGGCGCA 2100 CGCAGTCCCT CGCTGACCAG CGGCGGAATC TCGCCGCCGA AGCGGGTGTA GTCATCTCGG 2160 TCGTAGGTGA AGTGCACCTC GCCCTGCTTT CCGGTCAGCA GGGCAAGTGT GATCCCGGCA 2220 CTGTCCAGAA TACCTGAGCC GAGCGCGGCG AGCAGGGCAT TAGGCACGGG GCAGTTGCTG 2280 TAGGTGATCG TCGCGCGCGC CGCCGGCGAC GATGCGGCGC TCGCCGGGTG GATGTCTGTC 2340 GTCATGCGTA CTCTCCCAGG TATGCGGGTT CACGCAGTCC CAGATGGCTG CGCAGGGTGC 2400 GGCCTTCGTA ATCAGTGCGG AACAGTCCGC GGTGCTGCAG GATGGGCACC ACCTGATCGA 2460 CGAATTCCTC GTAAGATCCG GGCAGGAACG CCGGCGAGAT GATGAAGCCA TCGGCGCCGC 2520 CGGCCTTGAA ATGGATCTCG ATCAGGTCCG CGATCTGCTC GCGGGTTCCC GCCCACTGCG 2580 GGACGAAGCC CACGTTGGCG CCATAACGCC GGCCCATTTC TCCCACGGTC AGCGTCTCGG 2640 CCTGCAACAT CCTGGCGAAC ATGCCCAGTT GGGTGGGCAC GTTGCGCTGG GCGAGATCGC 2700 CCAGGACCTC GGTCAGCGGG GTATCGAGCG AATAGTCGGC AAGGTTGACC CCGACATGGC 2760 TGGACAACGT AGAAAGCCCG ACTTCGGGAT GCACCAGCGA ATTTATGTAT TCGAGACGCT 2820 GCCTGGCGAT CGCCTCGGTC TCGCCGAGGA TCGGCATCAC CGCGGCAAAC ACCTTGACCT 2880 GCTCGGGATC GCGTCCGGCG GCCTCGACCT GCGCCTTTAT GTCGCGGTAC GTGGCCTGCA 2940 TGATGTCCAG ATTGGGCGAA ATCGTGAACA CCGCGTCCGC CCAGCGCCCG GCGAAGCGCT 3000 TGCCCCGCGC CGAAAGCCCG GCCTGCAGAA TGACAGGCTC GCCCTGGGGG GAGCGCGGCA 3060 CCTGAAGCGG CCCGCGTACG TTGAGCCATT CGCCGCGGTG GTCGATGTAG CGCACCTTAG 3120 CCGGATCGGC GAACTGGCCG GTTGCCTTGT CGAGTGTCAG CGCATCGCGA TCCCAGCTGT 3180 TCCAGAGCTT GCGCACGACC TCGAGGAATT CATCGGCGCG ATCGTAGCGG GCATCGTGGT 3240 CGAGATGTTC ATCGAAGCCG AAGTTGCGCG CCTCTGCATT GCTGAGCGAG GTGACCACGT 3300 TCCACGACAC TCGCCCGGAG GACAGCTGGT CCAGCGAAGC GACGACCCGG GCTACATGGT 3360 AGGGCGGGTA GTAGGTGGTG GAAATGGTGG CGCCCAGCCC CAGATGTTCG GTCACCGAGG 3420 CCATCGCGGC GATAACTACG CCGGGCTCCA GCATCACCGC GCCTTGCCCG CCATACCGCA 3480 GACCGGTCTC CAGATTGTCG CCGTAGCTGT CCCACACGGC GAGCGCGTCG GGAAGGAACA 3540 GCAGGTCGAA CTTGCCGCGC TCGAGCGTGC GGGCAATCTG CTGGTAGTAC TCCTTGGTGA 3600 GGAAGCCGTT GGAGTCGTCG GCGTGGCGCC ACGCTCCGTG GGCGTGCGTG ACGTTGCCGG 3660 CACAGAAGAA TCCGGCCAGG TGCAGCTGAC GTGGATCGGT CATAGGCTCT TATCCTCGTG 3720 AGTTTGAAAC GAATAGTCGC GGCGCTACCG CCGTCAGTCG CCCACGCGAA CCGGCGTCGA 3780 AAAGACCTTG GCTATGAACG ATGGCGCGAA AGCGTAGGAA GCTGCCGTAC GTCGGTCCTC 3840 TTCATTGGCA TTCGCGAAAT GCACGGATCT TTCGTGCTAG GTGCGGATAG TGGTCATGCG 3900 CATCGTATCA TCTCATCATT GGTGTGAGGG AACGCAATGC CTAGCGCTGT CGCCCCAGCA 3960 TCGCGCGCTT TAGCTCGTGG CAACCCGGAG CGAGCACTTG AGACACACGG TCGGCCTGAT 4020 CACCGAACTT AGTGTCGAAT AAATATCTAT TTATTTAGTA GAGATTCCTA TGTCAAGTGC 4080 AAGAAGAGGC CCCAGAGGAT GGTTTGAACC GCGCCGGGTT CGACGGTCAG ATCGATCTCG 4140 AACC 4144