Abstract:
The present invention relates to heat tolerant phytases and DNA sequences which code therefor. The phytases are useful in hydrolyzing phytate to inositol and inorganic phosphates. The phytases are valuable feed additives.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. application Ser. No. 08/868,435 filed Jun. 3, 1997, now U.S. Pat. No. 6,291,221, which is a divisional of U.S. application Ser. No. 08/744,231 filed Nov. 5, 1996, now U.S. Pat. No. 6,358,722, which is a contiuation-in-part of U.S. application Ser. No. 08/424,757 filed Apr. 18, 1995 now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     Phosphorus is an essential element for the growth of all organisms. In livestock production, feed must be supplemented with inorganic phosphorus in order to obtain a good growth is performance of monogastric animals (for example, pigs, poultry and fish). 
     In contrast, no inorganic phosphate needs to be added to the feedstuffs of ruminant animals. Microorganisms, present in the rumen, produce enzymes which analyze the conversion of phytate (myo-inositolhexakis-phosphate) to inositol and inorganic phosphate. 
     Phytate occurs as a storage phosphorus source in virtually all feed substances originating from plants. Phytate comprises 1-3% of all nuts, cereals, legumes, oil seeds, spores and pollen. Complex salts of phytic acid are termed phytin. Phytic acid is considered to be an anti-nutritional factor since it chelates minerals such as calcium, zinc, magnesium, iron and may also react with proteins, thereby decreasing the bioavailability of protein and nutritionally important minerals. 
     Phytate phosphorous passes through the gastro-intestinal tract of monogastric animals and is excreted in the manure. Though some hydrolysis of phytate does occur in the colon, the thus-released inorganic phosphorus has no nutritional value since inorganic phosphorus is absorbed only in the small intestine. As a consequence, a significant amount of the nutritionally important phosphorus is not used by monogastric animals, despite its presence in the feed. 
     The excretion of phytate phosphorus in manure has further consequences. Intensive livestock production has increased enormously during the past decades. Consequently, the amount of manure produced has increased correspondingly and has caused environmental problems in various parts of the world. This is due, in part, to the accumulation of phosphate from manure in surface waters which has caused eutrophication. For other background information, see European Patent Application Publication No. 420 358. 
     Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8) are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate and are known to be valuable feed additives. 
     A phytase was first described in rice bran in 1907 [Suzuki et al., Bull. Coll. Agr. Tokyo Imp. Univ. 7, 495 (1907)] and phytases from Aspergillus species in 1911 [Dox and Golden, J. Biol. Chem. 10, 183-186 (1911)]. Phytases have also been found in wheat bran, plant seeds, animal intestines and in microorganisms [Howsen and Davis, Enzyme Microb. Technol. 5, 377-382 (1983), Lambrechts et al., Biotech. Lett. 14, 61-66 (1992), Shieh and Ware, Appl. Microbiol. 16, 1348-1351 (1968)]. 
     The cloning and expression of the phytase from  Aspergillus niger  (ficuum) has been described by Van Hartingsveldt et al., in Gene, 127, 87-94 (1993) and in European Patent Application, Publication No. 420 358 and from  Aspergillus niger  var awamori by Piddington et al. in Gene 133, 55-62 (1993). 
     Since phytases used so far in agriculture have certain disadvantages, it is an object of the present invention to provide new phytases or polypeptides having phytase activity with improved properties. Since it is known that phytases used so far lose activity during feed pelleting process due to heat treatment, improved heat tolerance would be such an improved property. 
     So far, phytases have not been reported in thermotolerant fungus with the exception of  Aspergillus fumigatus  [Dox and Golden et al., J. Biol. Chem. 10, 183-186 (1911)] and  Rhizopus oryzae  [Howson and Davies, Enzyme Microb. Technol. 5, 377-382 (1993)]. Thermotolerant phytases have been described originating from  Aspergillus terreus  Strain 9A-1 [Temperature optimum 70° C.; Yamada et al., Agr. Biol. Chem. 32, 1275-1282 (1968)] and  Schwanniomyces castellii  [Temperature optimum 77° C.; Segueilha et al., Bioeng. 74, 7-11 (1992)]. However for commercial use in agriculture such phytases must be available in large quantities. Accordingly it is an object of the present invention to provide DNA sequences coding for heat tolerant phytases. Improved heat tolerance of phytases encoded by such DNA sequences can be determined by assays known in the art, for example, by the processes used for feed pelleting or assays determining the heat dependence of the enzymatic activity itself as described, for example, by Yamada et al. (s.a.). 
     It is furthermore an object of the present invention to screen fungi which show a certain degree of thermotolerance for phytase production. Such screening can be made as described, for example, in Example 1. In this way heat tolerant fungal strains, listed in Example 1, have been identified for the first time to produce a phytase. 
     Heat tolerant fungal strains, see for example, those listed in Example 1, can then be grown as known in the art, for example, as indicated by their supplier, for example, the American Tissue Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Agricultural Research Service Culture Collection (NRRL) and the Centralbureau voor Schimmelcultures (CBS) from which such strains are available or as indicated, for example, in Example 2. 
     Further improved properties are, for example, an improved substrate specificity regarding phytic acid [myo-inositol(1,2,3,4,5,6)hexakisphosphate] which is a major storage form of phosphorous in plants and seeds. Since for the complete release of the six phosphate groups from phytic acid, a phytase and a pH 2.5. acid phosphatase activity are required, a polypeptide having phytase and pH 2.5 acid phosphatase activity would be highly desirable. For example, International Patent Application Publication No. 94/03072 discloses an expression system which allows the expression of a mixture of phytate degrading enzymes in desired ratios. However, it would be even more desirable to have both such activities in a single polypeptide. Therefore it is also an object of the present invention to provide DNA sequences coding for such polypeptides. Phytase and phosphatase activities can be determined by assays known in the state of the art or described, for example, in Example 9. 
     Another improved property is, for example, a so called improved pH-profile. This means, for example, two phytin degrading activity maxima, for example, one at around pH 2.5 which could be the pH in the stomach of certain animals and another at around pH 5.5 which could be the pH after the stomach in certain animals. Such pH profile can be determined by assays known in the state of the art or described, for example, in Example 9. Accordingly it is also an object of the present invention to provide DNA sequences coding for such improved polypeptides. 
     It is yet another object of the present invention to provide a DNA sequence coding for a polypeptide having phytase activity and which DNA sequence is derived from a fungus selected from the group consisting of  Acrophialophora levis, Aspergillus terreus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus sojae, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus , Humicola sp.,  Mycelia sterilia, Myrococcum thermophilum, Myceliophthora thermophila, Rhizomucor miehei, Sporotrichum cellulophilum, Sporotrichum thermophile, Scytalidium indonesicum  and  Talaromyces thermophilus  or a DNA sequence coding for a fragment of such a polypeptide which fragment still has phytase activity, or more specifically such a DNA sequence wherein the fungus is selected from the group consisting of  Acrophialophora levis, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Sporotrichum cellulophilum, Sporotrichum thermophile, Mycelia sterilia, Myceliophthora thermophila  and  Talaromyces thermophilus , or more specifically such a DNA sequence wherein the fungus is selected from the group consisting of  Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Aspergillus nidulans  and  Talaromyces thermophilus . DNA sequences coding for a fragment of a polypeptide of the present invention can, for example, be between 1350 and 900, preferably between 900 and 450 and most preferably between 450 and 150 nucleotides long and can be prepared on the basis of the DNA sequence of the complete polypeptide by recombinant methods or by chemical synthesis with which one skilled in the art is familiar with. 
     Furthermore it is an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from the following: 
     (a) the DNA sequence of FIG. 1 [SEQ ID NO:1] or its complementary strand; 
     (b) a DNA sequence which hybridizes under standard conditions with sequences defined under (a) or preferably with the coding region of such sequences or more preferably with a region between positions 491 to 1856 of such DNA sequences or even more preferably with a genomic probe obtained by preferably random priming using DNA of Aspergillus terreus 9A1 as described in Example 12. 
     (c) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with sequences of (a) or (b), but which codes For polypeptides having exactly the same amino acid sequences as the polypeptides encoded by these DNA sequences; and 
     (d) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b) or (c). 
     “Standard conditions” for hybridization mean in this context the conditions which are generally used by one skilled in the art to detect specific hybridization signals and which are described, for example, by Sambrook et al., “Molecular Cloning” second edition, Cold Spring Harbor Laboratory Press 1989, New York, or preferably so called stringent hybridization and non-stringent washing conditions or more preferably so called stringent hybridization and stringent washing conditions one skilled in the art is familiar with and which are described, for example, in Sambrook et al. (s.a.) or even more preferred the stringent hybridization and non-stringent or stringent washing conditions as given in Example 12. “Fragment of the DNA sequences” means in this context a fragment which codes for a polypeptide still having phytase activity as specified above. 
     It is also an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from the following: 
     (a) the DNA sequence of FIG. 2 [SEQ ID NO:3] or its complementary strand; 
     (b) a DNA sequence which hybridizes under standard conditions with sequences defined under (a) or preferably a region which extends to about at least 80% of the coding region optionally comprising about between 100 to 150 nucleotides of the 5′ end of the non-coding region of such DNA sequences or more preferably with a region between positions 2068 to 3478 of such DNA sequences or even more preferably with a genomic probe obtained by preferably random priming using DNA of Myceliophthora thermophila as described in Example 12. 
     (c) a DNA sequence which, because of the degeneracy of the genetic code, does not hybrida&amp;e with sequences of (a) or (b), but which codes for polypeptides having exactly the same amino acid sequences as the polypeptides encoded by these DNA sequences; and 
     (d) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b) or (c). 
     “Fragments” and “standard conditions” have the meaning as given above. 
     It is also an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from the following: 
     (a) a DNA sequence comprising one of the DNA sequences of FIGS. 4 [SEQ ID NO:5],  5  [SEQ ID NO:7],  6  [SEQ ID NO:9] or  10 A and B [“aterr21”, SEQ ID NO:13; “aterr58”: SEQ ID NO:14] or its complementary strand; 
     (b) a DNA sequence which hybridizes under standard conditions with sequences defined under (a) or preferably with such sequences comprising the DNA sequence of FIG. 4 [SEQ ID NO:5] isolatable from  Talaromyces thermophilus , or of FIG. 5 [SEQ ID NO:7] isolatable from  Aspergillus fumigatus , or of FIG. 6 [SEQ ID NO:9] isolatable from  Aspergillus nidulans  or of one or both of the sequences given in FIGS. 10A and B [“aterr21”, SEQ ID NO:13; “aterr58”: SEQ ID NO:14] isolatable from  Aspergillus terreus  (CBS 116.46) or more preferably with a region of such DNA sequences spanning at least 80% of the coding region or most preferably with a genomic probe obtained by random priming using DNA of  Talaromyces thermophilus  or  Aspergillus fumigatus  or  Aspergillus nidulans  or  Aspergillus terreus  (CBS 116.46) as described in Example 12; 
     (c) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with sequences of (a) or (b) but which codes for polypeptides having exactly the same amino acid sequences as the polypeptides encoded by these DNA sequences; and 
     (d) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b) or (c). 
     It is furthermore an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from a DNA sequence comprising the DNA sequence of FIG. 4 [SEQ ID NO:5] isolatable from  Talaromyces thermophilus , of FIG. 5 [SEQ ID NO:7] isolatable from  Aspergillus fumigatus , of FIG. 6 [SEQ ID NO:9] isolatable from  Aspergillus nidulans  or of FIGS. 10A and B [“aterr21”: SEQ ID NO:13; “aterr58”: SEQ ID NO:14] isolatable from  Aspergillus terreus  (CBS 116.46) or which DNA sequence is a degenerate variant or equivalent thereof. 
     “Fragments” and “standard conditions ” have the meaning as given above. “Degenerate variant ” means in this context a DNA sequence which because of the degeneracy of the genetic code has a different nucleotide sequence as the one referred to but codes for a polypeptide with the same amino acid sequence. “Equivalent” refers in this context to a DNA sequence which codes for polypeptides having phytase activity with an amino acid sequence which differs by deletion, substitution and/or addition of one or more amino acids, preferably up to 50, more preferably up to 20, even more preferably up to 10 or most preferably 5, 4, 3 or 2, from the amino acid sequence of the polypeptide encoded by the DNA sequence to which the equivalent sequence refers to. Amino acid substitutions which do not generally alter the specific activity are known in the state of the art and are described, for example, by H. Neurath and R. L. Hill in “The Proteins” (Academic Press, New York, 1979, see especially FIG. 6, page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well as these in reverse (the three letter abbreviations are. used for amino acids and are standard and known in the art). 
     Such equivalents can be produced by methods known in the state of the art and described, for example, in Sambrook et al. (s.a.). Whether polypeptides encoded by such equivalent sequences still have a phytase activity can be determined by one of the assays known in the art or, for example, described in Example 9. 
     It is also an object of the present invention to provide one of the aforementioned DNA sequences which code for a polypeptide having phytase activity which DNA sequence is derived from a fungus, or more specifically such a fungus selected from one of the above mentioned specific groups of fungi. 
     Furthermore it is an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence hybridizes under standard conditions with a probe which is a product of a PCR reaction with DNA isolated from a fungus of one of the above mentioned groups of fungi and the following pair of PCR primer: 
     “ATGGA(C/T)ATGTG(C/T)TC(N)TT(C/T)GA” [SEQ ID NO:15] as sense primer and 
     “TT(A/G)CC(A/G)GC(A/G)CC(G/A)TG(N)CC(A/G)TA” [SEQ ID NO: 16] as anti-sense primer. 
     “Standard conditions” have the meaning given above. “Product of a PCR reaction” means preferably a product obtainable or more preferably as obtained by a reaction described in Example 12 referring back to Example 11. 
     Furthermore it is an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence hybridizes under standard conditions with a probe which is a product of a PCR reaction with DNA isolated from Aspergillus terreus (CBS 116.46) and the following two pairs of PCR primers: 
     (a) “ATGGA(C/T)ATGTG(C/T)TC(N)TT(C/T)GA” [SEQ ID NO:15] as the sense primer and 
     “TT(A/G)CC(A/G)GC(A/G)CC(G/A)TG(N)CC(A/G)TA” [SEQ ID NO:16] as the anti-sense primer; and 
     (b) “ITA(C/T)GC(N)GA(C/T)TT(C/T)TC(N)CA(C/T)GA-” [SEQ ID NO:17] as the sense primer and 
     “CG(G/A)TC(G/A)TT(N)AC(N)AG(N)AC(N)CO [SEQ ID NO:18] as the anti-sense primer. 
     “Standard conditions” are as defined above and the term “product of a PCR reaction” means preferably a product obtainable gor more preferably as obtained by a reaction described in Example 11. 
     It is furthermore an object of the present invention to provide a DNA sequence coding for a chimeric construct having phytase activity which chimeric construct comprises a fragment of a DNA sequence as specified above. The chimeric construct can comprise a fragment of a DNA sequence derived from a fungus. The fragment of a DNA sequence from a fungus can be fused to the fragment of another DNA sequence from another fungus. The N-terminal end of a DNA sequence from a fungus can be fused at its C-terminal end to the fragment of another DNA sequence from different fungus. The fungus from which the fragments can be selected include those from  Acrophialophora levis, Aspergillus terreus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus sojae, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus , Humicola sp.,  Mycelia sterilia, Myrococcum thermophilum, Myceliophthora thermophila, Rhizomucor miehei, Sporotrichum cellulophilum, Sporotrichum thermophile, Scytalidium indonesicum  and  Talaromyces thermophilus , preferably selected from the group consisting of  Acrophialophora levis, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Sporotrichum cellulophilum, Sporotrichum thermophile, Mycelia sterilia, Myceliophthora thermophila  and  Talaromyces thermophilus , more preferably selected from the group consisting of  Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Aspergillus nidulans  and  Talaromyces thermophilus , and even more preferably such a DNA sequence wherein the chimeric construct consists at its N-terminal end of a fragment of the  Aspergillus niger  phytase fused at its C-terminal end to a fragment of the  Aspergillus terreus  phytase, or more preferably such a DNA sequence with the specific nucleotide sequence as shown in FIG. 7 [SEQ ID NO:11] and a degenerate variant or equivalent thereof, wherein “degenerate variant” and “equivalent” have the meanings as given above. 
     It is furthermore an object of the present invention to provide for the partial sequence of a 6 kb HindIII/KpnI insert of clone 1 (see FIG. 13, discussed herein) (SEQ ID NO:28), which includes the complete phytase-encoding gene of  Aspergillus nidulans , a protein of 463 amino acids (SEQ ID NO:29). 
     It is furthermore an object of the present invention to provide for the partial sequences of a 5.5 kb EcoRI/SacI insert of clone Tt29-132 (see FIG. 17, discussed herein) (SEQ ID NO:30), which includes the complete phytase-encoding gene of  Talaromyces thermophilus , a protein of 466 amino acids (SEQ ID NO:31). 
     It is furthermore an object of the present invention to provide for the partial sequence of a 6 kb BamHI fragment (see FIG. 19 discussed herein) (SEQ ID NO: 32), which included the complete phytase-encoding gene of  Aspergillus fumigatus , a protein of 465 amino acids (SEQ ID NO:33). 
     It is furthermore an object of the present invention to provide for the partial sequence of a 2 kb KpnI insert of clone 227 (see FIG. 21 discussed herein) (SEQ ID NO:34), which includes the complete phytase-encoding gene of  Aspergillus terreus  (CBS116.46), a protein of 466 amino acids (SEQ ID NO:35). 
     Furthermore it is an object of the present invention to provide a DNA sequence as specified above wherein the encoded polypeptide is a phytase. 
     Furthermore, it is an object of the present invention to provide the polypeptides encoded by the above described DNA sequences which have phytase activity and fragments of the polypeptides which retain phytase activity, and in particular those polypeptides of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:33, and SEQ ID NO:35. 
     Genomic DNA or cDNA from fungal strains can be prepared as known in the art [see for example, Yelton et al., Procd. Natl. Acad. Sci. USA, 1470-1474 (1984) or Sambrook et al., s.a., or other standard reference for preparing CDNA from fungi] or, for example, as specifically described in Example 2. 
     The cloning of the DNA-sequences of the present invention from such genomic DNA can then be effected, for example, by using the well known polymerase chain reaction (PCR) method. The principles of this method are outlined for example, by White et al. (1989), whereas improved methods are described for example, in Innis et al. [PCR Protocols: A guide to Methods and Applications, Academic Press, Inc. (1990)]. PCR is an in vitro method for producing large amounts of a specific DNA of defined length and sequence from a mixture of different DNA-sequences. Thereby, PCR is based on the enzymatic amplification of the specific DNA fragment of interest which is flanked by two oligonucleotide primers which are specific for this sequence and which hybridize to the opposite strands of the target sequence. The primers are oriented with their 3′ ends pointing toward each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences and extension of the annealed primers with a DNA polymerase result in the amplification of the segment between the PCR primers. Since the extension product of each primer can serve as a template for the other, each cycle essentially doubles the amount of the DNA fragment produced in the previous cycle. By utilizing the thermostable Taq DNA polymerase, isolated from the thermophilic bacteria Thermus aquaticus, it has been possible to avoid denaturation of the polymerase which necessitated the addition of enzyme after each heat denaturation step. This development has led to the automation of PCR by a variety of simple temperature-cycling devices. In addition, the specificity of the amplification reaction is increased by allowing the use of higher temperatures for primer annealing and extension. The increased specificity improves the overall yield of amplified products by minimizing the competition by non-target fragments for enzyme and primers. In this way the specific sequence of interest is highly amplified and can be easily separated from the non-specific sequences by methods known in-the art, for example, by separation on an agarose gel and cloned by methods known in the art using vectors as described for example, by Holten and Graham in Nucleic Acid Res. 19, 1156 (1991), Kovalic et. al. in Nucleic Acid Res. 19, 4560 (1991), Marchuk et al. in Nucleic Acid Res. 19, 1154 (1991) or Mead et al. in Bio/Technology 9, 657-663 (1991). 
     The oligonucleotide primers used in the PCR procedure can be prepared as known in the art and described for example, in Sambrook et al. (1989 “Molecular cloning” 2nd edt., Cold Spring Harbor Laboratory Press, Cold Spring Harbor). 
     The specific primers used in the practice of the present invention have been designed as degenerate primers on the basis of DNA-sequence comparisons of known sequences of the  Aspergillus niger  phytase, the  Aspergillus niger  acid phosphatase, the  Saccharomyces cerevisiae  acid phosphatase and the  Schizosaccharomyces pombe  acid phosphatase (for sequence information see, for example, European Bioinformatics Institute (Hinxton Hall, Cambridge, GB). The degeneracy of the primers was reduced by selecting some codons according to a codon usage table of  Aspergillus niger  prepared on the basis of known sequences from  Aspergillus niger . Furthermore it has been found that the amino acid at the C-terminal end of the amino acid sequences used to define the specific probes should be a conserved amino acid in all acid phosphatases including phytases specified above but the rest of the amino acids should be more phytase than phosphatase specific. 
     Such amplified DNA-sequences can than be used to screen DNA libraries of DNA of, for example, fungal origin by methods known in the art (Sambrook et al., s.a.) or as specifically described in Examples 5-7. 
     Once complete DNA-sequences of the present invention have been obtained they can be integrated into vectors by methods known in the art and described for example, in Sambrook et al. (s.a.) to overexpress the encoded polypeptide in appropriate host systems. However, one skilled in the art knows that also the DNA-sequences themselves can be used to transform the suitable host systems of the invention to get overexpression of the encoded polypeptide. Appropriate host systems are for example fungi, like Aspergilli, for example, Aspergillus niger ATCC 91421 or  Aspergillus ficuum  [NRRL 31351 or like Trichoderma, for example,  Trichoderma reesei  or yeasts, like Saccharomyces, for example,  Saccharomyces cerevisiae  or Pichia, like  Pichia pastoris , all available from ATCC. Bacteria which can be used are for example,  E. coli , Bacilli as, for example,  Bacillus subtilis  or Streptomyces, for example,  Streptomyces lividans  (see for example, Anné and Mallaert in FEMS Microbiol. Letters 114, 121 (1993).  E. coli , which could be used are  E. coli  K12 strains for example, M15 [described as DZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974)], HB 101 [ATCC No. 33694] or  E. coli  SG13009 (Gottesman et al., J. Bacteriol. 148, 265-273 (1981)]. 
     Vectors which can be used for expression in fungi are known in the art and described for example, in EP 420 358, or by Cullen et al. [Bio/Technology 5, 369-376 (1987)] or Ward in Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York (1991), Upshall et al. [Bio/Technology 5, 1301-1304 (1987)] Gwynne et al. [Bio/Technology 5, 71-79 (1987)], Punt et al. [J. of Biotechnology 17, 19-34 (1991)] and for yeast by Sreekrishna et al. [J. Basic Microbiol. 28, 265-278 (1988), Biochem. 28, 4117-4125 (1989)], Hitzemann et al. [Nature A, 717-722 (1981)] or in EP 183 070, EP 183 071, EP 248 227, EP 263 311. Suitable vectors which can be used for expression in  E. coli  are mentioned, for example, by Sambrook et al. [s.a.] or by Fiers et al. in Procd. 8 th Int. Biotechnology Symposium” [Soc. Franc. de Microbiol., Paris (Durand et al., eds.), pp. 680-697 (1988)] or by Bujard et al. in Methods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc. Vol. 155, 416-433 (1987) and Stüber et al. in Immunological Methods, eds. Lefkovits and Pernis, Academic Press, Inc., Vol. IV, 121-152 (1990). Vectors which could be used for expression in Bacilli are known in the art and described, for example, in EP 405 370, Procd. Nat. Acad. Sci. USA, 81, 439 (1984) by Yansura and Henner, Meth. Enzym. 185, 199-228 (1990) or EP 207 459. 
     Either such vectors already carry regulatory elements, for example, promotors or the DNA-sequences of the present invention can be engineered to contain such elements. Suitable promotor-elements which can be used are known in the art and are, for example, for  Trichoderma reesei  the cbh1- [Haarki et al., Biotechnology 7, 596-600 (1989)] or the pki1-promotor [Schindler et al., Gene 130, 271-275 (1993)], for  Aspergillus oryzae  the amy-promotor [Christensen et al., Abstr. 19 th Lunteren Lectures on Molecular Genetics F23 (1987), Christensen et al., Biotechnology 6, 1419-1422 (1988), Tada et al., Mol. Gen. Genet. 229, 301 (1991)], for  Aspergillus niger  the glaA- [Cullen et al., Bio/Technology 5, 369-376 (1987), Gwynne et al., Bio/Technlogy 5, 713-719 (1987), Ward in Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York, 83-106 (1991)], alcA- [Gwynne et al., Bio/Technology 5, 71-719 (1987)], suc1- [Boddy et al. Current Genetics, 24, 60-66 (1993)], aphA- [MacRae et al., Gene 71, 339-348 (1988), MacRae et al., Gene 132, 193-198 (1993)], tpiA- [McKnight et al., Cell 46, 143-147 (1986), Upshall et al., Bio/Technology 5, 1301-1304 (1987)], gpdA- [Punt et al., Gene 69, 49-57 (1988), Punt et al., J. of Biotechnology 17, 19-37 (1991)] and the pkiA-promotor [de Graaff et al., Curr. Genet. 22, 21-27 (1992)]. Suitable promotor-elements which could be used for expression in yeast are known in the art and are, for example, the pho5-promotor [Vogel et al., Molecular and Cellular Biology, 2050-2057 (1989); Rudolf and Hinnen, Proc. Natl. Acad. Sci. 84, 1340-1344 (1987)] or the gap-promotor for expression in Saccharamyces cerevisiae und for  Pichia pastoris , for example, the aox1-promotor [Koutz et al. Yeast 5, 167-177 (1989); Sreekrishna et al., J. Basic Microbiol. 28, 265-278 (1988)]. 
     Accordingly vectors comprising DNA sequences of the present invention, preferably for the expression of said DNA sequences in bacteria or a fungal or a yeast host and such transformed bacteria or fungal or yeast hosts are also an object of the present invention. 
     Once such DNA-sequences have been expressed in an appropriate host cell in a suitable medium the encoded phytase can be isolated either from the medium in the case the phytase is secreted into the medium or from the host organism in case such phytase is present intracellularly by methods known in the art of protein purification or described, for example, in EP 420 358. Accordingly a process for the preparation of a polypeptide of the present invention characterized in that transformed bacteria or a host cell as described above is cultured under suitable culture conditions and the polypeptide is recovered therefrom and a polypeptide when produced by such a process or a polypeptide encoded by a DNA sequence of the present invention are also an object of the present invention. 
     Once obtained the polypeptides of the present invention can be characterized regarding their activity by assays known in the state of the art or as described, for example, by Engelen et al. [J. AOAC Intern. 77, 760-764 (1994)] or in Example 9. Regarding their properties which make the polypeptides of the present invention useful in agriculture any assay known in the art and described for example, by Simons et al. (British Journal of Nutrition 64, 525-540 (1990)], Schöner et al. [J. Anim. Physiol. a. Anim. Nutr. 66, 248-255 (1991)], Vogt [Arch. Geflügelk. 56, 93-98 (1992)], Jongbloed et al. [J. Anim. Sci., 70, 1159-1168 (1992)], Perney et al. [Poultry Science 72, 2106-2114 (1993)], Farrell et al., [J. Anim. Physiol. a. Anim. Nutr. 69, 278-283 (1993), Broz et al., [British Poultry Science 35, 273-280 (1994)] and Düngelhoef et al. [Animal Feed Science and Technology 49, 1-10 (1994)] can be used. Regarding their thermotolerance any assay known in the state of the art and described, for example, by Yamada et al. (s.a.), and regarding their pH and substrate specificity profiles any assays known in the state of the art and described, for example, in Example 9 or by Yamada et al., s.a., can be used. 
     In general the polypeptides of the present invention can be used without being limited to a specific field of application for the conversion of phytate to inositol and inorganic phosphate. 
     Furthermore the polypeptides of the present invention can be used in a process for the preparation of compound food or feeds wherein the components of such a composition, for example, feed and other nutrients, are mixed with one or more polypeptides of the present invention. The feed can then be fed to those animals, especially monogastic animals (for example, pigs and poultry). Accordingly compound food or feeds comprising one or more polypeptides of the present invention are also an object of the present invention. One skilled in the art is familiar with their process of preparation. Such compound foods or feeds can further comprise additives or components generally used for such purpose and known in the state of the art. 
     It is furthermore an object of the present invention to provide a method for the reduction of levels of phytate in animal manure characterized in that an animal is fed such a feed composition in an amount effective in converting phytate contained in the feedstuff to inositol and inorganic phosphate. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows that amino acid sequence of the phytase from  Aspergillus terreus  strain 9A1 and its encoding DNA sequence (SEQ ID NO: 1). 
     FIG. 2 shows the amino acid sequence of the phytase from  Myceliophthora thermophila  and its encoding DNA sequence (SEQ ID NO: 3). 
     FIG. 3A shows a restriction map for the DNA of  Aspergillus terreus.    
     FIG. 3B shows a restriction map for the DNA of  Myceliophthora thermophila.    
     FIG. 4 shows the amino acid sequence of the phytase from  Talaromyces thermophilus  and its encoding DNA sequence (SEQ ID NO: 5). 
     FIG. 5 shows the amino acid sequence of the phytase from  Aspergillus fumigatus  and its encoding DNA sequence (SEQ ID NO: 7). 
     FIG. 6 shows the amino acid sequence of the phytase from  Aspergillus nidulans  and its encoding DNA sequence (SEQ ID NO: 9). 
     FIG. 7 shows the amino acid sequence of the phytase from the fusion construct of  Aspergillus niger  and  Aspergillus terreus  and its encoding DNA sequence (SEQ ID NO: 11). 
     FIG. 8 shows physical map of vector pFPAN1. 
     FIG. 9 shows physical map of plasmid pPAT1. 
     FIG. 10 shows DNA sequences of two different PCR fragments obtained and their comparison to relevant parts of the phytase gene of  Aspergillus terreus  9A1. Relevant parts of the phytase gene of  Aspergillus terreus  9A1 “9A1” (top lines) (1) and the PCR fragments of  Aspergillus terreus  CBS 116.46 “aterr21” (SEQ ID NO: 13) (bottom lines). Panel A: Fragment obtained with primer pair 8 plus 9 (aterr2 l. Panel B: Fragment obtained with primer pair 10 plus 11 (aterr58(SEQ ID NO: 14). 
     FIG.  11 : DNA fragments of phytase genes from different fungi obtained by PCR using primers 8 [SEQ ID NO:16] and 9 [SEQ ID NO:16], a:  T. thermophilus  (PCRTth); b:  A. fumigatus  (PCRAfu); c)  A. nidulans  (PCRAni); d:  A. terreus  CBS116.46 (PCRAteCBS89). PCR amplified DNA fragment of  A. terreus  CBS116.46 obtained with primers 10 [SEQ ID NO:17] and 11 (SEQ ID NO:18]: e) (PCRAteCBS1011). The underlined sequence in panel d) shows the position of the Aterr21 primer. The underlined sequence in e) shows the antisense sequence of primer Aterr58. The sequence originating from the primers used to obtain these fragments is not included. 
     FIG.  12 : Southern blot hybridization analysis of  A. nidulans  genomic DNA digested with the restriction enzymes shown on top of each lane and hybridized to the radiolabelled PCRAni probe. 
     FIG.  13 : Clone 1 was obtained by screening partial, size selected HindIII/KpnI libraries (5-7 kb), with the PCRAni probe as outlined herein. Partial sequence of the 6 kb HindIII/KpnI insert of clone 1, including the complete phytase-encoding gene of  A. nidulans . The intron is indicated by lower-case letters. Potential-N-glycosylation sites are marked with a +. The position of the PCR fragment is indicated by the underlined sequence (SEQ. ID NO: 28). 
     FIG.  14 : Southern blot hybridization analysis of  T. thermophilus  genomic DNA digested with the restriction enzymes shown on top of each lane and hybridized to the radiolabelled PCRTth probe. The 4.7 kb XbaI/BamHI fragment was obtained by screening a partial, size selected XbaI/BamHI library (4-5 kb), with the PCRTth probe as outlined herein resulting in clone Tt29. 
     FIG.  15 : Southern blot hybridization analysis of  T. thermophilus  genomic DNA digested with the restriction enzymes shown on top of each lane and hybridized to the radiolabelled BamHI/BstEII probe. To get the indicated 4.5 kb EcoRI/BstEII fragment, size selected (4-5 kb) genomic DNA digested with EcoRI and BstEII was isolated and cloned into the BstEII/EcoRI site of clone Tt29 resulting in clone Tt29-132. 
     FIG.  16 : Map of the region covered by the inserts of clones Tt29, Tt29-1 and Tt29-132 spanning 9.2 kb of the genomic DNA of  T. thermophilus . The position and direction of the transcription of the phytase gene is indicated. 
     FIG.  17 : Partial sequence of the 5.5 kb EcoRI/SacI insert of Tt29-132, carrying the complete phytase-encoding gene of  T. thermophilus . The intron is indicated by lower-case letters. Potential N-glycosylation sites are marked with a +. The position of the PCR fragment is indicated by the underlined sequence (SEQ ID NO: 30). 
     FIG.  18 : Map of 15 kb NotI insert of the positive Lambda clone isolated from the FIXII  A. fumigatus  genomic DNA library containing the phytase gene. The position of the subcloned 6 kb BamHI fragment containing the the phytase gene and the direction of the transcription are indicated. 
     FIG.  19 : Partial sequence of the 6 kb BamHI fragment including the complete phytase-encoding gene of  A. fumigatus . The intron is indicated by lower-case letters. Potential N-glycosylation sites are marked with a +(SEQ ID NO: 32). 
     FIG.  20 : Map of the  A. terreus  strain 9A1 phytase showing the position of the different primers used for PCR amplifications on genomic DNA of  A. terreus  CBS116.46. The expected size of the two PCR products are also indicated. 
     FIG.  21 : Partial sequence of the 2 kb KpnI insert of clone 227 including the complete phytase-encoding gene of  A. terreus  CBS116.46. The intron is indicated by lower-case letters. Potential N-glycosylation sites are marked with a +. The position of the PCR fragment is indicated by the underlined sequence (SEQ ID NO: 34). 
     FIG.  22 : Purification of  A. fumigatus  phytase. The OD 280  and conductivity traces are shown. All contaminants eluted either in the break-through fractions or early in the gradient (around 10 ml).  A. fumigatus  phytase was eluted as a symmetrical and homogeneous peak at approx. 15 ml. 
     FIG.  23 : Substrate specificities of purified  A. fumigatus, A. nidulans  and  A. terreus  CBS phytase. The activities found with these substrates were expressed relative (in %) to the activity found with phytic acid. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLES 
     Deposit of Biological Material 
     The  Aspergillus terreus  CBS 116.46 strain was deposited under the terms of the Budapest Treaty on Mar. 3, 1995 at the Centralbureau voor Schimmel-cultures, Oosterstraat 1, P.O. Box 273, 3740 AG BAARN, The Netherlands, and it was given the deposit No: CBS 220.95. 
     The  Aspergillus terreus  9A1 strain was deposited under the terms of the Budapest Treaty on Mar. 17, 1994 at the DSM-DEUTSCHE SAMMLUNG VON, MIKROORGANISMEN UND ZELLKULTUREN, GmbH, Maacheroder Weg 1b, D-38124 Braunschweig, and it was given the deposit No.: DSM 9076. 
     
       
         
               
             
               
               
               
             
               
             
               
               
             
               
               
               
             
               
               
             
           
               
                   
               
               
                 Specific media and solutions used 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Complete medium (Clutterbuck) 
                   
                   
               
               
                 Glucose 
                 10 
                 g/l 
               
               
                 —CN solution 
                 10 
                 ml/l 
               
               
                 Sodium nitrate 
                 6 
                 g/l 
               
               
                 Bacto peptone (Difco Lab., Detroit, MI, USA) 
                 2 
                 g/l 
               
               
                 Yeast Extract (Difco) 
                 1 
                 g/l 
               
               
                 Casamino acids (Difco) 
                 1.5 
                 g/l 
               
               
                 Modified trace element solution 
                 1 
                 ml/l 
               
               
                 Vitamin solution 
                 1 
                 ml/l 
               
               
                 M3 Medium 
               
               
                 Glucose 
                 10 
                 g/l 
               
               
                 —CN Solution 
                 10 
                 ml/l 
               
               
                 Modified trace element solution 
                 1 
                 ml/l 
               
               
                 Ammonium nitrate 
                 2 
                 g/l 
               
             
          
           
               
                 M3 Medium - Phosphate 
               
               
                 M3 medium except that —CN is replaced with —CNP 
               
               
                 M3 Medium - Phosphate + Phytate 
               
               
                 M3 Medium - Phosphate with the addition of 5 g/l of Na 12  Phytate 
               
               
                 (Sigma #P-3168; Sigma, St. Louis, MO, USA) 
               
               
                 Modified trace element solution 
               
             
          
           
               
                 CuSO4 
                 0.04% 
               
               
                 FeSO4.7H 2 O 
                 0.08% 
               
               
                 Na 2 MoO 4 .2H 2 O 
                 0.08% 
               
               
                 ZnSO 4 .7H 2 O 
                 0.8% 
               
               
                 B 4 Na 2 O 7 .10H 2 O 
                 0.004% 
               
               
                 MnSO 4 .H 2 O 
                 0.08% 
               
               
                 Vitamin Solution 
               
               
                 Riboflavin 
                 0.1% 
               
               
                 Nicotinamide 
                 0.1% 
               
               
                 p-amino benzoic acid 
                 0.01% 
               
               
                 Pyridoxine/HCl 
                 0.05% 
               
               
                 Aneurine/HCl 
                 0.05% 
               
               
                 Biotin 
                 0.001% 
               
               
                 —CN Solution 
               
             
          
           
               
                  KH 2 PO 4   
                 140 
                 g/l 
               
               
                 K 2 PO 4 .3H 2 O 
                 90 
                 g/l 
               
               
                 KCl 
                 10 
                 g/l 
               
               
                 MgSO 4 .7H 2 O 
                 10 
                 g/l 
               
               
                 —CNP Solution 
               
             
          
           
               
                 HEPES 
                 47.6 g/200 mls    
               
               
                 KCl 
                 2 g/200 mls 
               
               
                 MgSO 4 .7H 2 O 
                 2 g/200 mls 
               
               
                   
               
             
          
         
       
     
     Example 1 
     Screening Fungi for Phytase Activity 
     Fungi were screened on a three plate system, using the following three media: “M3” a defined medium containing phosphate); “M3-P” (M3 medium lacking phosphate); and “M3 P+Phytate” (M3 medium lacking phosphate but containing phytate as a sole phosphorus source). Plates were made with agarose to decrease the background level of phosphate. 
     Fungi were grown on the medium and at the temperature recommended by the supplier. Either spores or mycelium were transferred to the test plates and incubated at the recommended temperature until growth was observed. 
     The following thermotolerant strains were found to exhibit growth consistent with the production of an extracellular phytase: 
       Myceliophthora thermophila  [ATCC 48102] 
       Talaromyces thermophilus  [ATCC 20186] 
       Aspergillus fumigatus  [ATCC 346251] 
     Example 2 
     Growth of Fungi and Dreparation of Genomic DNA 
     Strains of  Myceliophthora thermophila, Talaromyces thermophilus, Aspergillus fumigatus, Aspergillus nidulans  and  Aspergillus terreus  9A-1 were grown in Potato Dextrose Broth (Difco Lab., Detroit, Mich., USA) or complete medium (Clutterbuck).  Aspergillus terreus  9A-1 and  Aspergillus nidulans  have been deposited under the Budapest Treaty for patent purposes at the DSM in Braunschweig, BRD at Mar. 17, 1994 under accession number DSM 9076 and at Feb. 17, 1995 under accession number DSM 9743, respectively. 
     Genomic DNA was Prepared as Follows: 
     Medium was innoculated at a high density with spores and grown overnight (O/N) with shaking. This produced a thick culture of small fungal pellets. The mycelium was recovered by filtration blotted dry and weighed. Up to 2.0 g was used per preparation. The mycelium was ground to a fine powder in liquid nitrogen and immediately added to 10 mls of extraction buffer (200 mM Tris/HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS, pH 8.5) and mixed well. Phenol (7 mls) was added to the slurry and mixed and then chloroform (3 mls) was also added and mixed well. The mixture was centrifuged (20,000 g) and the aqueous phase recovered. RNase A was added to a final concentration of 250 μ/ml and incubated at 37° C. for 15 minutes. The mixture was then extracted with 1 volume of chloroform and centrifuged (10,000 g, 10 minutes). The aqueous phase was recovered and the DNA precipitated with 0.54 volumes of RT isopropanol for 1 hour at room temperature (RT). The DNA was recovered by spooling and resuspended in water. 
     The Resultant DNA was Further Purified as Follows: 
     A portion of the DNA was digested with proteinase K for 2 hrs at 37 °C. and then extracted repeatedly (twice to three times) with an equal volume of phenol/chloroform and then ethanol precipitated prior to resuspension in water to a concentration of approximately 1 μg/μl. 
     Example 3 
     Degenerate PCR 
     PCR was performed essentially according to the protocol of Perkin Elmer Cetus [(PEC); Norwalk, Conn., USA]. The following two primers were used (bases indicated in brackets are either/or): 
     Phyt 8: 5′ ATG GA(C/T) ATG TG(C/T) TC(N) TT(C/T) GA 3′ [SEQ ID NO:15] 
     Degeneracy =32 
     Tm High =60° C./Tm Low 52° C. 
     Phyt 9: 5′ TT(A/G) CC(A/G) GC(A/G) CC(G/A) TG(N) CC(G/A) TA 3′ 
     [SEQ ID NO:16] 
     Tm High =70° C./Tm Low 58° C. 
     A typical Reaction was Performed as Follows: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 H 2 O 
                 24.5 
                 μl 
               
               
                   
                 10× PEC GeneAmp Buffer 
                 5 
                 μl 
               
               
                   
                 GeneAmp dNTP&#39;s (10 mM) 
                 8 
                 μl 
               
               
                   
                 Primer 1 (Phyt 8, 100 μM) 
                 5 
                 μl 
               
               
                   
                 Primer 2 (Phyt 9, 100 μM) 
                 5 
                 μl 
               
               
                   
                 DNA (˜1 μg/μl) 
                 1 
                 μl 
               
               
                   
                 Taq Polymerase (PEC) 
                 0.5 
                 μl 
               
               
                   
                   
                 50 
                 μl 
               
               
                   
                   
               
             
          
         
       
     
     All components with the exception of the Taq polymerase were incubated at 95 ° C. for 10 minutes and then 50° C. for 10 minutes and then the reaction placed on ice. The Taq polymerase (Amplitaq, F. Hoffmann-La Roche, Basel, CH) was then added and 35 cycles of PCR performed in a Triothermoblock (Biometra, Göttingen, DE) according to the following cycle profile: 
     95° C./60″ 
     50° C./90″ 
     72° C./120″ 
     An aliquot of the reaction was analyzed on 1.5% agarose gel. 
     Example 4 
     Subcloning and Sequencing of PCR Fragments 
     PCR products of the expected size (approximately 146 bp predicted from the  Aspergillus niger  DNA-sequence) were excised from low melting point agarose and purified from a NACS—PREPAC—column (BRL Life Technologies Inc., Gaithersburg, Md., USA) essentially according to the manufacturer&#39;s protocol. The fragment was polyadenylated in 50 μl 100 mM Sodiumcacodylate pH6.6, 12.5 mM Tris/HCI pH 7.0, 0.1 mM Dithiothreitol, 125 μg/ml bovine serum albumin, 1 mM CoCl 2 , 20 μMdATP, 10 units terminal deoxytransferase (Boehringer Mannheim, Mannheim, DE) for 5 minutes at 37° C. and cloned into the p123T vector [Mitchell et al., PCR Meth. App. 2, 81-82 (1992)]. 
     Alternatively, PCR fragments were purified and cloned using the “Sure Clone” ligation kit (Pharmacia) following the manufacturers instructions. 
     Sequencing was performed on dsDNA purified on a Quiagen-column (Diagen GmbH, Hilden, DE) using the dideoxy method and the Pharmacia T7 kit (Pharmacia, LKB Biotechnology AB, Uppsala, SE) according to the protocol supplied by the manufacturer. 
     Example 5 
     Construction and Screening of Lambda Fix II Libraries 
     The fragments from  Aspergillus terreus  Strain 9A-1 and  Myceliophthora thermophila  were used to probe BamHI and BglII southerns to determine the suitable restriction enzyme to use to construct genomic libraries in the Lambda Fix II vector (Strategene, La Jolla, Calif., USA). Lambda Fix II can only accept inserts from 9-23 kb. Southerns were performed according to the following protocol. Genomic DNA (10 gg) was digested in a final volume of 200 μl. The reaction without enzyme was prepared and incubated on ice for 2 hours. The enzyme (50 units) was added and the reaction incubated at the appropriate temperature for 3 hours. The reaction was then extracted with an equal volume of phenol/chloroform and ethanol precipitated. The resuspended DNA in loading buffer was heated to 65° C. for 15 minutes prior to separation on a 0.7% agarose gel (O/N 30 V). Prior to transfer the gel was washed twice in 0.2 M HCl/10′/room temperature (RT) and then twice in 1 M NaCl/0.4 M NaOH for 15′ at RT. The DNA was transferred in 0.4 M NaOH in a capillary transfer for 4 hours to Nytran 13 N nylon membrane (Schleicher and Schuell AG, Feldbach, Zürich, CH). Following transfer the membrane was exposed to UV. [Auto cross-link, UV Stratalinker 2400, Stratagene (La Jolla, Calif., USA)]. 
     The membrane was prehybridized in hybridization buffer [50% formamide, 1% sodium dodecylsulfate (SDS), 10% dextransulfate, 4×SSPE (180 mM NaCl, 10 mM NaH 2  PO 4 , 1 mM EDTA, ph 7.4)] for 4 hours at 42° C. and following addition of the denatured probe O/N at 42° C. The blot was washed: 
     1×SSPE/0.5% SDS/RT/30 minutes 
     0.1×SSPE/0.1% SDS/RT/30 minutes 
     0.1×SSPE/0.1% SDS/65° C./30 minutes 
     Results indicate that  Aspergillus terreus  Strain 9A-1 genomic DNA digested with BamHI and  Myceliophthora thermophila  genomic DNA digested with BglII produce fragments suitable for cloning into the lambda Fix II vector. 
     The construction of genomic libraries of  Aspergillus terreus  Strain 9A-1 and  Myceliophthora thermophila  in Lambda Fix II was performed according to the manufacturer&#39;s protocols (Stratagene). 
     The lambda libraries were plated out on 10 137 mm plates for each library. The plaques were lifted to Nytran 13N round filters and treated for 1 minute in 0.5 M NaOH/1.5 M NaCl followed by 5 minutes in 0.5 M Tris-HCl pH 8.0/1.5 M NaCl. The filters were then treated in 2×SSC for 5 minutes and air dried. They were then fixed with UV (1 minute, UV Stratalinker 2400, Stratagene). The filters were hybridized and washed as above. Putative positive plaques were cored and the phage soaked out in SM buffer (180 mM NaCl, 8 MM MgSO 4 .7H 2 O, 20mM Tris/HCl pH 7.5, 0.01% gelatin). This stock was diluted and plated out on 137 mm plates. Duplicate filters were lifted and treated as above. A clear single positive plaque from each plate was picked and diluted in SM buffer. Three positive plaques were picked. Two from  Aspergillus terreus  Strain 9A-1 (9A1λ17 and 9A1λ22) and one from  Myceliophthora thezmophila  (MTλ27). 
     Example 6 
     Preparation of Lambda DNA and Confirmation of the Clones 
     Lambda DNA was prepared from the positive plaques. This was done using the “Magic Lambda Prep” system (Promega Corp., Madison, Wis., USA) and was according to the manufactures specifications. To confirm the identity of the clones, the lambda DNA was digested with PstI and SalI and the resultant blot probed with the PCR products. In all cases this confirmed the clones as containing sequences complementary to the probe. 
     Example 7 
     Subcloning and Sequencing of Phytase Genes 
     DNA from 9A1λ17 was digested with PstI and the resultant mixture of fragments ligated into pBluescript II SK+ (Stratagene) cut with PstI and treated with shrimp alkaline phosphatase (United States Biochemical Corp., Cleveland, Ohio, USA). The ligation was O/N at 16° C. The ligation mixture was transformed into XL-1 Blue Supercompetent cells (Stratagene) and plated on LB Plates containing 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), 40 μg/ml 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (Xgal), 50 μg/ml ampicillin. 
     DNA from 9Aλ17 was digested with BglII and XbaI and the resultant mixture ligated into pBluescript II SK+ digested with BamHI/XbaI. Ligation, transformation and screening were performed as described above. 
     DNA from MTλ27 was digested with SalI and the resultant mixture of fragments ligated into pBluescript II SK+ cut with SalI and treated with shrimp alkaline phosphatase. The ligation was O/N at 16° C. The ligation mixture was transformed into XL1 Blue Supercompetent cells and plated on LB Plates containing Xgal/IPTG and ampicillin. 
     Colonies from the above transformations were picked and “gridded” approximately 75 to a single plate. Following O/N incubation at 37° C. the colonies were lifted to a nylon filter (“Hybond-N”, Amersham Corp., Arlington Heights, Ill., USA) and the filters treated with 0.5 M NaOH for 3 minutes, 1 M Tris/HCl pH7.5 twice for 1 minute, then 0.5 M Tris/HCl pH7.5/1.5 M NaCl for 5 minutes. The filters were air dried and then fixed with UV (2 minutes, UV Stratalinker 2400, Stratagene). The filters were hybridized with the PCR products of Example 5. Positive colonies were selected and DNA prepared. The subclones were sequenced as previously described in Example 4. Sequences determined are shown in FIG. 1 (FIG. 1) for the phytase from  Aspergillus terreus  strain 9A1 and its encoding DNA sequence, FIG. 2 for the phytase from  Myceliophthora thermophila  and its encoding DNA-sequence, FIG. 3A shows a restriction map for the DNA of  Aspergillus terreus  (wherein the arrow indicates the coding region, and the strips the regions sequenced in addition to the coding region) and 3B for  M. thermophila , and FIG. 4 for the phytase from  Talaromyces thermophilus  and its encoding DNA sequence, FIG. 5 for the phytase from  Aspergillus fumigatus  and its encoding DNA-sequence and FIG. 6 for the phytase from  Aspergillus nidulans  and its encoding DNA-sequence. The sequences for the phytases and its encoding DNA-sequences from  Talaromyces thermophilus, Aspergillus fumigatus  and  Aspergillus nidulans  were obtained in the same way as described for those of  Aspergillus terreus  strain 9A1 and  Myceliophthora thermophila  in Examples 2-7. Bases are given for both strands in small letters by the typically used one letter code abbreviations. Derived amino acid sequences of the phytase are given in capital letters by the typically used one letter code below the corresponding DNA-sequence. 
     Example 8 
     Construction of a Chimeric Construct Between  A. niger  and  A. terreus  Phytase DNA-sequences 
     All constructions were made using standard molecular biological procedures as described by Sambrook et al., (1989) (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY). 
     The first 146 amino acids (aa) of the  Aspergillus niger  phytase, as described in EP 420 358, were fused to the 320 Cterminal aa of the  Aspergillus terreus  9A1 gene. A NcoI site was introduced at the ATG start codon when the  A. niger  phytase gene was cloned by PCR. The intron found in the  A. niger  phytase was removed by site directed mutagenesis (Bio-Rad kit, Cat Nr 170-3581; Bio-Rad, Richmond, Calif., USA) using the following primer (wherein the vertical dash indicates that the sequence to its left hybridizes to the 3′ end of the first exon and the sequence to its right hybridizes to the 5′ end of the second exon): 
     5′-AGTCCGGAGGTGACT|CCAGCTAGGAGATAC-3′ [SEQ ID NO:19]. 
     To construct the chimeric construct of phytases from  A. niger  and  A. terreus  an Eco 47III site was introduced into the  A. niger  coding sequence to aid cloning. PCR with a mutagenic primer (5′ CGA TTC GTA gCG CTG GTA G 3′) in conjunction with the T3 primer was used to produce a DNA fragment that was cleaved with BamHI and Eco 47III. The BamHI/Eco 47III fragment was inserted into BamHI/Eco 47III cut p9A1Pst (Example 7). FIG. 7 shows the amino acid sequence of the fusion construct and its encoding DNA-sequence. 
     Example 9 
     Expression of Phytases 
     Construction of Expression Vectors 
     For expression of the fusion construct in  A. niger  an expression cassette was chosen where the fusion gene was under control of the inducible  A. niger  glucoamylase (glaA) promoter. 
     For the complete  A. terreus  9A1 gene, expression cassettes with the constitutive  A. nidulans  glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter were made. 
     All genes used for expression in  A. niger  carried their own signal sequence for secretion. 
     Construction of Vector pFPAN1 
     The  A. niger  glucoamylase (glaA) promoter was isolated as a 1960 bp XhoI/ClaI fragment from plasmid pDH33 [Smith et al. (1990), Gene 88: 259-262] and cloned into pBluescriptSK + -vector (pBS) [Stratagene, La Jolla, Calif., USA] containing the 710 bp BamHI/XbaI fragment of the  A. nidulans  trpC terminator. The plasmid with the cassette was named pGLAC. The fusion gene, as described in Example 8, was put under control of the  A. niger  glaA promoter by ligating the blunt ended NcoI/EcoRI fragment to the blunt ended ClaI site and the EcoRV site of plasmid pGLAC. The correct orientation was verified by restriction enzyme digests. The entire cassette was transferred as a KpnI/XbaI fragment to pUC19 (New England Biolabs, GmbH, Schwalbach, BRD), that carried the Neurospora crassa pyr4 gene (pUC19-pyr4), a selection marker in uridine auxotrophic Aspergilli, resulting in vector pFPAN1 (see FIG. 8 with restriction sites and coding regions as indicated; crossed out restriction sites indicate sites with blunt end ligation). 
     Construction of Vector pPAT1 
     The  A. nidulans  glyceraldehyd-3-phosphate dehydrogenase (gpdA) promoter was isolated as a ˜2.3 kb EcoRI/NcoI fragment from plasmid pAN52-1 [Punt et al. (1987), Gene 56: 117-124], cloned into pUC19-NcoI (pUC19 having a SmaI-site replaced by a NcoI-site), reisolated as EcoRI/BamHI fragment and cloned into pBS with the trpC terminator as described above. The obtained cassette was named pGPDN. The  A. terreus  gene was isolated as a NcoI/EcoRI fragment, where the EcoRI site was filled in to create blunt ends. Plasmid pGPDN was cut with BamHI and NcoI. The BamHI site was filled in to create blunt ends. The NcoI/EcoRI(blunt) fragment of the  A. terreus  gene was cloned between the gpda promoter and trpC terminator. The expression cassette was isolated as KpnI/XbaI fragment and cloned into pUC19-pyr4 resulting in plasmid pPAT1 (see FIG. 9; for explanation of abbreviations see legend to FIG.  8 ). 
       
     Expression of the Fusion Protein in  Aspergillus niger    
     A) Transformation 
     The plasmid pFPAN1 was used to transform  A. niger  by using the transformation protocol as described by Ballance et al. [(1983), Biochem. Biophys. Res. Commun 112, 284-289] with some modifications: 
     YPD medium (1% yeast extract, 2% peptone, 2% dextrose) was inoculated with 10 6  spores per ml and grown for 24 hours at 30° C. and 250 rpm 
     cells were harvested using Wero-Lene N tissue (No. 8011.0600 Wernli AG Verbandstoffabrik, 4852 Rothrist, CH) and once washed with buffer (0.8 M KCl, 0.05 M CaCl 2 , in 0.01 M succinate buffer; pH 5.5) 
     for protoplast preparation only lysing enzymes (SIGMA L2265, St. Louis, Mo., USA) were used 
     the cells were incubated for 90 min at 30° C. and 100 rpm, and the protoplasts were separated by filtration (Wero-Lene N tissue) 
     the protoplasts were once washed with STC (1 M sorbitol, 0.05 M CaCl 2 , 0.01 M Tris/HCl pH 7.5) and resuspended in the same buffer 
     150 μl protoplasts (˜10 8 /ml) were gently mixed with 10-15 μg plasmid DNA and incubated at room temperature (RT) for 25 min 
     polyethylene glycol (60% PEG 4000, 50 mM CaCl 2 , 10 mM Tris/HCl pH 7.5) was added in three steps, 150 μl, 200 μl and 900 μl, and the sample was further incubated at room temperature (RT) for 25 min 
     5 ml STC were added, centrifuged and the protoplasts were resuspended in 2.5 ml YGS (0.5% yeast extract, 2% glucose, 1.2 M sorbitol) 
     the sample was incubated for 2 hours at 30° C. (100 rpm) centrifuged and the protoplasts were resuspended in 1 ml 1.2 M sorbitol 
     the transformed protoplasts were mixed with 20 ml minimal regeneration medium (0.7% yeast nitrogen base without amino acids, 2% glucose, 1 M sorbitol, 1.5% agar, 20 mM Tris/HCl pH 7.5 supplemented with 0.2 g arginine and 10 mg nicotinamide per liter) 
     the plates were incubated at 30° C. for 3-5 days 
     B) Expression 
     Single transformants were isolated, purified and tested for overproduction of the fusion protein. 100 ml M25 medium (70 g maltodextrin (Glucidex 17 D, Sugro Basel, CH), 12.5 g yeast extract, 25 g casein-hydrolysate, 2 g KH 2 PO 4 , 2 g K 2 SO 4 , 0.5 g MgSO 4 .7H 2 O, 0.03 g ZnCl 2 , 0.02g CaCl 2 , 0.05 g MnSO 4 .4H 2 O, 0.05 g FeSO 4  per liter pH 5.6) were inoculated with 10 6  spores per ml from transformants FPAN1#11, #13, #16, #E25, #E30 respectively #E31 and incubated for 5 days at 30° C. and 270 rpm. Supernatant was collected and the activity determined. The fusion protein showed the highest activity with phytic acid as substrate at pH 2.5, whereas with 4-nitrophenyl phosphate as substrate it showed two activity optima at pH 2.5 and 5.0 (Table 1). 
     C) Activity Assay 
     a) Phytic Acid 
     A 1 ml enzyme reaction contained 0.5 ml dialyzed supernatant (diluted if necessary) and 5.4 mM phytic acid (SIGMA P-3168). The enzyme reactions were made in 0.2 M sodium acetate buffer pH 5.0, respectively 0.2 M glycine buffer pH 2.5. The samples were incubated for 15 min at 37° C. The reactions were stopped by adding 1 ml 15% TCA (trichloroacetic acid). 
     For the colour reaction 0.1 ml of the stopped sample was diluted with 0.9 ml distilled water and mixed with 1 ml reagent solution (3 volumes 1 M H 2 SO 4 , 1 volume 2.5% (NH 4 ) 6 Mo 7 O 24 , 1 volume 10% ascorbic acid). The samples were incubated fot 20 min at 50° C. and the blue color was measured spetrophotometrically at 820 nm. Since the assay is based on the release of phosphate a phosphate standard curve, 11-45 nmol per ml, was used to determine the activity of the samples. 
     b) 4-Nitrophenyl Phosphate 
     A 1 ml enzyme reaction contained 100 μl dialyzed supernatant (diluted if necessary) and 1.7 mM 4-nitrophenyl phosphate (Merck, 6850, Darmstadt, BRD). The enzyme reactions were made in 0.2 M sodium acetate buffer pH 5.0, respectively 0.2 M glycine buffer pH 2.5. The samples were incubated for 15 min at 37° C. The reactions were stopped by adding 1 ml 15% TCA. 
     For the determination of the enzyme activity the protocol described above was used. 
     
       
         
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 SUBSTRATE 
                   
               
             
          
           
               
                   
                   
                   
                   
                 *4-Nitrophenyl 
                   
               
               
                   
                 Trans- 
                 *Phytic Acid 
                   
                 phosphate 
               
             
          
           
               
                   
                 formant 
                 pH 5.0 
                 pH 2.5 
                 pH 5.0 
                 pH 2.5 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   A. niger 
                   1) 
                 
                 0.2 
                 1 
                 1 
                 2 
               
               
                   
                 FPAN1 #11 
                 6 
                 49 
                 173 
                 399 
               
               
                   
                 FPAN1 #13 
                 2 
                 21 
                 60 
                 228 
               
               
                   
                 FPAN1 #16 
                 1 
                 16 
                 46 
                 153 
               
               
                   
                 FPAN1 #E25 
                 3 
                 26 
                 74 
                 228 
               
               
                   
                 FPAN1 #E30 
                 3 
                 43 
                 157 
                 347 
               
               
                   
                 FPAN1 #E31 
                 3 
                 39 
                 154 
                 271 
               
               
                   
                   
               
               
                   
                 *Units per ml: 1 unit = 1 μmol phosphate released per min at 37° C.  
               
               
                   
                   1) not transformed  
               
             
          
         
       
     
     Expression of the  Aspergillus terreus  9A1 gene in  Aspergillus niger    
       A. niger  NW205 was transformed with plasmid pPAT1 as described above. Single transformants were isolated, purified and screened for overproduction of the  A. terreus  protein. 50 ml YPD medium were inoculated with 106 spores per ml from transformants PAT1#3, #10, #11, #13 and #16 and incubated for 3 days at 30° C. and 270 rpm. Supernatant was collected and the activity determined as described above except that the pH for the enzyme reactions were different. The enzyme showed its main activity at pH 5.5 with phytic acid as substrate and at pH 3.5 with 4-nitrophenyl phosphate as substrate (Table 2). 
     
       
         
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
             
             
               
                   
                   
               
               
                   
                 SUBSTRATE 
                   
               
             
          
           
               
                   
                   
                   
                   
                 *4-Nitrophenyl 
                   
               
               
                   
                 Trans- 
                 *Phytic Acid 
                   
                 phosphate 
               
             
          
           
               
                   
                 formant 
                 pH 5.5 
                 pH 3.5 
                 pH 5.5 
                 pH 3.5 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   A. niger 
                   1) 
                 
                 0 
                 0 
                 0 
                 0.1 
               
               
                   
                 PAT1 #3 
                 10 
                 0 
                 0.2 
                 0.7 
               
               
                   
                 PAT1 #10 
                 9 
                 0 
                 0.2 
                 0.8 
               
               
                   
                 PAT1 #11 
                 5 
                 0 
                 0.1 
                 0.5 
               
               
                   
                 PAT1 #13 
                 9 
                 0 
                 0.2 
                 0.7 
               
               
                   
                 PAT1 #16 
                 5 
                 0 
                 0.1 
                 0.5 
               
               
                   
                   
               
               
                   
                 *Units per ml: 1 unit = 1 μmol phosphate released per min at 37° C.  
               
               
                   
                   1) not transformed  
               
             
          
         
       
     
     Example 10 
     Fermentation of  Aspergillus niger  NW 205 Transformants 
     A) Transformant FPAN1#11 
     Preculture medium [30 g maltodextrin (Glucidex 17 D), 5 g yeast extract, 10 g casein-hydrolysate, 1 g KH 2 PO 4 , 0.5 g MgSO 4 .7H 2 O, 3 g Tween 80 per liter; pH 5.5] was inoculated with 10 6  spores per ml in a shake flask and incubated for 24 hours at 34° C. and 250 rpm. 
     A 10 liter fermenter was inoculated with the pre-culture to a final dilution of the pre-culture of 1:100. The batch fermentation was run at 30° C. with an automatically controlled dissolved oxygen concentration of minimum 25% (PO 2 ≧25%). The pH was kept at 3.0 by automatic titration with 5 M NaOH. The medium used for the fermentation was: 35 g maltodextrin, 9.4 g yeast extract, 18.7 g casein-hydrolysate, 2 g KH 2 PO 4 , 0.5 g MgSO 4 .7H 2 O, 2 g K 2 SO 4 , 0.03 g ZnCl 2 , 0.02 g CaCl 2 , 0.05 g MnSO 4 .4H 2 O, 0.05 g FeSO 4  per liter; pH 5.6. 
     Enzyme activities reached after 3 days under these conditions were 35 units/ml respectively 16 units/ml at pH 2.5 respectively pH 5.0 with phytic acid as substrate and 295 units/ml respectively 90 units/ml at pH 2.5 respectively pH 5.0 and 4-nitrophenyl phosphate as substrate. 
     B) Transformant PAT1#11 
     Preculture, inoculation of the fermenter and the fermentation medium were as described above, except that the pH was kept at 4.5 by automatic titration with 5 M NaOH. 
     Enzyme activities reached after 4 days under these conditions were 17.5 units/ml at pH 5.5 with phytic acid as substrate and 2 units/ml at pH 3.5 with 4-nitrophenyl phosphate as substrate. 
     Example 11 
     Isolation of PCR Fragments of a Phytase Gene of  Asperaillus terreus  (CBS 116.46) 
     Two different primer pairs were used for PCR amplification of fragments using DNA of  Aspergillus terreus  [CBS 116.46]. The primers used are shown in the Table below. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Fragment 
                 Pri- 
                   
               
               
                 amplified 
                 mers 
                 Oligonucleotide sequences (5′ to 3′) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 8 plus 9 
                 8 
                   ATGGA ( C/T ) ATGTG ( C/T ) TC ( N ) TT ( C/T ) GA   
               
               
                 about 
                   
                 [SEQ ID NO:15] 
               
               
                 150 bp 
                   
                 Amino acids 254-259: MDMCSF 
               
               
                   
                 9 
                   TT ( A/G ) CC ( A/G ) GC ( A/G ) CC ( G/A ) TG ( N ) CC ( A/G ) TA   
               
               
                   
                   
                 [SEQ ID NO:16] 
               
               
                   
                   
                 Amino acids 296-301: YGHGAG 
               
               
                 10 plus 11 
                 10 
                   TA ( C/T ) GC ( N ) GA ( C/T ) TT ( C/T ) TC ( N ) CA ( C/T ) GA   
               
               
                 about 
                   
                 [SEQ ID NO:17] 
               
               
                 250 bp 
                   
                 Amino acids 349-354: YADFSH 
               
               
                   
                 11 
                   CG ( G/A ) TC ( G/A ) TT ( N ) AC ( N ) AG ( N ) AC ( N ) C   
               
               
                   
                   
                 [SEQ ID NO:18] 
               
               
                   
                   
                 Amino acids 416-422: RVLVNDR 
               
               
                   
               
             
          
         
       
     
     DNA sequences in bold show the sense primer and in italics the antisense primer. The primers correspond to the indicated part of the coding sequence of the  Aspergillus niger  gene. The combinations used are primers 8 plus 9 and 10 plus 11. The Taq-Start antibody kit from Clontech (Palo Alto, Calif., USA) was used according to the manufacturer&#39;s protocol. Primer concentrations for 8 plus 9 were 0.2 mM and for primers 10 plus 11 one mM. Touch-down PCR was-used for amplification [Don, R. H. et al. (1991), Nucleic Acids Res. 19, 4008]. First the DNA was denatured for 3 min at 95° C. Then two cycles were done at each of the following annealing temperatures: 60° C., 59° C., 58° C., 57° C., 56° C., 55° C., 54° C., 53° C., 52° C. and 51° C., with an annealing time of one min. each. Prior to annealing the incubation was heated to 95° C. for one min and after annealing elongation was performed for 30 sec at 72° C. Cycles 21 to 35 were performed as follows: denaturation one min at 95° C., annealing one min at 50° C. and elongation for 30 sec at 72° C. 
     Two different PCR fragments were obtained. The DNA sequences obtained and their comparison to relevant parts of the phytase gene of  Aspergillus terreus  9A1 are shown in FIG. 10 [relevant parts of the phytase gene of  Aspergillus terreus  9A1 “9A1 ”(top lines) (1) and the PCR fragments of  Aspergillus terreus  CBS 116.46 “aterr2” (bottom lines). Panel A: Fragment obtained with primer pair 8 plus. 9 (aterr21). Panel B: Fragment obtained with primer pair 10 plus 11 (aterr58). DNA sequences of  Aspergillus terreus  CBS 116.46 (top lines) are compared with those of  Aspergillus terreus  9A1 (1) (bottom lines). PCR amplifications were performed as described in the legend to Table 4. Panel A: The bold gc sequence (bases 16 plus 17) in the aterr21 fragment could possibly be cg (DNA sequencing uncertainty). Panel B: The N at position 26 of the aterr58 PCR fragment could possibly represent any of the four nucleotides]. 
     Example 12 
     Cross Hybridizations under Non-stringent and Stringent Washing Conditions 
     Five mg&#39;s of genomic DNA of each strain listed in Table 3 were incubated with 4 units of HindIII or PstI, respectively, per mg of DNA at 37° C. for 4 hours. After digestion, the mixtures were extracted with phenol and DNAs were precipitated with ethanol. Samples were then analyzed on 0.8% agarose gels. DNA-s were transferred to Nytran membranes (Schleicher &amp; Schuell, Keene, N.H., USA) using 0.4M NaOH containing 1M NaCl as transfer solution. Hybridizations were performed for 18 hours at 42° C. The hybridization solution contained 50% formamide, 1% SDS, 10% dextran sulphate, 4×SSPE (1×SSPE=0.18M NaCl, 1 mM EDTA, 10 mM NaH2 PO 4 , pH 7.4), 0.5% blotto (dried milk powder in H 2 O) and 0.5 mg salmon sperm DNA per ml. The membranes were washed under non-stringent conditions using as last and most-stringent washing condition incubation for 30 min at room temperature in 0.1×SSPE containing 0.1% SDS. The probes (labeled at a specific activity of around 10 9  dpm/mg DNA) used were the PCR fragments generated with primers 8 plus 9 (see Example 11) using genomic DNA of  Myceliophthora thermophila; Mycelio. thermo.; Aspergillus nidulans, Asperg. nidul.; Aspergillus fumigatus, Asperg. fumig.,; Aspergillus terreus  9A1 , Asperg. torrous  9A1 . Talaromyces thermophilus, Talarom. thermo . The MT2 genomic probe was obtained by random priming (according to the protocol given by Pharmacia, Uppsala, Sweden) and spans 1410 bp, from the BspEI site upstream of the N-terminus of the  Mycelo. thezmo . phytase gen to the PvuII site in the C-terminus (positions 2068 is to 3478). The AT2 genomic probe was obtained by random priming and spans 1365 bp, from the ApaI site to the NdeI site of the Auperg. terreus 9A1 phytase gene (positions 491 to 1856). The AN2 DNA probe was obtained by random priming and spans the complete coding sequence (1404 bp) of the  Asperg. niger  gene (EP 420 358). Results are given in Table 3. [“*” except for weak signal corresponding to a non-specific 20 kb fragment; In case of the very weak cross-hybridization signal at 20 kb seen with DNA from  Aspergillus niger  using the PCR fragment from  Talaromyces thermophilus  this signal is unspecific, since it differs significantly from the expected 10 kb HindIII-fragment, containing the phytase gene; “**” signal due to only particle digest of DNA]. 
     For cross-hybridizations with stringent washing conditions membranes were further washed for 30 min. at 65° C. in 0.1×SSPE containing 0.1% SDS. Results are shown in Table 4 [ (1)  only the 10.5-kb HindIII fragment is still detected, the.6.5-kb HindIII fragment disappeared (see Table 3)]. 
     
       
         
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 PCR Probes 
                 Genomic Probes 
                 DNA Probes 
               
             
          
           
               
                   
                   
                   
                 Band (kb) 
                   
                   
                 Band (kb) 
                 Band (kb) 
                 Band (kb) 
               
               
                   
                 Band (kb) 
                 Band (kb) 
                 detected with 
                 Band (kb) 
                 Band (kb) 
                 detected with 
                 detected with 
                 detected with 
               
               
                   
                 detected with 
                 detected with 
                 Probe of 
                 detected with 
                 detected with 
                 genomic 
                 genomic 
                 cDNA 
               
               
                 Source of DNA used 
                 Probe of 
                 Probe of 
                 
                   Asperg. 
                 
                 Probe of 
                 Probe of 
                 Probe MT2 
                 Probe AT2 of 
                 Probe AN2 of 
               
               
                 for 
                 
                   Asperg. 
                 
                 
                   Asperg. 
                 
                 
                   terreus 
                 
                 
                   Mycelio. 
                 
                 
                   Talarom. 
                 
                 of  Mycelio.   
                 
                   Asperg. terreus 
                 
                 
                   Asperg. niger 
                 
               
               
                 cross-hybrization 
                 
                   fumig. 
                 
                 
                   nidul. 
                 
                 9A1 
                 
                   thermo. 
                 
                 
                   thermo. 
                 
                 
                   thermo. 
                 
                 9A1 
                 (control) 
               
               
                   
               
               
                 
                   Acrophialophora levis 
                 
                 no 
                 no 
                 no 
                 no 
                 no 
                 8-kb 
                 no 
                 no 
               
               
                 [ATCC 48380] 
               
               
                 
                   Aspergillus niger 
                 
                 no 
                 no 
                 no 
                 no 
                 no* 
                 no 
                 no 
                 10 kb 
               
               
                 [ATCC 9142] 
                   
                   
                   
                   
                   
                   
                   
                 HindIII 
               
               
                 (control) 
               
               
                 
                   Aspergillus terreus 
                 
                 no 
                 no 
                 11-kb 
                 no 
                 no 
                 no 
                 11-kb 
                 no 
               
               
                 [CBS 116.46] 
                   
                   
                 HindIII 
                   
                   
                   
                 HindIII 
               
               
                 
                   Aspergillus sojae 
                 
                 no 
                 no 
                 no 
                 no 
                 no* 
                 no 
                 3.7-kb 
                 no 
               
               
                 [CBS 126.59] 
                   
                   
                   
                   
                   
                   
                 HindIII 
               
               
                 
                   Calcarisporiella 
                 
                 no 
                 no 
                 10.5-kb 
                 no 
                 no 
                 10.5-kb 
                 10.5-kb 
                 no 
               
               
                   thermophila  [ATCC 22718] 
                   
                   
                 HindIII 
                   
                   
                 HindIII 
                 HindIII 
               
               
                 
                   Chaetomium rectopilium 
                 
                 no 
                 no 
                 no 
                 no 
                 no 
                 &gt;20-kb** 
                 &gt;20-kb** 
                 no 
               
               
                 [ATCC 22431] 
                   
                   
                   
                   
                   
                 HindIII 
                 HindIII 
               
               
                 
                   Corynascus thermophilus 
                 
                 no 
                 no 
                 no 
                 no 
                 no 
                 10.5-kb 
                 no 
                 no 
               
               
                 [ATCC 22066] 
                   
                   
                   
                   
                   
                 HindIII 
               
               
                 Humicola sp. 
                 no 
                 no 
                 no 
                 no 
                 no 
                 9.5-kb 
                 no 
                 no 
               
               
                 [ATCC 60849] 
                   
                   
                   
                   
                   
                 HindIII 
               
               
                 
                   Mycelia sterilia 
                 
                 no 
                 no 
                 no 
                 6-kb 
                 no 
                 6-kb 
                 6-kb 
                 no 
               
               
                 [ATCC 20350] 
                   
                   
                   
                 HindIII 
                   
                 HindIII 
                 HindIII 
               
               
                 
                   Myrococcum thermophilum 
                 
                 no 
                 no 
                 no 
                 no 
                 4.8-kb 
                 no 
                 no 
                 no 
               
               
                 [ATCC 22112] 
                   
                   
                   
                   
                 HindIII 
               
               
                 
                   Rhizomucor miehei 
                 
                 no 
                 3.8-kb 
                 no 
                 no 
                 no 
                 no 
                 no 
                 no 
               
               
                 [ATCC 22064] 
                   
                 HindIII 
               
               
                 
                   Sporotrichum cellulophilum 
                 
                 no 
                 no 
                 no 
                 6-kb 
                 no 
                 6-kb 
                 6-kb 
                 no 
               
               
                 [ATCC 20494] 
                   
                   
                   
                 HindIII 
                   
                 and 
                 and 
               
               
                   
                   
                   
                   
                 2.1/3.7- 
                   
                 10.5-kb 
                 10.5-kb 
               
               
                   
                   
                   
                   
                 kb PstI 
                   
                 HindIII 
                 HindIII 
               
               
                 
                   Sporotrichum thermophile 
                 
                 no 
                 no 
                 no 
                 6-kb 
                 6-kb 
                 6-kb 
                 6-kb 
                 no 
               
               
                 [ATCC 22482] 
                   
                   
                   
                 HindIII 
                 HindIII 
                 HindIII 
                 HindIII 
               
               
                   
                   
                   
                   
                 2.1/3.7- 
               
               
                   
                   
                   
                   
                 kb PstI 
               
               
                 
                   Scytalidium indonesicum 
                 
                 no 
                 no 
                 no 
                 no 
                 no 
                 9-kb 
                 no 
                 no 
               
               
                 [ATCC 46858] 
                   
                   
                   
                   
                   
                 HindIII 
               
               
                 
                   Aspergillus fumigatus 
                 
                 2.3-kb 
                 no 
                 no 
                 no 
                 no 
                 no 
                 no 
                 no 
               
               
                 [ATCC 34625] 
                 HindIII 
               
               
                 
                   Aspergillus nidulans 
                 
                 no 
                 9.5-kb 
                 no 
                 no 
                 no 
                 no 
                 9.5-kb 
                 no 
               
               
                 [DSM 9743] 
                   
                 HindIII 
                   
                   
                   
                   
                 HindIII 
               
               
                 
                   Aspergillus terreus 
                 
                 no 
                 no 
                 10.5-kb 
                 no 
                 6.5-kb 
                 10.5-kb 
                 10.5-kb 
                 no 
               
               
                 9A1 [DSM 9076] 
                   
                   
                 HindIII 
                   
                 HindIII 
                 HindIII 
                 HindIII 
               
               
                 
                   Myceliophthora 
                 
                 no 
                 no 
                 no 
                 6.5-kb 
                 no 
                 6.5-kb 
                 6.5-kb 
                 no 
               
               
                   thermophila  [ATCC 48102] 
                   
                   
                   
                 HindIII 
                   
                 HindIII 
                 HindIII 
               
               
                 
                   Talaromyces thermophilus 
                 
                 no 
                 no 
                 no 
                 no 
                 9.5-kb 
                 no 
                 no 
                 no 
               
               
                 [ATCC 20186] 
                   
                   
                   
                   
                 HindIII 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 Genomic 
                 DNA 
               
               
                   
                   
                   
                   
                   
                   
                 Genomic 
                 Probe of 
                 Probe of 
               
               
                   
                   
                   
                 Probe 
                   
                   
                 Probe 
                 AT2 
                 AN2 
               
               
                   
                 Probe 
                 Probe 
                 
                   Asperg. 
                 
                 Probe 
                 Probe 
                 of MT2 
                 
                   Asperg. 
                 
                 
                   Asperg. 
                 
               
               
                 Source of DNA used for 
                 
                   Asperg. 
                 
                 
                   Asperg. 
                 
                 
                   terreus 
                 
                 
                   Mycelio. 
                 
                 
                   Talarom. 
                 
                 
                   Mycelio. 
                 
                 
                   terreus 
                 
                 
                   niger 
                 
               
               
                 cross-hybriziation 
                 
                   fumig. 
                 
                 
                   nidul. 
                 
                 9A1 
                 
                   thermo. 
                 
                 
                   thermo. 
                 
                 
                   thermo. 
                 
                 9A1 
                 (control) 
               
               
                   
               
             
             
               
                 
                   Acrophiolophora levis 
                 
                   
                   
                   
                   
                   
                 yes 
                   
                   
               
               
                   Aspergillus niger  (control) 
                   
                   
                   
                   
                   
                   
                   
                 yes 
               
               
                   Aspergillus terreus  (CBS 116.46) 
                   
                   
                 yes 
                   
                   
                   
                 yes 
               
               
                 
                   Calcarisporiella thermophila 
                 
                   
                   
                 yes 
                   
                   
                   
                 yes 
               
               
                 
                   Chaetomium rectopilium 
                 
                   
                   
                   
                   
                   
                 yes 
               
               
                 
                   Corynascus thermophilus 
                 
                   
                   
                   
                   
                   
                 yes 
               
               
                 
                   Sporotrichum cellulophilum 
                 
                   
                   
                   
                 yes 
                   
                 yes 
                 yes (1)   
               
               
                 
                   Sporotrichum thermophile 
                 
                   
                   
                   
                 yes 
                   
                 yes 
               
               
                 
                   Aspergillus fumigatus 
                 
                 yes 
               
               
                 
                   Aspergillusd nidulans 
                 
                   
                 yes 
               
               
                   Aspergillus terreus  9A1 
                   
                   
                 yes 
                   
                   
                   
                 yes 
               
               
                 
                   Mycelia sterilia 
                 
                   
                   
                   
                   
                   
                 yes 
               
               
                 
                   Myceliophthora thermophila 
                 
                   
                   
                   
                 yes 
                   
                 yes 
               
               
                 
                   Talaromyces thermophilus 
                 
                   
                   
                   
                   
                 yes 
               
               
                   
               
             
          
         
       
     
     The complete phytase encoding genes of  Aspergllus nidulans, Talaromyces thermophilus, Aspergillus fumigatus , and  Aspergillus terreus  (CBS116.46) are provided for in a manner set forth below. 
     Organisms and growth conditions:  Aspergillus nidulans  (DSM 9743),  Aspergillus fumigatus  (ATCC 34625),  Aspergillus terreus  (CBS 116.46) and  Talaromyces thermophilus  (ATCC 20186) were grown on potato dextrose broth (Difco Lab., Detroit, Mich., USA) at 28° C. except for  T. thermophilus , a thermotolerant fungus, which was grown at 45° C. Transformed  E. coli  (TG-1) were grown in Luria broth (LB) at 37° C. with 100 μg/ml ampicillin for selection. 
     Gencmic DNA: Fungal mycelium was obtained by incubating potato dextrose medium at a high density with spores O/N (200 rpm) at the temperatures indicated above. Up to 2 grams of the mycelium, obtained by filtration through a Whatmann filter, were used for the isolation of genomic DNA as described in the present application. 
     DNA amplification: Genomic DNA of the coding regions of the different phytase genes was amplified using PCR on a Gene Amp Kit (Perkin Elmer Cetus) according to the manufacturer&#39;s instructions using degenerate primers: 
     Primer 8: 5′ -ATGGA(C/T)ATGTG(C/T)TC(N)TT(C/T)GA-3′ [SEQ ID NO:15] 
     Primer 9: 5′-TT(A/G)CC(A/G)GC (A/G)CC(G/A)TG(N)CC(A/G)TA-3′ [SEQ ID NO:16] 
     Primer 10: 5′-TA(C/T)GC(N)GA(C/T)TT(C/T)TC(N)CA(C/T) GA-3′ [SEQ ID NO:17] 
     Primer 11: 5′-CG(G/A)TC(G/A)TT(N)AC(N)AG(N)AC(N)C-3′ [SEQ ID NO:18] 
     For  T. thermophilus  all components of the reaction, including the primers 8 [SEQ ID NO:15] and 9 [SEQ ID NO:16] at a final concentration of 10 mM, but with the exception of the Taq polymerase, were incubated at 95° C. for 10 min and 50° C. for 1 min before the reaction was placed on ice. The Taq polymerase was then added and 35 cycles of PCR performed according to the following cycle profiles: 60 sec, 95° C./60 sec, 50° C./90 sec, 72° C./120 sec. 
     For  A. nidulans, A. terreus  CBS116.46 and  A. fumigatus  a “touch-down” PCR with a final primer concentration of 0.2 mM for primers 8 [SEQ ID NO:151 and 9 [SEQ ID NO:16] was performed as described in Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., Mattick, J. S., “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res., 19:4008 (1991).]. In the case of  A. terreus  CBS116.46 additional amplifications with primers 10 [SEQ ID NO:17] and 11 [SEQ ID NO:18] at a final concentration of 1 mM were also done. In all “touch-down” reactions first the DNA was denatured for 3 min at 95° C., followed by two cycles at each of the following annealing temperatures: 60° C., 59° C., 58° C., 57° C., 56° C., 55° C., 54° C., 53° C., 52 and 51° C., with an annealing time of one min each. Prior to annealing the incubation was heated to 95° C. for one min and after annealing elongation was performed for 30 sec at 72° C. Cycles 21 to 35 were performed as follows: denaturation one min at 95° C., annealing one min at 50° C. and elongation for 30 sec at 72° C. As template, 1 μg of the genomic DNA was used in a total reaction mixture of 50 μl. 
     Primers Aterr21, Aterr58, Terr380 and Terr1810 were used to isolate the complete  A. terreus  CBS 116.46 phytase gene by PCR (see results). The  A. terreus  CBS 116.46 specific primer, Aterr21 (5′-CTGTCGCCGTTCTGCGACC TC-3′) [SEQ ID NO:20] and Aterr 58 (5′CGGTGCCGTTGTACAGACCCAGC-3′) [SEQ ID NO:21] were designed using the nucleotide sequence of the two PCR fragments obtained with primers 8 [SEQ ID NO:15] and 9 [SEQ ID NO:16] and primers 10 [SEQ ID NO:17] and 11 [SEQ ID NO:18] on genomic DNA of  A. terreus  CBS116.46 (see FIG.  11 ). Primer Terr380 (5′ATGGGCTTTCTTGCCATTGT-3′) [SEQ ID NO:22] and Terrl8 lO (5′TCAGAAACAATCCGCCCAAGTT-3′) [SEQ ID NO:23] are specific for the 5′ and 3′ of the coding sequence of the phytase gene of  A. terreus  9A1. 
     In all cases an aliquot of the reaction was analysed on 1.5% agarose gel. PCR products of the expected size were excised from the agarose and isolated by centrifugation of the gel slices through siliconized glass wool as described by Heery, D. M., Gannon, F. and Powell, R., A simple method for subcloning DNA fragments from gel slices. Trends. Genet., 6:173 (1990) or using a GENECLEAN Kit (BIO101.Inc.) essentially according to the manufacturer&#39;s protocol. The fragment was subsequently cloned 25 into pUC 18 using the Sure-Clone ligation kit (Pharmacia). 
     Southern blot analysis: Southern hybridization experiments were performed to construct genomic maps to find appropriate DNA fragments carrying the phytase gene. Genomic DNA (3 μg) was digested with the different restriction enzymes as indicated in the legends of the figures and electrophoresed on a 0.75% agarose gel. The transfer to Zeta-Probe blotting membranes (BIO-RAD) was done as described in Southern, E. M., Detection of specific sequences among DNA fraaments separated by gel electrophoresis. J. Mol. Biol., 98:503 (1975). Prehybridization and hybridization was in 7% SDS, 1% BSA (fraction V; Boehringer), 0.5M Na 2 HPO 4 , pH 7.2 at 65° C. Probes derived from PCR products of the respective phytase genes (see FIG. 11) were labeled with (α- 32 P)-dGTP (Amersham) by random-priming according to Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: a laboratory manual. Second Edition ed. 1989, Cold Spring Harbor N.Y., Cold Spring Harbor Laboratory Press, U.S.A. and used in the hybridization experiments. After hybridization the membranes were washed twice for 5 minutes in 2×SSC., 1% SDS at room temperature and twice for 15 minutes in 0.1% SSC., 0.1% SDS at 65° C. before exposure O/N on Kodak X-Omat AR film. 
     Library construction: Prior to the partial library construction, Southern blot analysis with a given probe was done in order to identify a specific restriction fragment of interest. Subsequently 10-20 μg of genomic DNA was digested with the appropriate restriction enzymes and electrophoresed on an agarose gel. According to comigrating DNA markers, the region of interest was cut out of the gel, the DNA isolated and subcloned into the pBluescriptII—(KS) vector. Transformation of the ligation mixture into  E. coli  TG-1 cells resulted in partial genomic libraries carrying the fragment of interest. The genomic  A. fumigatus  (NIH stock#5233) Lambda FIXII library was obtained from Stratagene (cat. Nr. 946055). The size of the cloned fragments, generated by partial Sau3AI digestion of genomic DNA were in the range of 9-22 kb, according to the manufacturer. 
     Screening of genomic libraries:  E. coli  transformants of the partial genomic libraries of  A. nidulans, A. terreus  CBS 116.46 and  T. thermophilus  were screened using the colony lift assay described in Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: a laboratory manual. Second Edition ed. 1989, Cold Spring Harbor N.Y., Cold Spring Harbor Laboratory Press, U.S.A. and the appropriate probe (see FIG.  11 ). The  A. fumigatus  Lambda FIXII library was screened according to the manufacturer&#39;s instructions using the DNA fragment PCRAfu as probe. Putative positive plaques were cored and subjected to a second round of purification. A clear single positive plaque was picked and used to make a large scale phage preparation as described in Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: a laboratory manual. Second Edition ed. 1989, Cold Spring Harbor N.Y., Cold Spring Harbor Laboratory Press, U.S.A. The analysis of the DNA insert and further subcloning steps are outlined herein. 
     DNA sequencing: The sequence was determined by the dideoxy chain termination technique described in Sanger, F., Nicklen, S. and Coulson, A. R., DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA., 74:5463-5467 (1977) using the Sequenase Kit (United States Biochemical). Both strands were completely sequenced and the sequence analyzed using the GCG sequence analysis software package (Version 8.0) by Genetics Computer, Inc. See Devereux, J., Haeberli, P. and Smithies, O., A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res., 12:387-395 (1984). 
     Cloning fragments of phytase genes by PCR: To have suitable probes for the library screening, specific DNA fragments of the different phytases were obtained by performing PCR on. genomic DNA of the individual fungi using degenerate primers as described above. These degenerate PCR amplifications on genomic DNA with primers 8 [SEQ ID NO:15] and 9 [SEQ ID NO:16] gave discrete bands of about 120 to 130 bp for  A. nidulans, A. fumigatus, T. thermophilus  and about 150 bp for  A. terreus  CBS 116.46. Furthermore amplification with primers 10 [SEQ ID NO:17] and 11 [SEQ ID NO:18] on genomic DNA of  A. terreus  CBS116.46 gave a amplification product of 220 bp. The sequences of these fragments are shown in FIG.  11 . 
     The  A. nidulans  phytase: To identify and isolate DNA fragments carrying the putative phytase gene the PCR fragment PCRAni (FIG. 11) was used to probe a Southern blot carrying chromosomal DNA of  A. nidulans  digested with different restriction enzymes (FIG.  12 ). The 6 kb HindIII/KpnI fragment hybridizing to the probe seemed the most suitable for cloning Genomic  A. nidulans  DNA was digested with HindIII and KpnI and electrophoresed on an agarose gel. According to comigrating DNA markers, the region of about 5-7 kb was cut out of the gel and the DNA isolated. One positive transformant, clone 1, was chosen and the sequence determined. FIG. 13 shows 1931 nts of the insert [SEQ ID NO:28] carrying the complete phytase gene. The encoded protein [SEQ ID NO:29] consists of 463 amino acids with a theoretical MW of 51785 Da and is interrupted by a single intron with a predicted length of 54 nts and positioned close to the 5′ of the gene. In the open reading frame (ORF) upstream of the intron there is one additional potential initiation codon at position 125-127 followed by a putative signal peptide, however when the amino acid sequences of all known phytases are aligned the ATG at position 158 -160 is the most likely translation start site. 
     The  T. themchiwus  phytase: A partial library with BamHI/XbaI fragments of about 4.7 kb was constructed based on the Southern blot analysis results with genomic DNA of  T. thermophilus  (FIG.  14 ). One positive transformant, clone Tt29, was chosen and sequencing reactions with primer and reverse primer performed. Sequencing data showed that the BamHI restriction site was present within the phytase gene and therefore only part of the complete gene had been isolated. Comparison of the amino acid sequence to known phytase proteins predicted that we had cloned the C-terminus of the putative phytase gene. To get the missing N-terminus a chromosome walking approach was taken using 5′ end of clone Tt29 (a 370 bp BamHI-BstEII fragment) as probe, to hybridize to  T. thermophilus  genomic DNA double digested with BstEII and a number of other restriction enzymes (FIG.  15 ). The 4.5 kb EcoRI/BstEII DNA fragment identified by the probe was the most appropriate for cloning. Genomic DNA digested with EcoRI and BstEII and having a size between 4 and 5 kb was isolated and subcloned into the EcoRI and BstEII sites of plasmid Tt29-1. Plasmid Tt29-1 is a shorter variant of construct Tt29, and was obtained by deleting a 3.6 kb. SacI fragment. This reduction of the plasmid size was done to avoid instability problems which potentially could arise when cloning the 4.5 kb EcoRI-BstEII fragment into the plasmid Tt29, already containing a 4.7 kb DNA insert. Transformant carrying the Tt29-132 construct were identified by hybridization to the BamHI-BstEII DNA probe. One positive clone carrying the EcoRI-SacI insert of approx. 5.5 kb was chosen and the phytase sequence determined. FIG. 16 shows a linear map of the position of the insert of the plasmid clones Tt29, Tt29-1 and Tt29-132. 
     The 1845 nts of the insert Tt29-132 [SEQ ID NO:30] are shown in FIG.  17 . The phytase gene of  T. thermophilus  encodes, interrupted by a single intron located similar to the above mentioned phytase genes, a protein [SEQ ID NO:31] of 466 amino acids. The theoretical molecular weight is 51450 Da. 
     One additional potential initiation codon is present upstream at position 236-238 followed by a putative signal sequence. However based on amino acid homology comparisons to the other phytases the ATG at position 288-290 is the most likely translation initiation site. 
     The  A. fumigatus  phytase: Screening of 5.3×10 6  plaques of the  A. fumigatus  FIXII library with the probe PCRAfu gave 115 hybridizing plaques. Two plaques were picked and subjected to a second round of purification. Bacteriophage DNA of the two candidates was isolated and digested with NotI. The Lambda clone having the largest insert (approx. 15 kb) was further mapped by restriction analysis and genomic Southern. FIG. 18 shows the position of the BamHI fragment within the insert of the bacteriophage lambda clone. The 6 kb BamHI fragment giving a strong signal with the above mentioned probe was isolated, subcloned and part of the sequence encoding the phytase gene was determined. FIG. 19 shows 1571 nts of the insert [SEQ ID NO:32] carrying the complete phytase gene. One single intron of 56 nts is found close to the 5′ of the gene. This is in accordance with the aforementioned phytase genes. The enzyme. consists of 465 amino acids [SEQ ID NO:33] with a theoretical MW of 50704 Da. 
     The  A. terres  CBS 116.46 phytase: Based on the high sequence identity seen between both strains of  A. terreus , 9A1 and CBS 116.46, we tried to isolate the phytase gene of the latter strain by PCR, using primers derived from the 5′ and 3′ of the  A. terreus  9A1 sequence (Terr 380 [SEQ ID NO:22] and Terr 1810 [SEQ ID NO:23] ) and two internal primers (Aterr 21 [SEQ ID NO:20] and Aterr 58 [SEQ ID NO:21]) derived from the  A. terreus  CBS 116.46 DNA fragments described in FIG.  11 . FIG. 20 outlines the position of the primers on the phytase gene of  A. terreus  strain 9A1 and the expected amplification products. Only primer Atterr 21 [SEQ ID NO:20] and Terr 1810 [SEQ ID NO:23] gave a product with an expected size of about 570 bp. The PCR fragment was cloned into the SmaI site of pUC18, resulting in plasmid pUC18-569. Sequencing of the insert confirmed that we had cloned the C-terminus of the phytase gene. The missing N-terminus of the gene was cloned basically as described for the other phytases. Southern blot analysis of genomic DNA of  A. terreus  CBS-116.46, with the 570 bp  A. terreus  CBS116.46 DNA piece as probe, identified a KpnI/KpnI fragment of 2 kb carrying the complete phytase gene. The region of about 2 kb was isolated and used to construct a partial genomic library. One  E. coli  transformant, clone 227, hybridizing to the probe was then used for further analysis. FIG. 21 shows 1567 nts of the insert [SEQ ID NO:34]. The encoded phytase has 466 amino acids [SEQ ID NO:35] and a theoretical molecular weight of 51054 Da. 
     The phytases from  Aspergillus fumigatus, A. nidulans, A. terreus  9A1 , A. terreus  CBS, and  Myceliophthora thermophila  were overexpressed in  A. niger  or  Hansenula polymozpha  and purified to apparent homogeneity. After removal of the cells, the clear culture supernatant was concentrated by ultrafiltration and subjected to buffer exchange on a Fast Desalting Column HR 10/10 (Pharmacia). Final purification was achieved by cation exchange chromatography on either a Mono S HR 5/5 (Pharmacia) or a Poros HS/M column (PerSeptive Biosystems) in the case of  A. fumigatus  phytase (FIG.  22 ). The sample was loaded in a buffer containing 10 mM sodium acetate, pH 5.0 . A. fumigatus  phytase was eluted ith a linear gradient from 10 mM sodium acetate, pH 5.0 to 10 mM sodium acetate, 1 M NaCl, pH 5.0. Anion exchange chromatography on a Poros HQ/M column (PerSeptive Biosystems) was used in the case of the other phytases. 
     In order to corroborate the identities of the purified proteins, samples were separated by SDS-PAGE and blotted onto PVDF membranes (Immobilon-P SQ , Millipore). N-terminal sequencing of the proteins was done by automated Edman degradation on an Applied Biosystems 494A sequencer with on-line microbore phenylthiohydantoin detection. The results of the N-terminal sequencing are set forth in Table 5. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 N-terminal sequences of the purified proteins 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   Aspergillus fumigatus  phytase: 
                 SKSXDTVDLGY 
               
               
                   
                   Aspergillus nidulans  phytase: 
                 VVQNHSX 
               
               
                   
                   
                 NHSXNTA 
               
               
                   
                   Aspergillus terreus  9A1 phytase: 
                 SDXNSVDHGY 
               
               
                   
                   Myceliophthora thermophila  phytase: 
                 SESRP 
               
               
                   
                   
               
             
          
         
       
     
     For the determination of the specific activity of the respective proteins, protein concentration was calculated from OD 280  according to the theoretical absorption calculated from the amino acid sequences. Phytase activity was measured in an assay mixture containing 0.5% phytic acid (˜5 mM), 200 mM sodium acetate, pH 5.0. After a 15 min-incubation period at 37° C., the reaction was stopped by addition of an equal volume of 15% TCA. The liberated phosphate ions were quantified by mixing 100 μl of the assay mixture with 900 μl H 2 O and 1 ml of 0.6 M H 2 SO 4 , 2% ascorbic acid and 0.5% ammonium molybdate. Standard solutions of potassium phosphate were used as reference. One unit (U) is defined as the amount of protein liberating 1 μmol of inorganic phosphate per minute at 37° C. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Specific activities (in U/mg protein) of the purified phytases 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   Aspergillus fumigatus  phytase: 
                    25.1 ± 5.1 
                 (n = 16) 
               
               
                   
                   Aspergillus nidulans  phytase: 
                 28.6 ± 4.4 
                 (n = 7) 
               
               
                   
                   Aspergillus terreus  9A1 phytase: 
                 141.6 ± 7.1  
                 (n = 3) 
               
               
                   
                   Aspergillus terreus  CBS phytase: 
                 203.8 ± 12.2 
                 (n = 5) 
               
               
                   
                   Myceliophthora thermophila  phytase: 
                 41.8 ± 4.4 
                 (n = 3) 
               
               
                   
                   
               
             
          
         
       
     
     For the investigation of the substrate specificities of  A. fumigatus, A. nidulans  and  A. terreus  CBS phytase, phytic acid was replaced in the activity assay by 5 mM-concentrations of pnitrophehyl phosphate, phenyl phosphate, fructose 1,6-diphosphate, fructose 6-phosphate, glucose 6-phosphate, ribose 5-phosphate, α-glycerophosphate, β-glycerophosphate, 3-phosphoglycerate, AMP, ADP, ATP, or NADH. The activities found with these substrates were expressed relative (in %) to the activity found with phytic acid. The results of. the substrate specificities are set forth in FIG.  23 . 
     
       
         
           
             35 
           
           
             
               2327 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                join(374..420, 469..1819)
 
             
              1
TCTAGAACAA TAACAGGTAC TCCCTAGGTA CCCGAAGGAC CTTGTGGAAA ATGTATGGAG     60
GTGGACACGG CACCAACCAC CACCCGCGAT GGCGCACGTG GTGCCCTAAC CCCTTGCTCC    120
CTCAGGATGG AATCCATGTC GACTCTTTAC CCTCACCATC GCCTGGATGA AACCTCCCCG    180
CTAAGCTCAC GACGATCGCT ATTTCCGACC GATTTGACCG TCATGGTGGA GGGCTGATTC    240
GGTCGATGCT CCTGCCTTCA TTTCGGAGTT CGGAGACATG AAAGGCTTAT ATGAGGACGT    300
CCCAGGTCGG GGACGAAATC CGCCCTGGGC TGTGCTCCTT CGTCGGAAAC ATCTGCTGTC    360
CGTGATGGCT ACC ATG GGC TTT CTT GCC ATT GTG CTC TCC GTC GCC TTG       409
               Met Gly Phe Leu Ala Ile Val Leu Ser Val Ala Leu
                 1               5                  10
CTC TTT AGA  AG  GTATGCACCC CTCTACGTCC AATTCTCTGG GCACTGACAA         460
Leu Phe Arg  Ser
         15
CGGCGCAG C ACA TCG GGC ACC CCG TTG GGC CCC CGG GGC AAA CAT AGC       508
           Thr Ser Gly Thr Pro Leu Gly Pro Arg Gly Lys His Ser
                        20                  25
GAC TGC AAC TCA GTC GAT CAC GGC TAT CAA TGC TTT CCT GAA CTC TCT      556
Asp Cys Asn Ser Val Asp His Gly Tyr Gln Cys Phe Pro Glu Leu Ser
 30                  35                  40                  45
CAT AAA TGG GGA CTC TAC GCG CCC TAC TTC TCC CTC CAG GAC GAG TCT      604
His Lys Trp Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln Asp Glu Ser
                 50                  55                  60
CCG TTT CCT CTG GAC GTC CCA GAG GAC TGT CAC ATC ACC TTC GTG CAG      652
Pro Phe Pro Leu Asp Val Pro Glu Asp Cys His Ile Thr Phe Val Gln
             65                  70                  75
GTG CTG GCC CGC CAC GGC GCG CGG AGC CCA ACC CAT AGC AAG ACC AAG      700
Val Leu Ala Arg His Gly Ala Arg Ser Pro Thr His Ser Lys Thr Lys
         80                  85                  90
GCG TAC GCG GCG ACC ATT GCG GCC ATC CAG AAG AGT GCC ACT GCG TTT      748
Ala Tyr Ala Ala Thr Ile Ala Ala Ile Gln Lys Ser Ala Thr Ala Phe
     95                 100                 105
CCG GGC AAA TAC GCG TTC CTG CAG TCA TAT AAC TAC TCC TTG GAC TCT      796
Pro Gly Lys Tyr Ala Phe Leu Gln Ser Tyr Asn Tyr Ser Leu Asp Ser
110                 115                 120                 125
GAG GAG CTG ACT CCC TTC GGG CGG AAC CAG CTG CGA GAT CTG GGC GCC      844
Glu Glu Leu Thr Pro Phe Gly Arg Asn Gln Leu Arg Asp Leu Gly Ala
                130                 135                 140
CAG TTC TAC GAG CGC TAC AAC GCC CTC ACC CGA CAC ATC AAC CCC TTC      892
Gln Phe Tyr Glu Arg Tyr Asn Ala Leu Thr Arg His Ile Asn Pro Phe
            145                 150                 155
GTC CGC GCC ACC GAT GCA TCC CGC GTC CAC GAA TCC GCC GAG AAG TTC      940
Val Arg Ala Thr Asp Ala Ser Arg Val His Glu Ser Ala Glu Lys Phe
        160                 165                 170
GTC GAG GGC TTC CAA ACC GCT CGA CAG GAC GAT CAT CAC GCC AAT CCC      988
Val Glu Gly Phe Gln Thr Ala Arg Gln Asp Asp His His Ala Asn Pro
    175                 180                 185
CAC CAG CCT TCG CCT CGC GTG GAC GTG GCC ATC CCC GAA GGC AGC GCC     1036
His Gln Pro Ser Pro Arg Val Asp Val Ala Ile Pro Glu Gly Ser Ala
190                 195                 200                 205
TAC AAC AAC ACG CTG GAG CAC AGC CTC TGC ACC GCC TTC GAA TCC AGC     1084
Tyr Asn Asn Thr Leu Glu His Ser Leu Cys Thr Ala Phe Glu Ser Ser
                210                 215                 220
ACC GTC GGC GAC GAC GCG GTC GCC AAC TTC ACC GCC GTG TTC GCG CCG     1132
Thr Val Gly Asp Asp Ala Val Ala Asn Phe Thr Ala Val Phe Ala Pro
            225                 230                 235
GCG ATC GCC CAG CGC CTG GAG GCC GAT CTT CCC GGC GTG CAG CTG TCC     1180
Ala Ile Ala Gln Arg Leu Glu Ala Asp Leu Pro Gly Val Gln Leu Ser
        240                 245                 250
ACC GAC GAC GTG GTC AAC CTG ATG GCC ATG TGT CCG TTC GAG ACG GTC     1228
Thr Asp Asp Val Val Asn Leu Met Ala Met Cys Pro Phe Glu Thr Val
    255                 260                 265
AGC CTG ACC GAC GAC GCG CAC ACG CTG TCG CCG TTC TGC GAC CTC TTC     1276
Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp Leu Phe
270                 275                 280                 285
ACG GCC ACT GAG TGG ACG CAG TAC AAC TAC CTG CTC TCG CTG GAC AAG     1324
Thr Ala Thr Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu Asp Lys
                290                 295                 300
TAC TAC GGC TAC GGC GGG GGC AAT CCG CTG GGT CCG GTG CAG GGG GTC     1372
Tyr Tyr Gly Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val Gln Gly Val
            305                 310                 315
GGC TGG GCG AAC GAG CTG ATG GCG CGG CTA ACG CGC GCC CCC GTG CAC     1420
Gly Trp Ala Asn Glu Leu Met Ala Arg Leu Thr Arg Ala Pro Val His
        320                 325                 330
GAC CAC ACC TGC GTC AAC AAC ACC CTC GAC GCG AGT CCG GCC ACC TTC     1468
Asp His Thr Cys Val Asn Asn Thr Leu Asp Ala Ser Pro Ala Thr Phe
    335                 340                 345
CCG CTG AAC GCC ACC CTC TAC GCC GAC TTC TCC CAC GAC AGC AAC CTG     1516
Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Ser Asn Leu
350                 355                 360                 365
GTG TCG ATC TTC TGG GCG CTG GGC CTG TAC AAC GGC ACC GCG CCG CTG     1564
Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu
                370                 375                 380
TCG CAG ACC TCC GTC GAG AGC GTC TCC CAG ACG GAC GGG TAC GCC GCC     1612
Ser Gln Thr Ser Val Glu Ser Val Ser Gln Thr Asp Gly Tyr Ala Ala
            385                 390                 395
GCC TGG ACG GTG CCG TTC GCC GCT CGC GCG TAC GTC GAG ATG ATG CAG     1660
Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln
        400                 405                 410
TGT CGC GCC GAG AAG GAG CCG CTG GTG CGC GTG CTG GTC AAC GAC CGG     1708
Cys Arg Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
    415                 420                 425
GTC ATG CCG CTG CAT GGC TGC CCT ACG GAC AAG CTG GGG CGG TGC AAG     1756
Val Met Pro Leu His Gly Cys Pro Thr Asp Lys Leu Gly Arg Cys Lys
430                 435                 440                 445
CGG GAC GCT TTC GTC GCG GGG CTG AGC TTT GCG CAG GCG GGC GGG AAC     1804
Arg Asp Ala Phe Val Ala Gly Leu Ser Phe Ala Gln Ala Gly Gly Asn
                450                 455                 460
TGG GCG GAT TGT TTC TGATGTTGAG AAGAAAGGTA GATAGATAGG TAGTACATAT     1859
Trp Ala Asp Cys Phe
            465
GGATTGCTCG GCTCTGGGTC GTTGCCCACA ATGCATATTA CGCCCGTCAA CTGCCTTGCG   1919
CCATCCACCT CTCACCCTGG ACGCAACCGA GCGGTCTACC CTGCACACGG CTTCCACCGC   1979
GACGCGCACG GATAAGGCGC TTTTGTTACG GGGTTGGGGC TGGGGGCAGC CGGAGCCGGA   2039
GAGAGAGACC AGCGTGAAAA ACGACAGAAC ATAGATATCA ATTCGACGCC AATTCATGCA   2099
GAGTAGTATA CAGACGAACT GAAACAAACA CATCACTTCC CTCGCTCCTC TCCTGTAGAA   2159
GACGCTCCCA CCAGCCGCTT CTGGCCCTTA TTCCCGTACG CTAGGTAGAC CAGTCAGCCA   2219
GACGCATGCC TCACAAGAAC GGGGGCGGGG GACACACTCC GCTCGTACAG CACCCACGAC   2279
GTGTACAGGA AAACCGGCAG CGCCACAATC GTCGAGAGCC ATCTGCAG                2327
 
           
           
             
               466 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              2
Met Gly Phe Leu Ala Ile Val Leu Ser Val Ala Leu Leu Phe Arg Ser
  1               5                  10                  15
Thr Ser Gly Thr Pro Leu Gly Pro Arg Gly Lys His Ser Asp Cys Asn
             20                  25                  30
Ser Val Asp His Gly Tyr Gln Cys Phe Pro Glu Leu Ser His Lys Trp
         35                  40                  45
Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln Asp Glu Ser Pro Phe Pro
     50                  55                  60
Leu Asp Val Pro Glu Asp Cys His Ile Thr Phe Val Gln Val Leu Ala
 65                  70                  75                  80
Arg His Gly Ala Arg Ser Pro Thr His Ser Lys Thr Lys Ala Tyr Ala
                 85                  90                  95
Ala Thr Ile Ala Ala Ile Gln Lys Ser Ala Thr Ala Phe Pro Gly Lys
            100                 105                 110
Tyr Ala Phe Leu Gln Ser Tyr Asn Tyr Ser Leu Asp Ser Glu Glu Leu
        115                 120                 125
Thr Pro Phe Gly Arg Asn Gln Leu Arg Asp Leu Gly Ala Gln Phe Tyr
    130                 135                 140
Glu Arg Tyr Asn Ala Leu Thr Arg His Ile Asn Pro Phe Val Arg Ala
145                 150                 155                 160
Thr Asp Ala Ser Arg Val His Glu Ser Ala Glu Lys Phe Val Glu Gly
                165                 170                 175
Phe Gln Thr Ala Arg Gln Asp Asp His His Ala Asn Pro His Gln Pro
            180                 185                 190
Ser Pro Arg Val Asp Val Ala Ile Pro Glu Gly Ser Ala Tyr Asn Asn
        195                 200                 205
Thr Leu Glu His Ser Leu Cys Thr Ala Phe Glu Ser Ser Thr Val Gly
    210                 215                 220
Asp Asp Ala Val Ala Asn Phe Thr Ala Val Phe Ala Pro Ala Ile Ala
225                 230                 235                 240
Gln Arg Leu Glu Ala Asp Leu Pro Gly Val Gln Leu Ser Thr Asp Asp
                245                 250                 255
Val Val Asn Leu Met Ala Met Cys Pro Phe Glu Thr Val Ser Leu Thr
            260                 265                 270
Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp Leu Phe Thr Ala Thr
        275                 280                 285
Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu Asp Lys Tyr Tyr Gly
    290                 295                 300
Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val Gln Gly Val Gly Trp Ala
305                 310                 315                 320
Asn Glu Leu Met Ala Arg Leu Thr Arg Ala Pro Val His Asp His Thr
                325                 330                 335
Cys Val Asn Asn Thr Leu Asp Ala Ser Pro Ala Thr Phe Pro Leu Asn
            340                 345                 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Ser Asn Leu Val Ser Ile
        355                 360                 365
Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Gln Thr
    370                 375                 380
Ser Val Glu Ser Val Ser Gln Thr Asp Gly Tyr Ala Ala Ala Trp Thr
385                 390                 395                 400
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Arg Ala
                405                 410                 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Met Pro
            420                 425                 430
Leu His Gly Cys Pro Thr Asp Lys Leu Gly Arg Cys Lys Arg Asp Ala
        435                 440                 445
Phe Val Ala Gly Leu Ser Phe Ala Gln Ala Gly Gly Asn Trp Ala Asp
    450                 455                 460
Cys Phe
465
 
           
           
             
               3995 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                join(2208..2263, 2321..3725)
 
             
              3
GTCGACGAGG CACACCACGC CCGTCCTCGG CGGGTCCGAG AGGGCCGGGC TCGGGTTCGA     60
CAAGGAGACG GGCGTCCCTT CGGGCGCGGC TGCGGGTGTG GGTGTTGCTG TGGACGGTGA    120
GGAGGGGGAC GGGCTGGGCG TTGATGACGG TACGAATGCG AACGGACACA GGCCGCTGAG    180
CGTGGGTGTT GCGTTCTAAT CTTTCTTTGT GTGGGTGTGT ACGTGTGGGT GTGTATGTGT    240
TTGGGGGGGG GAATGTTCTT GGTAATTATC TTTCTACCCT TCTTCTCTTT CCTTTATTCT    300
GTTCAGCAGG TATACCCCGT GTAAGTGTAC AGGATTATGG GACGGGTGGG TGGATGGACT    360
ACTTCTAGAA GGACGGATAA GGAAAAAGGG GAAACACGAA TATGGCGCCC TGGGTGGCGC    420
GTCGAGCTGG ATGCTTGACG CCGGTCTGGC AAACATTTTC TTCTTCTAGC ACCCAACCTA    480
GTACTTGATA GAGTGTTTCG GGGCCAGGCG GTTTGCGCTG TGTTTTTACC AATCACCAAC    540
TAGTGCTACT ACTATTATTG CGGCTGTTGA TGCAGCCGTG TACCAAAAAT GCCGCGGCAT    600
CTCCATTGAT ACTTGTAGTT TTGATAGATC AATATTTGGG AGGTTGCGCT GGGCTGCTCT    660
GAAACCCCTC TCTCTTGCTG TACGTAACGT ATGTGCACAG TATGTCACCG ACAAAGACGA    720
TTGCATGCGC ATCGTTTTTT GTTGTGTTTC AGGCCTCGCT CGTGTCTAGG GTATAAACAC    780
ATTGAAGACT ACATATGCGC AAGACGTTGA CATTAACGGG GTCCTGCAGC CGCCGCAGGT    840
GCATGTCGTG ATTAATACCA CGCGCCTGCG TAAATTAGCT AGCCGCCGCC CTGTTTCACT    900
CGGTTAGAGA CGGACAGGTG AGACGGGTCT CGGTTAAGCA AGCAAATTGG AATGCAAGGT    960
TGAAGGTGTA ATCTGCATAG CGTGGAAATG AGAGGGCTCT GTGGGCAGCC AGGAAGGTGA   1020
GACGAAATGA GGAAAGAGGC ACCAGAAGCT GTTGTTCTGA AGTGCCCGTG GTCATAGCTC   1080
CAGGATTAAG TACGGATGTC CCATGCCAAG CTGCTGGCTT CGAAAGCGAG TACGGAGTAG   1140
TGTCCATTGT TCACGAGGGA TCCCCAATGT GTTAGACATG CCTGAATCAA TTTTGTCCTA   1200
TTTTTGGATT TCAACTGTTT CTCTCGACTG TGCTCGGTAG CGACTATGCC GCAAGGTACA   1260
CTACATGTTG TACAATAATC ATACATCGAC CTTCCGTAGG AGTGCTGAAA TACCCGACCT   1320
GCTCTCTCTA GCAGGTGCCT AATGGCTTTC GTGTAACTCG ATCGAAACGG ATCAGCAAGT   1380
CCATTTGCTG TTGGTTGAGA TGTACGATTT ACAAACACGT GGAGAGGTGA GCCACAGCGA   1440
TAGGCTTCTG GAAGGATTCT GGCGTCTCGG AAAGAGGGCC ACTCGCCCCA CTAACCGGCG   1500
CCGATCTTGA CATGGGGCTC GCAGGGGGTT TAAGTGCACA CTACGGAGTA CGGATTACAC   1560
AGTAGTGTAT GGGTGGGGGC GAGTTTGGGT GGCCTTGTGT GGGGCTCACC GGCTGCCTGT   1620
TCTCGGGGAG TCTTGGCGGG CCGATTGGAC CCACCTAACC ACGGGTAGTC TTGGCCCGGC   1680
CAACTCACAC CGCCCTCATG TTTCGGAGCC AGTCAGGGAG GCAGGCACTA CTCAGTCAGG   1740
TACACACGTC GGGCTCCTCG ATGCTGGGTG ACATCGAGGC GATACTGCAT TCCAACTACG   1800
GTTGGCATAG GAGGTATCCT ATTCTAGAGC TGTTCTACGC CGGAACGTAA CCCGGGATAA   1860
CCCGGGATAT CGCTTCCCTG AGCGAGCGCG CTGCTGAGGA TCATACAACC CAACAACCGA   1920
CGACGGTGCA AGAAGGTTGG GGGAAGGAAG AAATCAAGGA AAAAAAAATA GGGGGGGTGG   1980
GGACCAAGAG AGAAAGAAAG GAGAAAAGGG TGGGGGGAGG GAAGAGAAAA AAAAAACGGA   2040
GGAATATGGC GTCGCTCTTC GACTGGTTCC GGAAGGGGGC ATCTGGGTAC ACATATGCAC   2100
CTCTTCCGCA CGGCAGGGAT ATAAACCGGG AGTGCAGTCC CACCGATCAT GCTGAGTCCG   2160
CCCGTCTCCA GACTTCACGG TCGCAGAGGA CTAGACGCGC GGTGAAG ATG ACT GGC     2216
                                                    Met Thr Gly
                                                      1
CTC GGA GTG ATG GTG GTG ATG GTC GGC TTC CTG GCG ATC GCC TCT  CT     2263
Leu Gly Val Met Val Val Met Val Gly Phe Leu Ala Ile Ala Ser  Leu
      5                  10                  15
GTAAGCAGCG ATTCCAGGGG TCCGGTGTGC GTTAAAAGAA AAAGCTAACG CCACCAG A    2321
CAA TCC GAG TCC CGG CCA TGC GAC ACC CCA GAC TTG GGC TTC CAG TGT     2369
Gln Ser Glu Ser Arg Pro Cys Asp Thr Pro Asp Leu Gly Phe Gln Cys
 20                  25                  30                  35
GGT ACG GCC ATT TCC CAC TTC TGG GGC CAG TAC TCG CCC TAC TTC TCC     2417
Gly Thr Ala Ile Ser His Phe Trp Gly Gln Tyr Ser Pro Tyr Phe Ser
                 40                  45                  50
GTG CCC TCG GAG CTG GAT GCT TCG ATC CCC GAC GAC TGC GAG GTG ACG     2465
Val Pro Ser Glu Leu Asp Ala Ser Ile Pro Asp Asp Cys Glu Val Thr
             55                  60                  65
TTT GCC CAA GTC CTC TCC CGC CAC GGC GCG AGG GCG CCG ACG CTC AAA     2513
Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Ala Pro Thr Leu Lys
         70                  75                  80
CGG GCC GCG AGC TAC GTC GAT CTC ATC GAC AGG ATC CAC CAT GGC GCC     2561
Arg Ala Ala Ser Tyr Val Asp Leu Ile Asp Arg Ile His His Gly Ala
     85                  90                  95
ATC TCC TAC GGG CCG GGC TAC GAG TTC CTC AGG ACG TAT GAC TAC ACC     2609
Ile Ser Tyr Gly Pro Gly Tyr Glu Phe Leu Arg Thr Tyr Asp Tyr Thr
100                 105                 110                 115
CTG GGC GCC GAC GAG CTC ACC CGG ACG GGC CAG CAG CAG ATG GTC AAC     2657
Leu Gly Ala Asp Glu Leu Thr Arg Thr Gly Gln Gln Gln Met Val Asn
                120                 125                 130
TCG GGC ATC AAG TTT TAC CGC CGC TAC CGC GCT CTC GCC CGC AAG TCG     2705
Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Arg Ala Leu Ala Arg Lys Ser
            135                 140                 145
ATC CCC TTC GTC CGC ACC GCC GGC CAG GAC CGC GTC GTC CAC TCG GCC     2753
Ile Pro Phe Val Arg Thr Ala Gly Gln Asp Arg Val Val His Ser Ala
        150                 155                 160
GAG AAC TTC ACC CAG GGC TTC CAC TCT GCC CTG CTC GCC GAC CGC GGG     2801
Glu Asn Phe Thr Gln Gly Phe His Ser Ala Leu Leu Ala Asp Arg Gly
    165                 170                 175
TCC ACC GTC CGG CCC ACC CTC CCC TAT GAC ATG GTC GTC ATC CCG GAA     2849
Ser Thr Val Arg Pro Thr Leu Pro Tyr Asp Met Val Val Ile Pro Glu
180                 185                 190                 195
ACC GCC GGC GCC AAC AAC ACG CTC CAC AAC GAC CTC TGC ACC GCC TTC     2897
Thr Ala Gly Ala Asn Asn Thr Leu His Asn Asp Leu Cys Thr Ala Phe
                200                 205                 210
GAG GAA GGC CCG TAC TCG ACC ATC GGC GAC GAC GCC CAA GAC ACC TAC     2945
Glu Glu Gly Pro Tyr Ser Thr Ile Gly Asp Asp Ala Gln Asp Thr Tyr
            215                 220                 225
CTC TCC ACC TTC GCC GGA CCC ATC ACC GCC CGG GTC AAC GCC AAC CTG     2993
Leu Ser Thr Phe Ala Gly Pro Ile Thr Ala Arg Val Asn Ala Asn Leu
        230                 235                 240
CCG GGC GCC AAC CTG ACC GAC GCC GAC ACG GTC GCG CTG ATG GAC CTC     3041
Pro Gly Ala Asn Leu Thr Asp Ala Asp Thr Val Ala Leu Met Asp Leu
    245                 250                 255
TGC CCC TTC GAG ACG GTC GCC TCC TCC TCC TCC GAC CCG GCA ACG GCG     3089
Cys Pro Phe Glu Thr Val Ala Ser Ser Ser Ser Asp Pro Ala Thr Ala
260                 265                 270                 275
GAC GCG GGG GGC GGC AAC GGG CGG CCG CTG TCG CCC TTC TGC CGC CTG     3137
Asp Ala Gly Gly Gly Asn Gly Arg Pro Leu Ser Pro Phe Cys Arg Leu
                280                 285                 290
TTC AGC GAG TCC GAG TGG CGC GCG TAC GAC TAC CTG CAG TCG GTG GGC     3185
Phe Ser Glu Ser Glu Trp Arg Ala Tyr Asp Tyr Leu Gln Ser Val Gly
            295                 300                 305
AAG TGG TAC GGG TAC GGG CCG GGC AAC CCG CTG GGG CCG ACG CAG GGG     3233
Lys Trp Tyr Gly Tyr Gly Pro Gly Asn Pro Leu Gly Pro Thr Gln Gly
        310                 315                 320
GTC GGG TTC GTC AAC GAG CTG CTG GCG CGG CTG GCC GGG GTC CCC GTG     3281
Val Gly Phe Val Asn Glu Leu Leu Ala Arg Leu Ala Gly Val Pro Val
    325                 330                 335
CGC GAC GGC ACC AGC ACC AAC CGC ACC CTC GAC GGC GAC CCG CGC ACC     3329
Arg Asp Gly Thr Ser Thr Asn Arg Thr Leu Asp Gly Asp Pro Arg Thr
340                 345                 350                 355
TTC CCG CTC GGC CGG CCC CTC TAC GCC GAC TTC AGC CAC GAC AAC GAC     3377
Phe Pro Leu Gly Arg Pro Leu Tyr Ala Asp Phe Ser His Asp Asn Asp
                360                 365                 370
ATG ATG GGC GTC CTC GGC GCC CTC GGC GCC TAC GAC GGC GTC CCG CCC     3425
Met Met Gly Val Leu Gly Ala Leu Gly Ala Tyr Asp Gly Val Pro Pro
            375                 380                 385
CTC GAC AAG ACC GCC CGC CGC GAC CCG GAA GAG CTC GGC GGG TAC GCG     3473
Leu Asp Lys Thr Ala Arg Arg Asp Pro Glu Glu Leu Gly Gly Tyr Ala
        390                 395                 400
GCC AGC TGG GCC GTC CCG TTC GCC GCC AGG ATC TAC GTC GAG AAG ATG     3521
Ala Ser Trp Ala Val Pro Phe Ala Ala Arg Ile Tyr Val Glu Lys Met
    405                 410                 415
CGG TGC AGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GAG GGG CGG CAG     3569
Arg Cys Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Gly Arg Gln
420                 425                 430                 435
GAG AAG GAT GAG GAG ATG GTC AGG GTG CTG GTG AAC GAC CGG GTG ATG     3617
Glu Lys Asp Glu Glu Met Val Arg Val Leu Val Asn Asp Arg Val Met
                440                 445                 450
ACG CTG AAG GGG TGC GGC GCC GAC GAG AGG GGG ATG TGT ACG CTA GAA     3665
Thr Leu Lys Gly Cys Gly Ala Asp Glu Arg Gly Met Cys Thr Leu Glu
            455                 460                 465
CGG TTC ATC GAA AGC ATG GCG TTT GCG AGG GGG AAC GGC AAG TGG GAT     3713
Arg Phe Ile Glu Ser Met Ala Phe Ala Arg Gly Asn Gly Lys Trp Asp
        470                 475                 480
CTC TGC TTT GCT TGATATGCCC ACGCCCGAGA TTGAACAGAA CTTGTGATGG         3765
Leu Cys Phe Ala
    485
GGGTAGAGTG TGGTATTCGA GATGATAGTT CACAGTTTTC GGGAATCAAA AATCGGTTAG   3825
ACTGGCGAAA TTCAAGTCTG GGGCCTGCGG CGTCTGCATT CTCCGTTCCC TGTTGTTACC   3885
TTCTTAATGG TTTTTTTTTA TTTTTTATTT TTCTTAAATT TTCACACAAA CCTTTTATTG   3945
TCTTTTTTTC TTCTTTTTCT TCTTCTGCAC ATCGGATGGG AATTGTCGAC              3995
 
           
           
             
               487 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              4
Met Thr Gly Leu Gly Val Met Val Val Met Val Gly Phe Leu Ala Ile
  1               5                  10                  15
Ala Ser Leu Gln Ser Glu Ser Arg Pro Cys Asp Thr Pro Asp Leu Gly
             20                  25                  30
Phe Gln Cys Gly Thr Ala Ile Ser His Phe Trp Gly Gln Tyr Ser Pro
         35                  40                  45
Tyr Phe Ser Val Pro Ser Glu Leu Asp Ala Ser Ile Pro Asp Asp Cys
     50                  55                  60
Glu Val Thr Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Ala Pro
 65                  70                  75                  80
Thr Leu Lys Arg Ala Ala Ser Tyr Val Asp Leu Ile Asp Arg Ile His
                 85                  90                  95
His Gly Ala Ile Ser Tyr Gly Pro Gly Tyr Glu Phe Leu Arg Thr Tyr
            100                 105                 110
Asp Tyr Thr Leu Gly Ala Asp Glu Leu Thr Arg Thr Gly Gln Gln Gln
        115                 120                 125
Met Val Asn Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Arg Ala Leu Ala
    130                 135                 140
Arg Lys Ser Ile Pro Phe Val Arg Thr Ala Gly Gln Asp Arg Val Val
145                 150                 155                 160
His Ser Ala Glu Asn Phe Thr Gln Gly Phe His Ser Ala Leu Leu Ala
                165                 170                 175
Asp Arg Gly Ser Thr Val Arg Pro Thr Leu Pro Tyr Asp Met Val Val
            180                 185                 190
Ile Pro Glu Thr Ala Gly Ala Asn Asn Thr Leu His Asn Asp Leu Cys
        195                 200                 205
Thr Ala Phe Glu Glu Gly Pro Tyr Ser Thr Ile Gly Asp Asp Ala Gln
    210                 215                 220
Asp Thr Tyr Leu Ser Thr Phe Ala Gly Pro Ile Thr Ala Arg Val Asn
225                 230                 235                 240
Ala Asn Leu Pro Gly Ala Asn Leu Thr Asp Ala Asp Thr Val Ala Leu
                245                 250                 255
Met Asp Leu Cys Pro Phe Glu Thr Val Ala Ser Ser Ser Ser Asp Pro
            260                 265                 270
Ala Thr Ala Asp Ala Gly Gly Gly Asn Gly Arg Pro Leu Ser Pro Phe
        275                 280                 285
Cys Arg Leu Phe Ser Glu Ser Glu Trp Arg Ala Tyr Asp Tyr Leu Gln
    290                 295                 300
Ser Val Gly Lys Trp Tyr Gly Tyr Gly Pro Gly Asn Pro Leu Gly Pro
305                 310                 315                 320
Thr Gln Gly Val Gly Phe Val Asn Glu Leu Leu Ala Arg Leu Ala Gly
                325                 330                 335
Val Pro Val Arg Asp Gly Thr Ser Thr Asn Arg Thr Leu Asp Gly Asp
            340                 345                 350
Pro Arg Thr Phe Pro Leu Gly Arg Pro Leu Tyr Ala Asp Phe Ser His
        355                 360                 365
Asp Asn Asp Met Met Gly Val Leu Gly Ala Leu Gly Ala Tyr Asp Gly
    370                 375                 380
Val Pro Pro Leu Asp Lys Thr Ala Arg Arg Asp Pro Glu Glu Leu Gly
385                 390                 395                 400
Gly Tyr Ala Ala Ser Trp Ala Val Pro Phe Ala Ala Arg Ile Tyr Val
                405                 410                 415
Glu Lys Met Arg Cys Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu
            420                 425                 430
Gly Arg Gln Glu Lys Asp Glu Glu Met Val Arg Val Leu Val Asn Asp
        435                 440                 445
Arg Val Met Thr Leu Lys Gly Cys Gly Ala Asp Glu Arg Gly Met Cys
    450                 455                 460
Thr Leu Glu Arg Phe Ile Glu Ser Met Ala Phe Ala Arg Gly Asn Gly
465                 470                 475                 480
Lys Trp Asp Leu Cys Phe Ala
                485
 
           
           
             
               103 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                2..103
 
             
              5
G ACC TTG GCT CGC AAC CAC ACA GAC ACG CTG TCT CCG TTC TGC GCT         46
  Thr Leu Ala Arg Asn His Thr Asp Thr Leu Ser Pro Phe Cys Ala
    1               5                  10                  15
CTT TCC ACG CAA GAG GAG TGG CAA GCA TAT GAC TAC TAC CAA AGT CTG       94
Leu Ser Thr Gln Glu Glu Trp Gln Ala Tyr Asp Tyr Tyr Gln Ser Leu
                 20                  25                  30
GGG AAA TAC                                                          103
Gly Lys Tyr
 
           
           
             
               33 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              6
Thr Leu Ala Arg Asn His Thr Asp Thr Leu Ser Pro Phe Cys Ala Leu
  1               5                  10                  15
Ser Thr Gln Glu Glu Trp Gln Ala Tyr Asp Tyr Tyr Gln Ser Leu Gly
             20                  25                  30
Asn
 
           
           
             
               106 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                2..106
 
             
              7
T ACG GTA GCG CGC ACC AGC GAC GCA AGT CAG CTG TCA CCG TTC TGT         46
  Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys
    1               5                  10                  15
CAA CTC TTC ACT CAC AAT GAG TGG AAG AAG TAC AAC TAC CTT CAG TCC       94
Gln Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser
                 20                  25                  30
TTG GGC AAG TAC                                                      106
Leu Gly Lys Tyr
             35
 
           
           
             
               35 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              8
Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln
  1               5                  10                  15
Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu
             20                  25                  30
Gly Lys Tyr
         35
 
           
           
             
               109 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                2..109
 
             
              9
C ACC ATG GCG CGC ACC GCC ACT CGG AAC CGT AGT CTG TCT CCA TTT         46
  Thr Met Ala Arg Thr Ala Thr Arg Asn Arg Ser Leu Ser Pro Phe
    1               5                  10                  15
TGT GCC ATC TTC ACT GAA AAG GAG TGG CTG CAG TAC GAC TAC CTT CAA       94
Cys Ala Ile Phe Thr Glu Lys Glu Trp Leu Gln Tyr Asp Tyr Leu Gln
                 20                  25                  30
TCT CTA TCA AAG TAC                                                  109
Ser Leu Ser Lys Tyr
             35
 
           
           
             
               36 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              10
Thr Met Ala Arg Thr Ala Thr Arg Asn Arg Ser Leu Ser Pro Phe Cys
  1               5                  10                  15
Ala Ile Phe Thr Glu Lys Glu Trp Leu Gln Tyr Asp Tyr Leu Gln Ser
             20                  25                  30
Leu Ser Lys Tyr
         35
 
           
           
             
               1912 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                1..1396
 
             
             
               CDS 
                1..1398
 
             
              11
ATG GGC GTC TCT GCT GTT CTA CTT CCT TTG TAT CTC CTA GCT GGA GTC       48
Met Gly Val Ser Ala Val Leu Leu Pro Leu Tyr Leu Leu Ala Gly Val
  1               5                  10                  15
ACC TCC GGA CTG GCA GTC CCC GCC TCG AGA AAT CAA TCC ACT TGC GAT       96
Thr Ser Gly Leu Ala Val Pro Ala Ser Arg Asn Gln Ser Thr Cys Asp
             20                  25                  30
ACG GTC GAT CAA GGG TAT CAA TGC TTC TCC GAG ACT TCG CAT CTT TGG      144
Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser Glu Thr Ser His Leu Trp
         35                  40                  45
GGT CAA TAC GCG CCG TTC TTC TCT CTG GCA AAC GAA TCG GTC ATC TCC      192
Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala Asn Glu Ser Val Ile Ser
     50                  55                  60
CCT GAT GTG CCC GCC GGT TGC AGA GTC ACT TTC GCT CAG GTC CTC TCC      240
Pro Asp Val Pro Ala Gly Cys Arg Val Thr Phe Ala Gln Val Leu Ser
 65                  70                  75                  80
CGT CAT GGA GCG CGG TAT CCG ACC GAG TCC AAG GGC AAG AAA TAC TCC      288
Arg His Gly Ala Arg Tyr Pro Thr Glu Ser Lys Gly Lys Lys Tyr Ser
                 85                  90                  95
GCT CTC ATT GAG GAG ATC CAG CAG AAC GTG ACC ACC TTT GAT GGA AAA      336
Ala Leu Ile Glu Glu Ile Gln Gln Asn Val Thr Thr Phe Asp Gly Lys
            100                 105                 110
TAT GCC TTC CTG AAG ACA TAC AAC TAC AGC TTG GGT GCA GAT GAC CTG      384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser Leu Gly Ala Asp Asp Leu
        115                 120                 125
ACT CCC TTC GGA GAG CAG GAG CTA GTC AAC TCC GGC ATC AAG TTC TAC      432
Thr Pro Phe Gly Glu Gln Glu Leu Val Asn Ser Gly Ile Lys Phe Tyr
    130                 135                 140
CAG CGC TAC AAC GCC CTC ACC CGA CAC ATC AAC CCC TTC GTC CGC GCC      480
Gln Arg Tyr Asn Ala Leu Thr Arg His Ile Asn Pro Phe Val Arg Ala
145                 150                 155                 160
ACC GAT GCA TCC CGC GTC CAC GAA TCC GCC GAG AAG TTC GTC GAG GGC      528
Thr Asp Ala Ser Arg Val His Glu Ser Ala Glu Lys Phe Val Glu Gly
                165                 170                 175
TTC CAA ACC GCT CGA CAG GAC GAT CAT CAC GCC AAT CCC CAC CAG CCT      576
Phe Gln Thr Ala Arg Gln Asp Asp His His Ala Asn Pro His Gln Pro
            180                 185                 190
TCG CCT CGC GTG GAC GTG GCC ATC CCC GAA GGC AGC GCC TAC AAC AAC      624
Ser Pro Arg Val Asp Val Ala Ile Pro Glu Gly Ser Ala Tyr Asn Asn
        195                 200                 205
ACG CTG GAG CAC AGC CTC TGC ACC GCC TTC GAA TCC AGC ACC GTC GGC      672
Thr Leu Glu His Ser Leu Cys Thr Ala Phe Glu Ser Ser Thr Val Gly
    210                 215                 220
GAC GAC GCG GTC GCC AAC TTC ACC GCC GTG TTC GCG CCG GCG ATC GCC      720
Asp Asp Ala Val Ala Asn Phe Thr Ala Val Phe Ala Pro Ala Ile Ala
225                 230                 235                 240
CAG CGC CTG GAG GCC GAT CTT CCC GGC GTG CAG CTG TCC ACC GAC GAC      768
Gln Arg Leu Glu Ala Asp Leu Pro Gly Val Gln Leu Ser Thr Asp Asp
                245                 250                 255
GTG GTC AAC CTG ATG GCC ATG TGT CCG TTC GAG ACG GTC AGC CTG ACC      816
Val Val Asn Leu Met Ala Met Cys Pro Phe Glu Thr Val Ser Leu Thr
            260                 265                 270
GAC GAC GCG CAC ACG CTG TCG CCG TTC TGC GAC CTC TTC ACG GCC ACT      864
Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp Leu Phe Thr Ala Thr
        275                 280                 285
GAG TGG ACG CAG TAC AAC TAC CTG CTC TCG CTG GAC AAG TAC TAC GGC      912
Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu Asp Lys Tyr Tyr Gly
    290                 295                 300
TAC GGC GGG GGC AAT CCG CTG GGT CCG GTG CAG GGG GTC GGC TGG GCG      960
Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val Gln Gly Val Gly Trp Ala
305                 310                 315                 320
AAC GAG CTG ATG GCG CGG CTA ACG CGC GCC CCC GTG CAC GAC CAC ACC     1008
Asn Glu Leu Met Ala Arg Leu Thr Arg Ala Pro Val His Asp His Thr
                325                 330                 335
TGC GTC AAC AAC ACC CTC GAC GCG AGT CCG GCC ACC TTC CCG CTG AAC     1056
Cys Val Asn Asn Thr Leu Asp Ala Ser Pro Ala Thr Phe Pro Leu Asn
            340                 345                 350
GCC ACC CTC TAC GCC GAC TTC TCC CAC GAC AGC AAC CTG GTG TCG ATC     1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Ser Asn Leu Val Ser Ile
        355                 360                 365
TTC TGG GCG CTG GGC CTG TAC AAC GGC ACC GCG CCG CTG TCG CAG ACC     1152
Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Gln Thr
    370                 375                 380
TCC GTC GAG AGC GTC TCC CAG ACG GAC GGG TAC GCC GCC GCC TGG ACG     1200
Ser Val Glu Ser Val Ser Gln Thr Asp Gly Tyr Ala Ala Ala Trp Thr
385                 390                 395                 400
GTG CCG TTC GCC GCT CGC GCG TAC GTC GAG ATG ATG CAG TGT CGC GCC     1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Arg Ala
                405                 410                 415
GAG AAG GAG CCG CTG GTG CGC GTG CTG GTC AAC GAC CGG GTC ATG CCG     1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Met Pro
            420                 425                 430
CTG CAT GGC TGC CCT ACG GAC AAG CTG GGG CGG TGC AAG CGG GAC GCT     1344
Leu His Gly Cys Pro Thr Asp Lys Leu Gly Arg Cys Lys Arg Asp Ala
        435                 440                 445
TTC GTC GCG GGG CTG AGC TTT GCG CAG GCG GGC GGG AAC TGG GCG GAT     1392
Phe Val Ala Gly Leu Ser Phe Ala Gln Ala Gly Gly Asn Trp Ala Asp
    450                 455                 460
TGT TTC TGATGTTGAG AAGAAAGGTA GATAGATAGG TAGTACATAT GGATTGCTCG      1448
Cys Phe
465
GCTCTGGGTC GTTGCCCACA ATGCATATTA CGCCCGTCAA CTGCCTTGCG CCATCCACCT   1508
CTCACCCTGG ACGCAACCGA GCGGTCTACC CTGCACACGG CTTCCACCGC GACGCGCACG   1568
GATAAGGCGC TTTTGTTACG GGGTTGGGGC TGGGGGCAGC CGGAGCCGGA GAGAGAGACC   1628
AGCGTGAAAA ACGACAGAAC ATAGATATCA ATTCGACGCC AATTCATGCA GAGTAGTATA   1688
CAGACGAACT GAAACAAACA CATCACTTCC CTCGCTCCTC TCCTGTAGAA GACGCTCCCA   1748
CCAGCCGCTT CTGGCCCTTA TTCCCGTACG CTAGGTAGAC CAGTCAGCCA GACGCATGCC   1808
TCACAAGAAC GGGGGCGGGG GACACACTCC GCTCGTACAG CACCCACGAC GTGTACAGGA   1868
AAACCGGCAG CGCCACAATC GTCGAGAGCC ATCTGCAGGA ATTC                    1912
 
           
           
             
               466 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              12
Met Gly Val Ser Ala Val Leu Leu Pro Leu Tyr Leu Leu Ala Gly Val
  1               5                  10                  15
Thr Ser Gly Leu Ala Val Pro Ala Ser Arg Asn Gln Ser Thr Cys Asp
             20                  25                  30
Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser Glu Thr Ser His Leu Trp
         35                  40                  45
Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala Asn Glu Ser Val Ile Ser
     50                  55                  60
Pro Asp Val Pro Ala Gly Cys Arg Val Thr Phe Ala Gln Val Leu Ser
 65                  70                  75                  80
Arg His Gly Ala Arg Tyr Pro Thr Glu Ser Lys Gly Lys Lys Tyr Ser
                 85                  90                  95
Ala Leu Ile Glu Glu Ile Gln Gln Asn Val Thr Thr Phe Asp Gly Lys
            100                 105                 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser Leu Gly Ala Asp Asp Leu
        115                 120                 125
Thr Pro Phe Gly Glu Gln Glu Leu Val Asn Ser Gly Ile Lys Phe Tyr
    130                 135                 140
Gln Arg Tyr Asn Ala Leu Thr Arg His Ile Asn Pro Phe Val Arg Ala
145                 150                 155                 160
Thr Asp Ala Ser Arg Val His Glu Ser Ala Glu Lys Phe Val Glu Gly
                165                 170                 175
Phe Gln Thr Ala Arg Gln Asp Asp His His Ala Asn Pro His Gln Pro
            180                 185                 190
Ser Pro Arg Val Asp Val Ala Ile Pro Glu Gly Ser Ala Tyr Asn Asn
        195                 200                 205
Thr Leu Glu His Ser Leu Cys Thr Ala Phe Glu Ser Ser Thr Val Gly
    210                 215                 220
Asp Asp Ala Val Ala Asn Phe Thr Ala Val Phe Ala Pro Ala Ile Ala
225                 230                 235                 240
Gln Arg Leu Glu Ala Asp Leu Pro Gly Val Gln Leu Ser Thr Asp Asp
                245                 250                 255
Val Val Asn Leu Met Ala Met Cys Pro Phe Glu Thr Val Ser Leu Thr
            260                 265                 270
Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp Leu Phe Thr Ala Thr
        275                 280                 285
Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu Asp Lys Tyr Tyr Gly
    290                 295                 300
Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val Gln Gly Val Gly Trp Ala
305                 310                 315                 320
Asn Glu Leu Met Ala Arg Leu Thr Arg Ala Pro Val His Asp His Thr
                325                 330                 335
Cys Val Asn Asn Thr Leu Asp Ala Ser Pro Ala Thr Phe Pro Leu Asn
            340                 345                 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Ser Asn Leu Val Ser Ile
        355                 360                 365
Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Gln Thr
    370                 375                 380
Ser Val Glu Ser Val Ser Gln Thr Asp Gly Tyr Ala Ala Ala Trp Thr
385                 390                 395                 400
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Arg Ala
                405                 410                 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Met Pro
            420                 425                 430
Leu His Gly Cys Pro Thr Asp Lys Leu Gly Arg Cys Lys Arg Asp Ala
        435                 440                 445
Phe Val Ala Gly Leu Ser Phe Ala Gln Ala Gly Gly Asn Trp Ala Asp
    450                 455                 460
Cys Phe
465
 
           
           
             
               112 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
              13
GACGGTCAGC CTGACCGACG ACGCGCACAC GCTGTCGCCG TTCTGCGACC TCTTCACCGC     60
CGCCGAGTGG ACGCAGTACA ACTACCTGCT CTCGCTGGAC AAGTACTACG TC            112
 
           
           
             
               91 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
              14
CAGTAACCTG GTGTCGATCT TCTGGNCGCT GGGTCTGTAC AACGGCACCA AGCCCCTGTC     60
GCAGACCACC GTGGAGGATA TCACCCGGAC G                                    91
 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
              15
ATGGAYATGT GYTCNTTYGA                                                 20
 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
              16
TTRCCRGCRC CRTGNCCRTA                                                 20
 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
              17
TAYGCNGAYT TYTCNCAYGA                                                 20
 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
              18
CGRTCRTTNA CNAGNACNC                                                  19
 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
              19
AGTCCGGAGG TGACTCCAGC TAGGAGATAC                                      30
 
           
           
             
               21 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
              20
CTGTCGCCGT TCTGCGACCT C                                               21
 
           
           
             
               23 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
              21
CGGTGCCGTT GTACAGACCC AGC                                             23
 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
              22
ATGGGCTTTC TTGCCATTGT                                                 20
 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
              23
TCAGAAACAA TCCGCCCAAG TT                                              22
 
           
           
             
               106 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               CDS 
                2..106
 
             
             
               Region 
                32..52 
                /note=“position of Aterr21 primer”
 
             
              24
G ACG GTC AGC CTG ACC GAC GAC GCG CAC ACG CTG TCG CCG TTC TGC         46
  Thr Val Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys
    1               5                  10                  15
GAC CTC TTC ACC GCC GCC GAG TGG ACG CAG TAC AAC TAC CTG CTC TCG       94
Asp Leu Phe Thr Ala Ala Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser
                 20                  25                  30
CTG GAC AAG TAC                                                      106
Leu Asp Lys Tyr
             35
 
           
           
             
               35 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              25
Thr Val Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp
  1               5                  10                  15
Leu Phe Thr Ala Ala Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu
             20                  25                  30
Asp Lys Tyr
         35
 
           
           
             
               181 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               CDS 
                2..181
 
             
             
               Region 
                28..50 
                /note=“position of Aterr58 primer”
 
             
              26
C AGT AAC CTG GTG TCG ATC TTC TGG GCG CTG GGT CTG TAC AAC GGC         46
  Ser Asn Leu Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly
    1               5                  10                  15
ACC AAG CCC CTG TCG CAG ACC ACC GTG GAG GAT ATC ACC CGG ACG GAC       94
Thr Lys Pro Leu Ser Gln Thr Thr Val Glu Asp Ile Thr Arg Thr Asp
                 20                  25                  30
GGG TAC GCG GCC GCC TGG ACG GTG CCG TTT GCC GCC CGC GCC TAC ATC      142
Gly Tyr Ala Ala Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Ile
             35                  40                  45
GAG ATG ATG CAG TGT CGC GCG GAG AAG CAG CCG CTG GTA                  181
Glu Met Met Gln Cys Arg Ala Glu Lys Gln Pro Leu Val
         50                  55                  60
 
           
           
             
               60 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              27
Ser Asn Leu Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr
  1               5                  10                  15
Lys Pro Leu Ser Gln Thr Thr Val Glu Asp Ile Thr Arg Thr Asp Gly
             20                  25                  30
Tyr Ala Ala Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Ile Glu
         35                  40                  45
Met Met Gln Cys Arg Ala Glu Lys Gln Pro Leu Val
     50                  55                  60
 
           
           
             
               1931 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               CDS 
                join(158..204, 259..1600)
 
             
             
               intron 
                205..258
 
             
             
               misc_feature 
                1000..1105 
                /note= “Position of PCR fragment”
 
             
              28
TCTGTAACCG ATAGCGGACC GACTAGGCAT CGTTGATCCA CAATATCTCA GACAATGCAA     60
CTCAGTCGAA TATGAAGGGC TACAGCCAGC ATTTAAATAC GGCCGTCTAG GTCGGGCTCC    120
GGGGATGAGG AGGAGCAGGC TCGTGTTCAT TTCGGTC ATG GCT TTT TTC ACG GTC     175
                                         Met Ala Phe Phe Thr Val
                                           1               5
GCT CTT TCG CTT TAT TAC TTG CTA TCG  AG  GTGAGATCTC TACAATATCT       224
Ala Leu Ser Leu Tyr Tyr Leu Leu Ser  Arg
             10                  15
GTCTGCTTAG TTGAATTGGT ACTTATCTGT ACAG A GTC TCT GCT CAG GCC CCA      277
                                        Val Ser Ala Gln Ala Pro
                                                     20
GTG GTC CAG AAT CAT TCA TGC AAT ACG GCG GAC GGT GGA TAT CAA TGC      325
Val Val Gln Asn His Ser Cys Asn Thr Ala Asp Gly Gly Tyr Gln Cys
         25                  30                  35
TTC CCC AAT GTC TCT CAT GTT TGG GGT CAG TAC TCG CCG TAC TTC TCC      373
Phe Pro Asn Val Ser His Val Trp Gly Gln Tyr Ser Pro Tyr Phe Ser
     40                  45                  50
ATC GAG CAG GAG TCA GCT ATC TCT GAG GAC GTG CCT CAT GGC TGT GAG      421
Ile Glu Gln Glu Ser Ala Ile Ser Glu Asp Val Pro His Gly Cys Glu
 55                  60                  65                  70
GTT ACC TTT GTG CAG GTG CTC TCG CGG CAT GGG GCT AGG TAT CCG ACA      469
Val Thr Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr
                 75                  80                  85
GAG TCG AAG AGT AAG GCG TAC TCG GGG TTG ATT GAA GCA ATC CAG AAG      517
Glu Ser Lys Ser Lys Ala Tyr Ser Gly Leu Ile Glu Ala Ile Gln Lys
             90                  95                 100
AAT GCT ACC TCT TTT TGG GGA CAG TAT GCT TTT CTG GAG AGT TAT AAC      565
Asn Ala Thr Ser Phe Trp Gly Gln Tyr Ala Phe Leu Glu Ser Tyr Asn
        105                 110                 115
TAT ACC CTC GGC GCG GAT GAC TTG ACT ATC TTC GGC GAG AAC CAG ATG      613
Tyr Thr Leu Gly Ala Asp Asp Leu Thr Ile Phe Gly Glu Asn Gln Met
    120                 125                 130
GTT GAT TCG GGT GCC AAG TTC TAC CGA CGG TAT AAG AAT CTC GCC AGG      661
Val Asp Ser Gly Ala Lys Phe Tyr Arg Arg Tyr Lys Asn Leu Ala Arg
135                 140                 145                 150
AAA AAT ACT CCT TTT ATC CGT GCA TCA GGG TCT GAC CGT GTC GTT GCG      709
Lys Asn Thr Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Val Ala
                155                 160                 165
TCT GCG GAG AAG TTC ATT AAT GGA TTT CGC AAG GCT CAG CTC CAC GAC      757
Ser Ala Glu Lys Phe Ile Asn Gly Phe Arg Lys Ala Gln Leu His Asp
            170                 175                 180
CAT GGC TCC AAA CGT GCT ACG CCA GTT GTC AAT GTG ATT ATC CCT GAA      805
His Gly Ser Lys Arg Ala Thr Pro Val Val Asn Val Ile Ile Pro Glu
        185                 190                 195
ATC GAT GGG TTT AAC AAC ACC CTG GAC CAT AGC ACG TGC GTA TCT TTT      853
Ile Asp Gly Phe Asn Asn Thr Leu Asp His Ser Thr Cys Val Ser Phe
    200                 205                 210
GAG AAT GAT GAG CGG GCG GAT GAA ATT GAA GCC AAT TTC ACG GCA ATT      901
Glu Asn Asp Glu Arg Ala Asp Glu Ile Glu Ala Asn Phe Thr Ala Ile
215                 220                 225                 230
ATG GGA CCT CCG ATC CGC AAA CGT CTG GAA AAT GAC CTC CCT GGC ATC      949
Met Gly Pro Pro Ile Arg Lys Arg Leu Glu Asn Asp Leu Pro Gly Ile
                235                 240                 245
AAA CTT ACA AAC GAG AAT GTA ATA TAT TTG ATG GAT ATG TGC TCT TTC      997
Lys Leu Thr Asn Glu Asn Val Ile Tyr Leu Met Asp Met Cys Ser Phe
            250                 255                 260
GAC ACC ATG GCG CGC ACC GCC CAC GGA ACC GAG CTG TCT CCA TTT TGT     1045
Asp Thr Met Ala Arg Thr Ala His Gly Thr Glu Leu Ser Pro Phe Cys
        265                 270                 275
GCC ATC TTC ACT GAA AAG GAG TGG CTG CAG TAC GAC TAC CTT CAA TCT     1093
Ala Ile Phe Thr Glu Lys Glu Trp Leu Gln Tyr Asp Tyr Leu Gln Ser
    280                 285                 290
CTA TCA AAG TAC TAC GGC TAC GGT GCC GGA AGC CCC CTT GGC CCA GCT     1141
Leu Ser Lys Tyr Tyr Gly Tyr Gly Ala Gly Ser Pro Leu Gly Pro Ala
295                 300                 305                 310
CAG GGA ATT GGC TTC ACC AAC GAG CTG ATT GCC CGA CTA ACG CAA TCG     1189
Gln Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Gln Ser
                315                 320                 325
CCC GTC CAG GAC AAC ACA AGC ACC AAC CAC ACT CTA GAC TCG AAC CCA     1237
Pro Val Gln Asp Asn Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro
            330                 335                 340
GCC ACA TTT CCG CTC GAC AGG AAG CTC TAC GCC GAC TTC TCC CAC GAC     1285
Ala Thr Phe Pro Leu Asp Arg Lys Leu Tyr Ala Asp Phe Ser His Asp
        345                 350                 355
AAT AGC ATG ATA TCG ATA TTC TTC GCC ATG GGT CTG TAC AAC GGC ACC     1333
Asn Ser Met Ile Ser Ile Phe Phe Ala Met Gly Leu Tyr Asn Gly Thr
    360                 365                 370
CAG CCG CTG TCA ATG GAT TCC GTG GAG TCG ATC CAG GAG ATG GAC GGT     1381
Gln Pro Leu Ser Met Asp Ser Val Glu Ser Ile Gln Glu Met Asp Gly
375                 380                 385                 390
TAC GCG GCG TCT TGG ACT GTT CCG TTT GGT GCG AGG GCT TAC TTT GAG     1429
Tyr Ala Ala Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu
                395                 400                 405
CTC ATG CAG TGC GAG AAG AAG GAG CCG CTT GTG CGG GTA TTA GTG AAT     1477
Leu Met Gln Cys Glu Lys Lys Glu Pro Leu Val Arg Val Leu Val Asn
            410                 415                 420
GAT CGC GTT GTT CCT CTT CAT GGC TGC GCA GTT GAC AAG TTT GGA CGG     1525
Asp Arg Val Val Pro Leu His Gly Cys Ala Val Asp Lys Phe Gly Arg
        425                 430                 435
TGC ACT TTG GAC GAT TGG GTA GAG GGC TTG AAT TTT GCA AGG AGC GGC     1573
Cys Thr Leu Asp Asp Trp Val Glu Gly Leu Asn Phe Ala Arg Ser Gly
    440                 445                 450
GGG AAC TGG AAG ACT TGT TTT ACC CTA TAAAGGGCGT TTGCTCATTC           1620
Gly Asn Trp Lys Thr Cys Phe Thr Leu
455                 460
ATAAGTGTTG TGCAGGTATA GGAAGGTTAG GGAATTAGCT GTTTGGCTTT ACTCTTATTA   1680
GACCAAGAAT GATTTGTTTG TTCTCAAGGC CTTCTAGCAT ATCGTCAAGT GGGATAAATC   1740
ACCTATCCTC CATGTGTAGG TGAACCCGCT CTTGCATCAA CCTCTTGTGT TTCAGAGTAG   1800
TTTCACCAAA CATATCCTCG TGTCCTCTCT TCTGCTCTTC GGTCTCATAT TACACTGTTC   1860
TCTATCTATA TCGTCAACAA AACTACCACC CAAACACCAA ATGTCACACT TTCCAGCACG   1920
AAATTTCTTC G                                                        1931
 
           
           
             
               463 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               misc_feature 
                26 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                41 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                103 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                118 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                203 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                226 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                331 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                335 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                372 
                /note=“potential N-glycosylation site”
 
             
              29
Met Ala Phe Phe Thr Val Ala Leu Ser Leu Tyr Tyr Leu Leu Ser Arg
  1               5                  10                  15
Val Ser Ala Gln Ala Pro Val Val Gln Asn His Ser Cys Asn Thr Ala
             20                  25                  30
Asp Gly Gly Tyr Gln Cys Phe Pro Asn Val Ser His Val Trp Gly Gln
         35                  40                  45
Tyr Ser Pro Tyr Phe Ser Ile Glu Gln Glu Ser Ala Ile Ser Glu Asp
     50                  55                  60
Val Pro His Gly Cys Glu Val Thr Phe Val Gln Val Leu Ser Arg His
 65                  70                  75                  80
Gly Ala Arg Tyr Pro Thr Glu Ser Lys Ser Lys Ala Tyr Ser Gly Leu
                 85                  90                  95
Ile Glu Ala Ile Gln Lys Asn Ala Thr Ser Phe Trp Gly Gln Tyr Ala
            100                 105                 110
Phe Leu Glu Ser Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu Thr Ile
        115                 120                 125
Phe Gly Glu Asn Gln Met Val Asp Ser Gly Ala Lys Phe Tyr Arg Arg
    130                 135                 140
Tyr Lys Asn Leu Ala Arg Lys Asn Thr Pro Phe Ile Arg Ala Ser Gly
145                 150                 155                 160
Ser Asp Arg Val Val Ala Ser Ala Glu Lys Phe Ile Asn Gly Phe Arg
                165                 170                 175
Lys Ala Gln Leu His Asp His Gly Ser Lys Arg Ala Thr Pro Val Val
            180                 185                 190
Asn Val Ile Ile Pro Glu Ile Asp Gly Phe Asn Asn Thr Leu Asp His
        195                 200                 205
Ser Thr Cys Val Ser Phe Glu Asn Asp Glu Arg Ala Asp Glu Ile Glu
    210                 215                 220
Ala Asn Phe Thr Ala Ile Met Gly Pro Pro Ile Arg Lys Arg Leu Glu
225                 230                 235                 240
Asn Asp Leu Pro Gly Ile Lys Leu Thr Asn Glu Asn Val Ile Tyr Leu
                245                 250                 255
Met Asp Met Cys Ser Phe Asp Thr Met Ala Arg Thr Ala His Gly Thr
            260                 265                 270
Glu Leu Ser Pro Phe Cys Ala Ile Phe Thr Glu Lys Glu Trp Leu Gln
        275                 280                 285
Tyr Asp Tyr Leu Gln Ser Leu Ser Lys Tyr Tyr Gly Tyr Gly Ala Gly
    290                 295                 300
Ser Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr Asn Glu Leu Ile
305                 310                 315                 320
Ala Arg Leu Thr Gln Ser Pro Val Gln Asp Asn Thr Ser Thr Asn His
                325                 330                 335
Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asp Arg Lys Leu Tyr
            340                 345                 350
Ala Asp Phe Ser His Asp Asn Ser Met Ile Ser Ile Phe Phe Ala Met
        355                 360                 365
Gly Leu Tyr Asn Gly Thr Gln Pro Leu Ser Met Asp Ser Val Glu Ser
    370                 375                 380
Ile Gln Glu Met Asp Gly Tyr Ala Ala Ser Trp Thr Val Pro Phe Gly
385                 390                 395                 400
Ala Arg Ala Tyr Phe Glu Leu Met Gln Cys Glu Lys Lys Glu Pro Leu
                405                 410                 415
Val Arg Val Leu Val Asn Asp Arg Val Val Pro Leu His Gly Cys Ala
            420                 425                 430
Val Asp Lys Phe Gly Arg Cys Thr Leu Asp Asp Trp Val Glu Gly Leu
        435                 440                 445
Asn Phe Ala Arg Ser Gly Gly Asn Trp Lys Thr Cys Phe Thr Leu
    450                 455                 460
 
           
           
             
               1845 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               CDS 
                join(288..334, 390..1740)
 
             
             
               intron 
                335..389
 
             
             
               misc_feature 
                1134..1236 
                /note= “Position of PCR fragment”
 
             
              30
TTCCACGCTG AAAGCCTGAC TGCGATTTCC AAGCTGCATG CAGGCTGCTC AACTGCCTGC     60
TTATCTTCAT CAGACGCAGA TACACAACCT GGTCTGTAGA TGCACCCATG ACGGACGAAC    120
GCACCGCTCT CTTGGCCTCC AGGGACCCGG AGGTCGAGGG CGATGAGGTC GCGCCCTCGA    180
CGGCCTCCCA GTCCCTGTTG CAGTTGAGAT CTCGCTGCGA ACGTCGACCG CAGATATGGT    240
TGTCTTCGAC GTTTTCTCGC CTTCGAGGAA GAATTGCTGC TGTGACG ATG AGT CTG      296
                                                    Met Ser Leu
                                                      1
TTG TTG CTG GTG CTG TCC GGC GGG TTG GTC GCG TTA  TA  GTATGCTCCT      344
Leu Leu Leu Val Leu Ser Gly Gly Leu Val Ala Leu  Tyr
      5                  10                  15
TCTCTCTGGT CATATTGTTT TCTGCTAACG TTCTCATAAT TGAAG T GTC TCA AGA      399
                                                    Val Ser Arg
AAT CCG CAT GTT GAT AGC CAC TCT TGC AAT ACA GTG GAA GGA GGG TAT      447
Asn Pro His Val Asp Ser His Ser Cys Asn Thr Val Glu Gly Gly Tyr
 20                  25                  30                  35
CAG TGT CGT CCA GAA ATC TCC CAC TCC TGG GGC CAG TAT TCT CCA TTC      495
Gln Cys Arg Pro Glu Ile Ser His Ser Trp Gly Gln Tyr Ser Pro Phe
                 40                  45                  50
TTC TCC CTG GCA GAC CAG TCG GAG ATC TCG CCA GAT GTC CCA CAG AAC      543
Phe Ser Leu Ala Asp Gln Ser Glu Ile Ser Pro Asp Val Pro Gln Asn
             55                  60                  65
TGC AAG ATT ACG TTT GTC CAG CTG CTT TCT CGT CAC GGC GCT AGA TAC      591
Cys Lys Ile Thr Phe Val Gln Leu Leu Ser Arg His Gly Ala Arg Tyr
         70                  75                  80
CCT ACG TCT TCC AAG ACG GAG CTG TAT TCG CAG CTG ATC AGT CGG ATT      639
Pro Thr Ser Ser Lys Thr Glu Leu Tyr Ser Gln Leu Ile Ser Arg Ile
     85                  90                  95
CAG AAG ACG GCG ACT GCG TAC AAA GGC TAC TAT GCC TTC TTG AAA GAC      687
Gln Lys Thr Ala Thr Ala Tyr Lys Gly Tyr Tyr Ala Phe Leu Lys Asp
100                 105                 110                 115
TAC AGA TAC CAG CTG GGA GCG AAC GAC CTG ACG CCC TTT GGG GAA AAC      735
Tyr Arg Tyr Gln Leu Gly Ala Asn Asp Leu Thr Pro Phe Gly Glu Asn
                120                 125                 130
CAG ATG ATC CAG TTG GGC ATC AAG TTT TAT AAC CAT TAC AAG AGT CTC      783
Gln Met Ile Gln Leu Gly Ile Lys Phe Tyr Asn His Tyr Lys Ser Leu
            135                 140                 145
GCC AGG AAT GCC GTC CCA TTC GTT CGT TGC TCC GGC TCT GAT CGG GTC      831
Ala Arg Asn Ala Val Pro Phe Val Arg Cys Ser Gly Ser Asp Arg Val
        150                 155                 160
ATT GCC TCG GGG AGA CTT TTC ATC GAA GGT TTC CAG AGC GCC AAA GTG      879
Ile Ala Ser Gly Arg Leu Phe Ile Glu Gly Phe Gln Ser Ala Lys Val
    165                 170                 175
CTG GAT CCT CAT TCA GAC AAG CAT GAC GCT CCT CCC ACG ATC AAC GTG      927
Leu Asp Pro His Ser Asp Lys His Asp Ala Pro Pro Thr Ile Asn Val
180                 185                 190                 195
ATC ATC GAG GAG GGT CCG TCC TAC AAT AAC ACG CTC GAC ACC GGC AGC      975
Ile Ile Glu Glu Gly Pro Ser Tyr Asn Asn Thr Leu Asp Thr Gly Ser
                200                 205                 210
TGT CCA GTC TTT GAG GAC AGC AGC GGG GGA CAT GAC GCA CAG GAA AAG     1023
Cys Pro Val Phe Glu Asp Ser Ser Gly Gly His Asp Ala Gln Glu Lys
            215                 220                 225
TTC GCA AAG CAA TTC GCA CCA GCT ATC CTG GAA AAG ATC AAG GAC CAT     1071
Phe Ala Lys Gln Phe Ala Pro Ala Ile Leu Glu Lys Ile Lys Asp His
        230                 235                 240
CTT CCC GGC GTG GAC CTG GCC GTG TCG GAT GTA CCG TAC TTG ATG GAC     1119
Leu Pro Gly Val Asp Leu Ala Val Ser Asp Val Pro Tyr Leu Met Asp
    245                 250                 255
TTG TGT CCG TTT GAG ACC TTG GCT CGC AAC CAC ACA GAC ACG CTG TCT     1167
Leu Cys Pro Phe Glu Thr Leu Ala Arg Asn His Thr Asp Thr Leu Ser
260                 265                 270                 275
CCG TTC TGC GCT CTT TCC ACG CAA GAG GAG TGG CAA GCA TAT GAC TAC     1215
Pro Phe Cys Ala Leu Ser Thr Gln Glu Glu Trp Gln Ala Tyr Asp Tyr
                280                 285                 290
TAC CAA AGT CTG GGG AAA TAC TAT GGC AAT GGC GGG GGT AAC CCG TTG     1263
Tyr Gln Ser Leu Gly Lys Tyr Tyr Gly Asn Gly Gly Gly Asn Pro Leu
            295                 300                 305
GGG CCA GCC CAA GGC GTG GGG TTT GTC AAC GAG TTG ATT GCT CGC ATG     1311
Gly Pro Ala Gln Gly Val Gly Phe Val Asn Glu Leu Ile Ala Arg Met
        310                 315                 320
ACC CAT AGC CCT GTC CAG GAC TAC ACC ACG GTC AAC CAC ACT CTT GAC     1359
Thr His Ser Pro Val Gln Asp Tyr Thr Thr Val Asn His Thr Leu Asp
    325                 330                 335
TCG AAT CCG GCG ACA TTC CCT TTG AAC GCG ACG CTG TAC GCA GAT TTC     1407
Ser Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe
340                 345                 350                 355
AGC CAC GAC AAC ACA ATG ACG TCA ATT TTC GCG GCC TTG GGC CTG TAC     1455
Ser His Asp Asn Thr Met Thr Ser Ile Phe Ala Ala Leu Gly Leu Tyr
                360                 365                 370
AAC GGG ACC GCG AAG CTG TCC ACG ACC GAG ATC AAG TCC ATT GAA GAG     1503
Asn Gly Thr Ala Lys Leu Ser Thr Thr Glu Ile Lys Ser Ile Glu Glu
            375                 380                 385
ACG GAC GGC TAC TCG GCG GCG TGG ACC GTT CCG TTC GGG GGG CGA GCC     1551
Thr Asp Gly Tyr Ser Ala Ala Trp Thr Val Pro Phe Gly Gly Arg Ala
        390                 395                 400
TAT ATC GAG ATG ATG CAG TGT GAT GAT TCG GAT GAG CCA GTC GTT CGG     1599
Tyr Ile Glu Met Met Gln Cys Asp Asp Ser Asp Glu Pro Val Val Arg
    405                 410                 415
GTG CTG GTC AAC GAC CGG GTG GTG CCA CTG CAT GGC TGC GAG GTG GAC     1647
Val Leu Val Asn Asp Arg Val Val Pro Leu His Gly Cys Glu Val Asp
420                 425                 430                 435
TCC CTG GGG CGA TGC AAA CGA GAC GAC TTT GTC AGG GGA CTG AGT TTT     1695
Ser Leu Gly Arg Cys Lys Arg Asp Asp Phe Val Arg Gly Leu Ser Phe
                440                 445                 450
GCG CGA CAG GGT GGG AAC TGG GAG GGG TGT TAC GCT GCT TCT GAG         1740
Ala Arg Gln Gly Gly Asn Trp Glu Gly Cys Tyr Ala Ala Ser Glu
            455                 460                 465
TAGGTTTATT CAGCGAGTTT CGACCTTTCT ATCCTTCAAA CACTGCACAA AGACACACTG   1800
CATGAAATGG TAACAGGCCT GGAGCGTTTT AGAAGGAAAA AAGTT                   1845
 
           
           
             
               466 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               misc_feature 
                204 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                269 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                335 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                348 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                372 
                /note=“potential N-glycosylation site”
 
             
              31
Met Ser Leu Leu Leu Leu Val Leu Ser Gly Gly Leu Val Ala Leu Tyr
  1               5                  10                  15
Val Ser Arg Asn Pro His Val Asp Ser His Ser Cys Asn Thr Val Glu
             20                  25                  30
Gly Gly Tyr Gln Cys Arg Pro Glu Ile Ser His Ser Trp Gly Gln Tyr
         35                  40                  45
Ser Pro Phe Phe Ser Leu Ala Asp Gln Ser Glu Ile Ser Pro Asp Val
     50                  55                  60
Pro Gln Asn Cys Lys Ile Thr Phe Val Gln Leu Leu Ser Arg His Gly
 65                  70                  75                  80
Ala Arg Tyr Pro Thr Ser Ser Lys Thr Glu Leu Tyr Ser Gln Leu Ile
                 85                  90                  95
Ser Arg Ile Gln Lys Thr Ala Thr Ala Tyr Lys Gly Tyr Tyr Ala Phe
            100                 105                 110
Leu Lys Asp Tyr Arg Tyr Gln Leu Gly Ala Asn Asp Leu Thr Pro Phe
        115                 120                 125
Gly Glu Asn Gln Met Ile Gln Leu Gly Ile Lys Phe Tyr Asn His Tyr
    130                 135                 140
Lys Ser Leu Ala Arg Asn Ala Val Pro Phe Val Arg Cys Ser Gly Ser
145                 150                 155                 160
Asp Arg Val Ile Ala Ser Gly Arg Leu Phe Ile Glu Gly Phe Gln Ser
                165                 170                 175
Ala Lys Val Leu Asp Pro His Ser Asp Lys His Asp Ala Pro Pro Thr
            180                 185                 190
Ile Asn Val Ile Ile Glu Glu Gly Pro Ser Tyr Asn Asn Thr Leu Asp
        195                 200                 205
Thr Gly Ser Cys Pro Val Phe Glu Asp Ser Ser Gly Gly His Asp Ala
    210                 215                 220
Gln Glu Lys Phe Ala Lys Gln Phe Ala Pro Ala Ile Leu Glu Lys Ile
225                 230                 235                 240
Lys Asp His Leu Pro Gly Val Asp Leu Ala Val Ser Asp Val Pro Tyr
                245                 250                 255
Leu Met Asp Leu Cys Pro Phe Glu Thr Leu Ala Arg Asn His Thr Asp
            260                 265                 270
Thr Leu Ser Pro Phe Cys Ala Leu Ser Thr Gln Glu Glu Trp Gln Ala
        275                 280                 285
Tyr Asp Tyr Tyr Gln Ser Leu Gly Lys Tyr Tyr Gly Asn Gly Gly Gly
    290                 295                 300
Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val Asn Glu Leu Ile
305                 310                 315                 320
Ala Arg Met Thr His Ser Pro Val Gln Asp Tyr Thr Thr Val Asn His
                325                 330                 335
Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr
            340                 345                 350
Ala Asp Phe Ser His Asp Asn Thr Met Thr Ser Ile Phe Ala Ala Leu
        355                 360                 365
Gly Leu Tyr Asn Gly Thr Ala Lys Leu Ser Thr Thr Glu Ile Lys Ser
    370                 375                 380
Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ala Trp Thr Val Pro Phe Gly
385                 390                 395                 400
Gly Arg Ala Tyr Ile Glu Met Met Gln Cys Asp Asp Ser Asp Glu Pro
                405                 410                 415
Val Val Arg Val Leu Val Asn Asp Arg Val Val Pro Leu His Gly Cys
            420                 425                 430
Glu Val Asp Ser Leu Gly Arg Cys Lys Arg Asp Asp Phe Val Arg Gly
        435                 440                 445
Leu Ser Phe Ala Arg Gln Gly Gly Asn Trp Glu Gly Cys Tyr Ala Ala
    450                 455                 460
Ser Glu
465
 
           
           
             
               1571 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               CDS 
                join(43..89, 147..1494)
 
             
             
               intron 
                90..146
 
             
             
               misc_feature 
                894..999 
                /note= “Position of PCR fragment”
 
             
              32
AGATTCAACG ACGGAGGAAT CGCAACCCTA ATTGTCGGTA TC ATG GTG ACT CTG        54
                                               Met Val Thr Leu
                                                 1
ACT TTC CTG CTT TCG GCG GCG TAT CTG CTT TCT  GG  GTGAGTGGCT           99
Thr Phe Leu Leu Ser Ala Ala Tyr Leu Leu Ser  Gly
  5                  10                  15
TGGATCTATT GCTCGGATAG GGCTGTGGTG CTGATTCTGA AACGGAG T AGA GTG        153
                                                      Arg Val
TCT GCG GCA CCT AGT TCT GCT GGC TCC AAG TCC TGC GAT ACG GTA GAC      201
Ser Ala Ala Pro Ser Ser Ala Gly Ser Lys Ser Cys Asp Thr Val Asp
     20                  25                  30
CTC GGG TAC CAG TGC TCC CCT GCG ACT TCT CAT CTA TGG GGC CAG TAC      249
Leu Gly Tyr Gln Cys Ser Pro Ala Thr Ser His Leu Trp Gly Gln Tyr
 35                  40                  45                  50
TCG CCA TTC TTT TCG CTC GAG GAC GAG CTG TCC GTG TCG AGT AAG CTT      297
Ser Pro Phe Phe Ser Leu Glu Asp Glu Leu Ser Val Ser Ser Lys Leu
                 55                  60                  65
CCC AAG GAT TGC CGG ATC ACC TTG GTA CAG GTG CTA TCG CGC CAT GGA      345
Pro Lys Asp Cys Arg Ile Thr Leu Val Gln Val Leu Ser Arg His Gly
             70                  75                  80
GCG CGG TAC CCA ACC AGC TCC AAG AGC AAA AAG TAT AAG AAG CTT GTG      393
Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr Lys Lys Leu Val
         85                  90                  95
ACG GCG ATC CAG GCC AAT GCC ACC GAC TTC AAG GGC AAG TTT GCC TTT      441
Thr Ala Ile Gln Ala Asn Ala Thr Asp Phe Lys Gly Lys Phe Ala Phe
    100                 105                 110
TTG AAG ACG TAC AAC TAT ACT CTG GGT GCG GAT GAC CTC ACT CCC TTT      489
Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu Thr Pro Phe
115                 120                 125                 130
GGG GAG CAG CAG CTG GTG AAC TCG GGC ATC AAG TTC TAC CAG AGG TAC      537
Gly Glu Gln Gln Leu Val Asn Ser Gly Ile Lys Phe Tyr Gln Arg Tyr
                135                 140                 145
AAG GCT CTG GCG CGC AGT GTG GTG CCG TTT ATT CGC GCC TCA GGC TCG      585
Lys Ala Leu Ala Arg Ser Val Val Pro Phe Ile Arg Ala Ser Gly Ser
            150                 155                 160
GAC CGG GTT ATT GCT TCG GGA GAG AAG TTC ATC GAG GGG TTC CAG CAG      633
Asp Arg Val Ile Ala Ser Gly Glu Lys Phe Ile Glu Gly Phe Gln Gln
        165                 170                 175
GCG AAG CTG GCT GAT CCT GGC GCG ACG AAC CGC GCC GCT CCG GCG ATT      681
Ala Lys Leu Ala Asp Pro Gly Ala Thr Asn Arg Ala Ala Pro Ala Ile
    180                 185                 190
AGT GTG ATT ATT CCG GAG AGC GAG ACG TTC AAC AAT ACG CTG GAC CAC      729
Ser Val Ile Ile Pro Glu Ser Glu Thr Phe Asn Asn Thr Leu Asp His
195                 200                 205                 210
GGT GTG TGC ACG AAG TTT GAG GCG AGT CAG CTG GGA GAT GAG GTT GCG      777
Gly Val Cys Thr Lys Phe Glu Ala Ser Gln Leu Gly Asp Glu Val Ala
                215                 220                 225
GCC AAT TTC ACT GCG CTC TTT GCA CCC GAC ATC CGA GCT CGC GCC GAG      825
Ala Asn Phe Thr Ala Leu Phe Ala Pro Asp Ile Arg Ala Arg Ala Glu
            230                 235                 240
AAG CAT CTT CCT GGC GTG ACG CTG ACA GAC GAG GAC GTT GTC AGT CTA      873
Lys His Leu Pro Gly Val Thr Leu Thr Asp Glu Asp Val Val Ser Leu
        245                 250                 255
ATG GAC ATG TGT TCG TTT GAT ACG GTA GCG CGC ACC AGC GAC GCA AGT      921
Met Asp Met Cys Ser Phe Asp Thr Val Ala Arg Thr Ser Asp Ala Ser
    260                 265                 270
CAG CTG TCA CCG TTC TGT CAA CTC TTC ACT CAC AAT GAG TGG AAG AAG      969
Gln Leu Ser Pro Phe Cys Gln Leu Phe Thr His Asn Glu Trp Lys Lys
275                 280                 285                 290
TAC AAC TAC CTT CAG TCC TTG GGC AAG TAC TAC GGC TAC GGC GCA GGC     1017
Tyr Asn Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly
                295                 300                 305
AAC CCT CTG GGA CCG GCT CAG GGG ATA GGG TTC ACC AAC GAG CTG ATT     1065
Asn Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr Asn Glu Leu Ile
            310                 315                 320
GCC CGG TTG ACT CGT TCG CCA GTG CAG GAC CAC ACC AGC ACT AAC TCG     1113
Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr Ser Thr Asn Ser
        325                 330                 335
ACT CTA GTC TCC AAC CCG GCC ACC TTC CCG TTG AAC GCT ACC ATG TAC     1161
Thr Leu Val Ser Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr Met Tyr
    340                 345                 350
GTC GAC TTT TCA CAC GAC AAC AGC ATG GTT TCC ATC TTC TTT GCA TTG     1209
Val Asp Phe Ser His Asp Asn Ser Met Val Ser Ile Phe Phe Ala Leu
355                 360                 365                 370
GGC CTG TAC AAC GGC ACT GAA CCC TTG TCC CGG ACC TCG GTG GAA AGC     1257
Gly Leu Tyr Asn Gly Thr Glu Pro Leu Ser Arg Thr Ser Val Glu Ser
                375                 380                 385
GCC AAG GAA TTG GAT GGG TAT TCT GCA TCC TGG GTG GTG CCT TTC GGC     1305
Ala Lys Glu Leu Asp Gly Tyr Ser Ala Ser Trp Val Val Pro Phe Gly
            390                 395                 400
GCG CGA GCC TAC TTC GAG ACG ATG CAA TGC AAG TCG GAA AAG GAG CCT     1353
Ala Arg Ala Tyr Phe Glu Thr Met Gln Cys Lys Ser Glu Lys Glu Pro
        405                 410                 415
CTT GTT CGC GCT TTG ATT AAT GAC CGG GTT GTG CCA CTG CAT GGC TGC     1401
Leu Val Arg Ala Leu Ile Asn Asp Arg Val Val Pro Leu His Gly Cys
    420                 425                 430
GAT GTG GAC AAG CTG GGG CGA TGC AAG CTG AAT GAC TTT GTC AAG GGA     1449
Asp Val Asp Lys Leu Gly Arg Cys Lys Leu Asn Asp Phe Val Lys Gly
435                 440                 445                 450
TTG AGT TGG GCC AGA TCT GGG GGC AAC TGG GGA GAG TGC TTT AGT         1494
Leu Ser Trp Ala Arg Ser Gly Gly Asn Trp Gly Glu Cys Phe Ser
                455                 460                 465
TGAGATGTCA TTGTTATGCT ATACTCCAAT AGACCGTTGC TTAGCCATTC ACTTCACTTT   1554
GCTCGAACCG CCTGCCG                                                  1571
 
           
           
             
               465 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               misc_feature 
                104 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                119 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                205 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                228 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                337 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                374 
                /note=“potential N-glycosylation site”
 
             
              33
Met Val Thr Leu Thr Phe Leu Leu Ser Ala Ala Tyr Leu Leu Ser Gly
  1               5                  10                  15
Arg Val Ser Ala Ala Pro Ser Ser Ala Gly Ser Lys Ser Cys Asp Thr
             20                  25                  30
Val Asp Leu Gly Tyr Gln Cys Ser Pro Ala Thr Ser His Leu Trp Gly
         35                  40                  45
Gln Tyr Ser Pro Phe Phe Ser Leu Glu Asp Glu Leu Ser Val Ser Ser
     50                  55                  60
Lys Leu Pro Lys Asp Cys Arg Ile Thr Leu Val Gln Val Leu Ser Arg
 65                  70                  75                  80
His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr Lys Lys
                 85                  90                  95
Leu Val Thr Ala Ile Gln Ala Asn Ala Thr Asp Phe Lys Gly Lys Phe
            100                 105                 110
Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu Thr
        115                 120                 125
Pro Phe Gly Glu Gln Gln Leu Val Asn Ser Gly Ile Lys Phe Tyr Gln
    130                 135                 140
Arg Tyr Lys Ala Leu Ala Arg Ser Val Val Pro Phe Ile Arg Ala Ser
145                 150                 155                 160
Gly Ser Asp Arg Val Ile Ala Ser Gly Glu Lys Phe Ile Glu Gly Phe
                165                 170                 175
Gln Gln Ala Lys Leu Ala Asp Pro Gly Ala Thr Asn Arg Ala Ala Pro
            180                 185                 190
Ala Ile Ser Val Ile Ile Pro Glu Ser Glu Thr Phe Asn Asn Thr Leu
        195                 200                 205
Asp His Gly Val Cys Thr Lys Phe Glu Ala Ser Gln Leu Gly Asp Glu
    210                 215                 220
Val Ala Ala Asn Phe Thr Ala Leu Phe Ala Pro Asp Ile Arg Ala Arg
225                 230                 235                 240
Ala Glu Lys His Leu Pro Gly Val Thr Leu Thr Asp Glu Asp Val Val
                245                 250                 255
Ser Leu Met Asp Met Cys Ser Phe Asp Thr Val Ala Arg Thr Ser Asp
            260                 265                 270
Ala Ser Gln Leu Ser Pro Phe Cys Gln Leu Phe Thr His Asn Glu Trp
        275                 280                 285
Lys Lys Tyr Asn Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly Tyr Gly
    290                 295                 300
Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr Asn Glu
305                 310                 315                 320
Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr Ser Thr
                325                 330                 335
Asn Ser Thr Leu Val Ser Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr
            340                 345                 350
Met Tyr Val Asp Phe Ser His Asp Asn Ser Met Val Ser Ile Phe Phe
        355                 360                 365
Ala Leu Gly Leu Tyr Asn Gly Thr Glu Pro Leu Ser Arg Thr Ser Val
    370                 375                 380
Glu Ser Ala Lys Glu Leu Asp Gly Tyr Ser Ala Ser Trp Val Val Pro
385                 390                 395                 400
Phe Gly Ala Arg Ala Tyr Phe Glu Thr Met Gln Cys Lys Ser Glu Lys
                405                 410                 415
Glu Pro Leu Val Arg Ala Leu Ile Asn Asp Arg Val Val Pro Leu His
            420                 425                 430
Gly Cys Asp Val Asp Lys Leu Gly Arg Cys Lys Leu Asn Asp Phe Val
        435                 440                 445
Lys Gly Leu Ser Trp Ala Arg Ser Gly Gly Asn Trp Gly Glu Cys Phe
    450                 455                 460
Ser
465
 
           
           
             
               1567 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               CDS 
                join(78..124, 177..1527)
 
             
             
               intron 
                125..176
 
             
             
               misc_feature 
                930..1035 
                /note= “Position of PCR fragment”
 
             
             
               misc_feature 
                1215..1394 
                /note= “Position of PCR fragment”
 
             
              34
ACGTCCCAGG TCGGGGACTA CATCCGCTAT GTGGTCCTCT ACTTCGTCGG AAGAATATAC     60
TGTCTCTTGT GGCTACC ATG GGG GTT TTC GTC GTT CTA TTA TCT ATC GCG       110
                   Met Gly Val Phe Val Val Leu Leu Ser Ile Ala
                     1               5                  10
ACT CTG TTC GGC  AG  GTATGTGCAC CGCTCTAGGT TCAACTCGCC TGGTAACTGA     164
Thr Leu Phe Gly  Ser
             15
CAAACAGCAC AG C ACA TCG GGC ACT GCG CTG GGC CCC CGT GGA AAT CAC      213
                Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn His
                             20                  25
AGC GAC TGC ACC TCA GTC GAC CGG GGG TAT CAA TGC TTC CCT GAG CTC      261
Ser Asp Cys Thr Ser Val Asp Arg Gly Tyr Gln Cys Phe Pro Glu Leu
     30                  35                  40
TCC CAT AAA TGG GGT CTC TAC GCG CCC TAT TTC TCC CTC CAG GAT GAA      309
Ser His Lys Trp Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln Asp Glu
 45                  50                  55                  60
TCT CCG TTT CCT CTG GAC GTC CCG GAT GAC TGC CAC ATC ACC TTT GTG      357
Ser Pro Phe Pro Leu Asp Val Pro Asp Asp Cys His Ile Thr Phe Val
                 65                  70                  75
CAG GTG CTG GCC CGA CAT GGA GCG CGG TCT CCA ACC GAT AGC AAG ACA      405
Gln Val Leu Ala Arg His Gly Ala Arg Ser Pro Thr Asp Ser Lys Thr
             80                  85                  90
AAG GCG TAT GCC GCG ACT ATT GCA GCC ATC CAG AAG AAT GCC ACC GCG      453
Lys Ala Tyr Ala Ala Thr Ile Ala Ala Ile Gln Lys Asn Ala Thr Ala
         95                 100                 105
TTG CCG GGC AAA TAC GCC TTC CTG AAG TCG TAC AAT TAC TCC ATG GGC      501
Leu Pro Gly Lys Tyr Ala Phe Leu Lys Ser Tyr Asn Tyr Ser Met Gly
    110                 115                 120
TCC GAG AAC CTG AAC CCC TTC GGG CGG AAC CAA CTG CAA GAT CTG GGC      549
Ser Glu Asn Leu Asn Pro Phe Gly Arg Asn Gln Leu Gln Asp Leu Gly
125                 130                 135                 140
GCC CAG TTC TAC CGT CGC TAC GAC ACC CTC ACC CGG CAC ATC AAC CCT      597
Ala Gln Phe Tyr Arg Arg Tyr Asp Thr Leu Thr Arg His Ile Asn Pro
                145                 150                 155
TTC GTC CGG GCC GCG GAT TCC TCC CGC GTC CAC GAA TCA GCC GAG AAG      645
Phe Val Arg Ala Ala Asp Ser Ser Arg Val His Glu Ser Ala Glu Lys
            160                 165                 170
TTC GTC GAG GGC TTC CAA AAC GCC CGC CAA GGC GAT CCT CAC GCC AAC      693
Phe Val Glu Gly Phe Gln Asn Ala Arg Gln Gly Asp Pro His Ala Asn
        175                 180                 185
CCT CAC CAG CCG TCG CCG CGC GTG GAT GTA GTC ATC CCC GAA GGC ACC      741
Pro His Gln Pro Ser Pro Arg Val Asp Val Val Ile Pro Glu Gly Thr
    190                 195                 200
GCC TAC AAC AAC ACG CTC GAG CAC AGC ATC TGC ACC GCC TTC GAG GCC      789
Ala Tyr Asn Asn Thr Leu Glu His Ser Ile Cys Thr Ala Phe Glu Ala
205                 210                 215                 220
AGC ACC GTC GGC GAC GCC GCG GCA GAC AAC TTC ACT GCC GTG TTC GCG      837
Ser Thr Val Gly Asp Ala Ala Ala Asp Asn Phe Thr Ala Val Phe Ala
                225                 230                 235
CCG GCG ATC GCC AAG CGT CTG GAG GCC GAT CTG CCC GGC GTG CAG CTG      885
Pro Ala Ile Ala Lys Arg Leu Glu Ala Asp Leu Pro Gly Val Gln Leu
            240                 245                 250
TCC GCC GAC GAC GTG GTC AAT CTG ATG GCC ATG TGT CCG TTC GAG ACG      933
Ser Ala Asp Asp Val Val Asn Leu Met Ala Met Cys Pro Phe Glu Thr
        255                 260                 265
GTC AGC CTG ACC GAC GAC GCG CAC ACG CTG TCG CCG TTC TGC GAC CTC      981
Val Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp Leu
    270                 275                 280
TTC ACC GCC GCC GAG TGG ACG CAG TAC AAC TAC CTG CTC TCG CTG GAC     1029
Phe Thr Ala Ala Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu Asp
285                 290                 295                 300
AAG TAC TAC GGC TAC GGC GGC GGC AAT CCG CTG GGC CCC GTG CAG GGC     1077
Lys Tyr Tyr Gly Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val Gln Gly
                305                 310                 315
GTG GGC TGG GCG AAC GAG CTG ATC GCG CGG CTG ACG CGC TCC CCC GTC     1125
Val Gly Trp Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val
            320                 325                 330
CAC GAC CAC ACC TGC GTC AAC AAC ACC CTC GAC GCC AAC CCG GCC ACC     1173
His Asp His Thr Cys Val Asn Asn Thr Leu Asp Ala Asn Pro Ala Thr
        335                 340                 345
TTC CCG CTG AAC GCC ACC CTC TAC GCG GAC TTT TCG CAC GAC AGT AAC     1221
Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Ser Asn
    350                 355                 360
CTG GTG TCG ATC TTC TGG GCG CTG GGT CTG TAC AAC GGC ACC AAG CCC     1269
Leu Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro
365                 370                 375                 380
CTG TCG CAG ACC ACC GTG GAG GAT ATC ACC CGG ACG GAC GGG TAC GCG     1317
Leu Ser Gln Thr Thr Val Glu Asp Ile Thr Arg Thr Asp Gly Tyr Ala
                385                 390                 395
GCC GCC TGG ACG GTG CCG TTT GCC GCC CGC GCC TAC ATC GAG ATG ATG     1365
Ala Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Ile Glu Met Met
            400                 405                 410
CAG TGT CGC GCG GAG AAG CAG CCG CTG GTG CGC GTG CTG GTC AAC GAC     1413
Gln Cys Arg Ala Glu Lys Gln Pro Leu Val Arg Val Leu Val Asn Asp
        415                 420                 425
CGT GTC ATG CCG CTG CAC GGC TGC GCG GTG GAT AAT CTG GGC AGG TGT     1461
Arg Val Met Pro Leu His Gly Cys Ala Val Asp Asn Leu Gly Arg Cys
    430                 435                 440
AAA CGG GAC GAC TTT GTG GAG GGA CTG AGC TTT GCG CGG GCA GGA GGG     1509
Lys Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ala Gly Gly
445                 450                 455                 460
AAC TGG GCC GAG TGT TTC TGATGTACAT GCTGTAGTTA GCTTTGAGTC            1557
Asn Trp Ala Glu Cys Phe
                465
CTGAGGTACC                                                          1567
 
           
           
             
               466 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               misc_feature 
                27 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                120 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                207 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                230 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                352 
                /note=“potential N-glycosylation site”
 
             
             
               misc_feature 
                376 
                /note=“potential N-glycosylation site”
 
             
              35
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
  1               5                  10                  15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn His Ser Asp Cys Thr
             20                  25                  30
Ser Val Asp Arg Gly Tyr Gln Cys Phe Pro Glu Leu Ser His Lys Trp
         35                  40                  45
Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln Asp Glu Ser Pro Phe Pro
     50                  55                  60
Leu Asp Val Pro Asp Asp Cys His Ile Thr Phe Val Gln Val Leu Ala
 65                  70                  75                  80
Arg His Gly Ala Arg Ser Pro Thr Asp Ser Lys Thr Lys Ala Tyr Ala
                 85                  90                  95
Ala Thr Ile Ala Ala Ile Gln Lys Asn Ala Thr Ala Leu Pro Gly Lys
            100                 105                 110
Tyr Ala Phe Leu Lys Ser Tyr Asn Tyr Ser Met Gly Ser Glu Asn Leu
        115                 120                 125
Asn Pro Phe Gly Arg Asn Gln Leu Gln Asp Leu Gly Ala Gln Phe Tyr
    130                 135                 140
Arg Arg Tyr Asp Thr Leu Thr Arg His Ile Asn Pro Phe Val Arg Ala
145                 150                 155                 160
Ala Asp Ser Ser Arg Val His Glu Ser Ala Glu Lys Phe Val Glu Gly
                165                 170                 175
Phe Gln Asn Ala Arg Gln Gly Asp Pro His Ala Asn Pro His Gln Pro
            180                 185                 190
Ser Pro Arg Val Asp Val Val Ile Pro Glu Gly Thr Ala Tyr Asn Asn
        195                 200                 205
Thr Leu Glu His Ser Ile Cys Thr Ala Phe Glu Ala Ser Thr Val Gly
    210                 215                 220
Asp Ala Ala Ala Asp Asn Phe Thr Ala Val Phe Ala Pro Ala Ile Ala
225                 230                 235                 240
Lys Arg Leu Glu Ala Asp Leu Pro Gly Val Gln Leu Ser Ala Asp Asp
                245                 250                 255
Val Val Asn Leu Met Ala Met Cys Pro Phe Glu Thr Val Ser Leu Thr
            260                 265                 270
Asp Asp Ala His Thr Leu Ser Pro Phe Cys Asp Leu Phe Thr Ala Ala
        275                 280                 285
Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser Leu Asp Lys Tyr Tyr Gly
    290                 295                 300
Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val Gln Gly Val Gly Trp Ala
305                 310                 315                 320
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val His Asp His Thr
                325                 330                 335
Cys Val Asn Asn Thr Leu Asp Ala Asn Pro Ala Thr Phe Pro Leu Asn
            340                 345                 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Ser Asn Leu Val Ser Ile
        355                 360                 365
Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Gln Thr
    370                 375                 380
Thr Val Glu Asp Ile Thr Arg Thr Asp Gly Tyr Ala Ala Ala Trp Thr
385                 390                 395                 400
Val Pro Phe Ala Ala Arg Ala Tyr Ile Glu Met Met Gln Cys Arg Ala
                405                 410                 415
Glu Lys Gln Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Met Pro
            420                 425                 430
Leu His Gly Cys Ala Val Asp Asn Leu Gly Arg Cys Lys Arg Asp Asp
        435                 440                 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ala Gly Gly Asn Trp Ala Glu
    450                 455                 460
Cys Phe
465