Regulatable growth of filamentous fungi

---

No:

6936449 -

Application no:

10100252 -

Filed date:

2002-03-14 -

Issue date:

2005-08-30

Kind:

B2

Claims:

9

Drawing sheets:

14

Abstract:

The present invention generally relates to hyphal growth in fungi and in particular describes the modulation of genes associated with hyphal growth in filamentous fungi. The present invention provides methods and systems for the production of proteins and/or chemicals from filamentous fungi which comprise modulation of genes associated with hyphal growth. Specifically, the present invention is directed to a full length cotA gene, its gene product and methods of use.

US Classes:

Inventors:

Agents:

Assignees:

Claims:

What is claimed is:

1. An isolated cotA protein comprising serine/threonine kinase activity and an amino acid sequence having at least 95% identity to the amino acid sequence disclosed in SEQ ID NO: 2, wherein the cotA polypeptide is from Humicola, Aspergillus or Trichoderma and a substitution of an amino acid of SEQ ID NO: 2 is a conservative substitution.

2. The protein of claim 1 having the amino acid sequence disclosed in SEQ ID NO: 2.

3. An isolated cotA protein comprising serine/threonine kinase activity and an amino acid sequence having at least 95% identity to the amino acid sequence disclosed in SEQ ID NO: 4, wherein the cotA polypeptide is from Humicola, Aspergillus or Trichoderma and a substitution of an amino acid of SEQ ID NO: 4 is a conservative substitution.

4. The protein of claim 3 having the amino acid sequence disclosed in SEQ ID NO: 4.

5. An isolated cotA protein comprising serine/threonine kinase activity and an amino acid sequence having at least 95% identity to the amino acid sequence disclosed in SEQ ID NO: 6, wherein the cotA polypeptide is from Humicola, Aspergillus or Trichoderma and a substitution of an amino acid SEQ ID NO: 6 is a conservative substitution.

6. The protein of claim 5 having the amino acid sequence disclosed in SEQ ID NO: 6.

7. An isolated mutant cotA polypeptide having serine/threonine kinase activity comprising a mutation in a cotA polypeptide, wherein said cotA polypeptide is from Humicola, Aspergillus or Trichoderma and has at least 95% sequence identity with SEQ ID NOs: 2, 4 or 6 wherein a substitution of an amino acid of SEQ ID NOs: 2, 4 or 6 which comprises the at least 95% sequence identity is a conservative substitution, and wherein the mutation corresponds to the histidine to arginine substitution found at position 252 of the N. crassa cot-1 variant.

8. The isolated mutant cotA polypeptide of claim 7, wherein the mutant is derived from any one of the cotA polypeptides presented as SEQ ID NOs: 2, 4 or 6.

9. An isolated full-length cotA polypeptide having serine/threonine kinase activity comprising an amino acid sequence of SEQ ID NOs: 2, 4 or 6.

Text:

BACKGROUND OF THE INVENTION

Growth morphology is an important factor affecting fermentation of filamentous fungi during production of proteins and fine chemicals. cot-1 of Neurospora crassa is a colonial temperature sensitive mutation that has been described in detail Steele, et al., Arch. Microbiol. 113:43 (1977) and Collinge, et al., Trans. Br. Mycol. Soc. 71:102 (1978)). Germination and growth of the mutant is normal at 26.degree. C., but a shift to 37.degree. C. causes the cessation of hyphal tip extension, and emergence of lateral branches at an abnormally high frequency to give hyperbranching germlings. An increase in the frequency of septation is also seen. Sequence analysis indicated the gene product belongs to the family of serine/threonine protein kinases (Yarden, et al., EMBO J. 11:2159 (1992). These kinases act in signal transduction pathways, but how cot-1 is integrated into the pathway(s) controlling hyphal growth polarity has yet to be elucidated. The specific mutation that causes the temperature sensitivity in N. crassa cot-1 has been found to be a histidine to arginine substitution (Gorovits, et al., Fungal Genetics and Biol. 27:264 (1999).

There remains a need in the art for genes that control growth morphology in filamentous fungal cells, like Trichoderma and Aspergillus, that are used as a source of recombinant proteins in an industrial setting and to enhance the production of proteins and fine chemicals. This invention meets this need as well as others.

SUMMARY OF THE INVENTION

One embodiment of this invention provides for an isolated polynucleotide selected from the group consisting of a nucleic acid sequence that encodes or is complementary to a sequence that encodes a cotA polypeptide having at least 85% sequence identity to the amino acid sequence presented in any one of FIGS. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); a nucleic acid sequence that encodes or is complementary to a sequence that encodes a cotA polypeptide having at least 90% sequence identity to the amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); a nucleic acid sequence that encodes or is complementary to a sequence that encodes a cotA polypeptide having at least 95% sequence identity to the amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); a nucleic acid sequence that encodes or is complementary to a sequence that encodes a cotA polypeptide having the amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); the nucleic acid sequence presented as any one of SEQ ID NOs:1, 3 or 5 (FIG. 1, 3 or 5, respectively) a portion greater than 200 bp thereof, or the complement thereof, and a nucleic acid sequence that hybridizes, under high stringency conditions to the sequence presented as any one of SEQ ID NOs:1, 3 or 5, or the complement or a fragment thereof, wherein said isolated polynucleotide, when induced in a fungal cell, causes said cell to grow more slowly.

In a first aspect of this embodiment, the % identity is calculated using the CLUSTAL-W program in MacVector version 6.5, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

In a second aspect of this embodiment, hybridization is conducted at 42.degree. C. in 50% formamide, 6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured carrier DNA followed by washing two times in 2.times.SSPE and 0.5% SDS at room temperature and two additional times in 0.1.times.SSPE and 0.5% SDS at 42.degree. C. In yet another embodiment, the isolated polynucleotide is an RNA molecule.

In a third aspect of this embodiment, the isolated polynucleotide is operably linked to a regulatable promoter. In a preferred aspect of this embodiment, the promoter is induced by maltose in the fungal cell environment. In another preferred aspect of this embodiment, the polynucleotide is in the antisense orientation.

In a fourth aspect of this embodiment, the polynucleotide is SEQ ID NO:1.

In a fifth aspect of this embodiment, the polynucleotide is SEQ ID NO:3.

In a sixth aspect of this embodiment, the polynucleotide is SEQ ID NO:5.

In second embodiment of this invention, a recombinant filamentous fungal host cell comprising a cotA polynucleotide is provided. In one aspect of this embodiment, the fungal host cell is a member of Aspergillus spp. In another aspect of this embodiment, the cell is an Aspergillus niger fungal cell. In yet another aspect of this invention, the cell is a member of Trichoderma, more preferred is T. reesei. In further aspect of this embodiment, the recombinant fungal host cell is transformed with the vector comprising any one of SEQ ID NOs:1, 3 or 5 operably linked to a regulatable promoter. In a particularly preferred aspect of this embodiment, the vector integrates into the wild-type cotA gene. In another aspect of this embodiment, the vector integrates ectopically. In an aspect of this embodiment, the polynucleotide integrates in the antisense orientation.

In a third embodiment of this invention, a substantially purified cotA polypeptide with the biological activity of a serine/threonine kinase is provided. The biologically active polypeptide comprises a sequence selected from the group consisting of an amino acid sequence having at least 85% sequence identity to the amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); an amino acid sequence having at least 90% sequence identity to the amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); an amino acid sequence having at least 95% sequence identity to the amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); the amino acid sequence presented as any one of SEQ ID NOs:2, 4 or 6, a substantially purified biologically active fragment of the amino acid sequence presented as any one of SEQ ID NO:2, 4 or 6, and a substantially purified full length protein comprising the amino acid sequence encoded by either of SEQ ID NOs:13 or 14.

In a fourth embodiment of this invention, a purified antibody that specifically binds to a cotA polypeptide is provided. In one aspect of this embodiment, a polynucleotide is provided that encodes a cotA polypeptide that specifically binds to an antibody.

In a fifth embodiment, a method is provided for the detection of a polynucleotide that encodes a filamentous fungal cotA in a biological sample. The method includes, but is not limited to, the following steps: (a) hybridizing, under moderate stringency, to a nucleic acid material of said biological sample, a polynucleotide fragment derived from any one of the sequences identified as SEQ ID NOs:1, 3 or 5, the fragment having a length of between about 15 and 250 nucleotides, thereby forming a hybridization complex; and (b) detecting said hybridization complex; wherein the presence of said hybridization complex correlates with the presence of a polynucleotide encoding the cotA protein in said biological sample. In a first aspect of this embodiment, the fragment is between 15 and 30 nucleotides in length. In another aspect, the fragment is between 30 and 100 nucleotides in length. In yet another aspect, the fragment is between 100 and 200 nucleotides in length, more preferred is a fragment between 200 and 250 nucleotides. In a final aspect, the fragment is about 241 nucleotides in length. In a second aspect of this embodiment, the biological sample is a filamentous fungal cell lysate. In third aspect of this embodiment, an agonist of cotA protein is identified. The method comprises the steps of (a) transfecting a fungal host cell with a polynucleotide that encodes a cotA protein; (b) inducing the expression of cotA; (c) contacting a test compound with the so induced fungal host cell, (d) measuring the effect of the test compound on the growth of the induced fungal cell; and (e) identifying the test compound as a candidate compound if it modulates the growth of the fungal cell beyond a selected threshold level.

In a final embodiment of this invention, a method of inducing a compact growth morphology of a filamentous fungal host cell is provided. In a preferred aspect of this embodiment, the fungal cell is a member of the Trichoderma genus, most preferred is Trichoderma reesei. In a more preferred aspect of this embodiment, the fungal cell is a member of the Aspergillus genus. In a most preferred aspect, the fungal cell is a A. niger cell. The method comprises the steps of transfecting said fungal host cell with a cotA polynucleotide or a fragment thereof operably linked to an inducible promoter, and exposing the transfected fungal host cell to a compound that induces expression of the cotA polynucleotide. In another preferred aspect of this embodiment, the cotA polynucleotide is as shown in any one of SEQ ID NOs:1, 3, 5, 13 or 14. In a particularly preferred aspect of this embodiment, the cotA polynucleotide is in the antisense orientation. In another particularly preferred aspect, the promoter is inducible by maltose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleic acid sequence of the truncated Aspergillus niger cotA of this invention (SEQ ID NO: 1).

FIG. 2 shows the deduced amino acid sequence of the truncated Aspergillus niger cotA of this invention (SEQ ID NO:2).

FIG. 3 is the nucleic acid sequence of the truncated Aspergillus nidulans cotA of this invention (SEQ ID NO:3).

FIG. 4 shows the deduced amino acid sequence of the truncated Aspergillus nidulans cotA of this invention (SEQ ID NO:4).

FIG. 5 is the nucleic acid sequence for an internal cotA fragment from Trichoderma reesei (SEQ ID NO:5).

FIG. 6 is the deduced amino acid sequence for the internal cotA fragment from Trichoderma reesei (SEQ ID NO:6).

FIG. 7 is a sequence alignment of cotA and related kinases. spo1 is the truncated cotA of Aspergillus niger (SEQ ID NO:17). andcot is the full length cotA from Aspergillus nidulans (SEQ ID NO:4). CT1-NEUCR is the full length cot-1 from Neurospora cresse (SEQ ID NO:18). S707706 is from Colletotrichum trifolii (SEQ ID NO:19). KNQ1_YEASST is from S. cerevisiae (SEQ ID NO:20). DMK_HUMAN is human myotonic dystrophy kinase (SEQ ID NO:21).

FIG. 8A is a schematic of the integration of the expression vector, pSMB5, into the cotA locus of Aspergillus niger. FIG. 8B is a schematic of the locus after transformation.

FIG. 9. Comparison of wt and glaAp-cotA strains on a variety of non-repressing (maltose) and repressing (xylose) carbon sources. Grown until same morphological age then stained with calcoflour. Bands=10 .mu.m. YEPX Yeast Extract, Peptone and Xylose. YEPD Yeast Extract, Peptone and Glucose.

FIGS. 10A and 10B are photographs of Aspergillus niger transfected with cotA in the antisense orientation under the control of the glaA promoter. As can be seen, a slowed growth phenotype is observed when transformed cells are grown in the presence of xylose or maltose (FIGS. 10a and b).

FIG. 11 is the nucleic acid sequence of the full length cotA from Aspergillus niger (SEQ ID NO:13). All introns are underlined. The start codon is in bold type. The functional truncated cotA gene ends at the italicized, underlined codon and is at the beginning of the second intron. The stop codon for the full-length cotA is shown in bold type.

FIG. 12 is the nucleic acid sequence of the full length cotA from Aspergillus nidulans (SEQ ID NO:14).

FIG. 13 is a 269 bp probe from Aspergillus niger (SEQ ID NO:15).

FIG. 14 is the deduced amino acid sequence of the full-length Aspergillus niger cotA of this invention (SEQ ID NO:16).

DETAILED DESCRIPTION OF THE INVENTION

Many proteins and other compounds with industrial or pharmaceutical use, e.g., cellulases, proteases, lipases, xylanases, are produced in filamentous fungal cell cultures. An ongoing problem is that as the fungal cells divide and the culture expands, the number of cells in the culture make the culture viscous. In a continuous culture, oxygen and other nutrients do not mix as readily and are therefore unavailable for all the cells. In a batch culture, nutrients are exhausted at a faster rate as the culture expands. In both cases, the growth of the culture as well as the production of the desired protein reaches a plateau and begins to drop. It has been found that transforming filamentous fungal cells with cotA-encoding nucleic acids under the control of a regulatable promoter causes the transformed cells to reduce the rate of growth when in the presence of a compound that regulates the promoter. Transformation can occur with the cotA-encoding nucleic acid integrating in the cotA locus or ectopically. The reduced growth phenotype is seen in both instances. Without being held to any theory, it is believed that if integration occurs in the cotA locus, expression of wild type cotA is under the control of the heterologous and regulatable promoter and becomes inducible.

Fungal protein synthesis is located at the fungal growing tips. Increasing the number of growing tips by isolating hyperbranching mutants has benefits in fermentation. The compact morphology seen in hyperbranching mutants such as cot-1 would be useful in fungal fermentations where reduced viscosity could allow better fermentation performance. Not to be limited by theory, it is believed that the low viscosity of the fermenation mixture allows for better oxygenation of the media, which in turn enhances cell protein production.

A temperature sensitive cotA mutant may be created in various ways. For example, putting the cotA gene under a temperature sensitive promoter or creating a temperature sensitive cotA mutant in the filamentous fungi cotA homolog similar to the N. crassa cot-1 variant would be especially desirable. In an embodiment the filamentous fungi cotA homolog has been altered to have a substitution corresponding to the histidine to arginine substitution found in the N. crassa cot-1 variant. Thus, a temperature sensitive mutant that produces a hyperbranching phenotype with a compact morphology at a higher temperature is particularly desirable.

In one embodiment the endogenous cotA gene is replaced with a temperature sensitive cotA mutant having a substitution at the histidine residue that corresponds H352 in N. crassa. In one aspect the alteration is a substitution of the histidine to arginine (as found in the temperature sensitive N. crassa cot-1 variant). Thus, once the temperature sensitive cotA mutant has integrated into the host genome by homologous recombination it will be under the regulation of the endogenous cotA control sequences.

The ability of cotA mutant to effect protein secretion may be examined by growing the cotA mutant on petri dishes with starch as the sole carbon source. Manipulation of the expression of the cotA gene product would have utility in increasing heterologous protein secretion.

I. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references are incorporated by reference for all purposes. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

The term "polypeptide" as used herein refers to a compound made up of a single chain of amino acid residues linked by peptide bonds. The term "protein" as used herein may be synonymous with the term "polypeptide" or may refer, in addition, to a complex of two or more polypeptides. A cotA polypeptide includes, but is not limited to, a polypeptide encoded by the cotA polynucleotides of this invention. Specifically, cotA polypeptides or proteins encompass Aspergillus and Trichoderma cotA full length proteins, including, but not limited to, signal or leader sequences, mature proteins and fragments thereof.

As used herein, the term "overexpressing" when referring to the production of a protein in a host cell means that the protein is produced in greater amounts than its production in its naturally occurring environment.

As used herein, the phrase "protein associated with hyphal growth" refers to a protein which is capable of modulating hyphal growth in fungus. Illustrative of such proteins are the cotA proteins disclosed herein.

The term "nucleic acid molecule" includes RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given proteins such as cotA may be produced. The present invention contemplates every possible variant nucleotide sequence, encoding cotA, all of which are possible given the degeneracy of the genetic code. A "heterologous" nucleic acid construct or sequence has a portion of the sequence which is not native to the cell in which it is expressed. Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, microinjection, electroporation, or the like. A "heterologous" nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence/DNA coding sequence combination found in the native cell.

As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

Accordingly, an "expression cassette" or "expression vector" is a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes.

As used herein, the term "selectable marker-encoding nucleotide sequence" refers to a nucleotide sequence which is capable of expression in fungal cells and where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of a corresponding selective agent.

As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. The promoter will generally be appropriate to the host cell in which the target gene is being expressed. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") are necessary to express a given gene or nucleic acid sequence. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

A "regulatable promoter" refers to a promoter that effects its regulatory control over a nucleic acid sequence under specific environmental conditions. For example, an inducible promoter is one that causes expression of the operably linked polynucleotide under certain environmental conditions, for example, blue light inducible promoters (bli-4), and copper metallothionein gene (cmt). In a more specific example, the glucoamylase A promoter (glaAp) induces expression in the presence of maltose.

"Chimeric gene" or "heterologous nucleic acid construct", as defined herein refers to a non-native gene (i.e., one that has been introduced into a host) that may be composed of parts of different genes, including regulatory elements. A chimeric gene construct for transformation of a host cell is typically composed of a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or, in a selectable marker chimeric gene, to a selectable marker gene encoding a protein conferring antibiotic resistance to transformed cells. A typical chimeric gene of the present invention, for transformation into a host cell, includes a transcriptional regulatory region that is constitutive or inducible, a protein coding sequence, and a terminator sequence. A chimeric gene construct may also include a second DNA sequence encoding a signal peptide if secretion of the target protein is desired.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

"Antisense" refers to sequences of nucleic acids that are complementary to the coding mRNA nucleic acid sequence of a gene. A nucleotide sequence linked to a promoter in an "antisense orientation" is linked to the promoter such that an RNA molecule complementary to the coding mRNA of the target gene is produced.

As used herein, the term "gene" means the segment of DNA involved in producing a polypeptide chain, that may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

In general, nucleic acid molecules that encode cotA or an analog or homologue thereof will hybridize, under moderate to high stringency conditions to any one of the sequences provided herein as SEQ ID NO:1, 3, 5, 13 or 14. However, in some cases a cotA-encoding nucleotide sequence is employed that possesses a substantially different codon usage, while the protein encoded by the cotA-encoding nucleotide sequence has the same or substantially the same amino acid sequence as the native protein. For example, the coding sequence may be modified to facilitate faster expression of cotA in a particular prokaryotic or eukaryotic expression system, in accordance with the frequency with which a particular codon is utilized by the host.

A nucleic acid sequence is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (T.sub.m) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about T.sub.m -5.degree. C. (5.degree. below the T.sub.m of the probe); "high stringency" at about 5-10.degree. below the T.sub.m ; "intermediate stringency" at about 10-20.degree. below the T.sub.m of the probe; and "low stringency" at about 20-25.degree. below the T.sub.m. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify sequences having about 80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known in the art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, and in Ausubel, F. M., et al., 1993, expressly incorporated by reference herein). An example of high stringency conditions includes hybridization at about 42.degree. C. in 50% formamide, 5.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured carrier DNA followed by washing two times in 2.times.SSC and 0.5% SDS at room temperature and two additional times in 0.1.times.SSC and 0.5% SDS at 42.degree. C.

The term "% homology" is used interchangeably herein with the term "% identity" herein and refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequence that encodes cotA or the cotA amino acid sequence, when aligned using a sequence alignment program.

For example, as used herein, 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence. Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., the coding sequence for cotA, as described herein.

Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See, also, Altschul, S. F. et al., 1990 and Altschul, S. F. et al., 1997.

Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. [See, Altschul, et al., 1997.]

A preferred alignment of selected sequences in order to determine "% identity" between two or more sequences, is performed using for example, the CLUSTAL-W program in MacVector version 6.5, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

In one exemplary approach, sequence extension of a nucleic acid encoding cotA may be may be carried out using conventional primer extension procedures as described in Sambrook et al., supra, to detect cotA precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA and/or to identify ORFs that encode a full length protein.

A nucleotide sequence encoding a cotA-type polypeptide, e.g., cot1 from Neurospora crassa, can also be used to construct hybridization probes for mapping the gene which encodes a cotA polypeptide and for further genetic analysis. Screening of a cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., 1989). Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

The probes or portions thereof may also be employed in PCR techniques to generate a pool of sequences for identification of closely related cotA sequences. When cotA sequences are intended for use as probes, a particular portion of a cotA encoding sequence, for example a highly conserved portion of the coding sequence may be used.

For example, a cotA nucleotide sequence may be used as a hybridization probe for a cDNA library to isolate genes, for example, those encoding naturally-occurring variants of cotA from other filamentous fungal species, which have a desired level of sequence identity to any one of the cotA nucleotide sequences disclosed in FIG. 1, 3, 5, 11 or 12 (SEQ ID NO:1, 3, 5, 13 or 14, respectively). Exemplary probes have a length of about 20 to about 50 bases but can go as long as 250 bp.

As used herein, "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.

As used herein, the terms "transformed", "stably transformed" or "transgenic" with reference to a cell means the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome that is maintained through two or more generations.

As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).

As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation. It follows that the term "cotA expression" refers to transcription and translation of the cotA gene, the products of which include precursor RNA, mRNA, polypeptide, post-translation processed polypeptide, and derivatives thereof, including cotA homologs from other fungal species. By way of example, assays for cotA expression include examination of fungal colonies when exposed to the appropriate conditions, western blot for cotA protein, as well as northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assays for cotA mRNA.

"Alternative splicing" is a process whereby multiple polypeptide isoforms are generated from a single gene, and involves the splicing together of nonconsecutive exons during the processing of some, but not all, transcripts of the gene. Thus a particular exon may be connected to any one of several alternative exons to form messenger RNAs. The alternatively-spliced mRNAs produce polypeptides ("splice variants") in which some parts are common while other parts are different.

By "host cell" is meant a cell that contains a vector and supports the replication, and/or transcription or transcription and translation (expression) of the expression construct. Host cells for use in the present invention can be prokaryotic cells, such as E. coli, or eukaryotic cells such as filamentous fungal, yeast, plant, insect, amphibian, or mammalian cells. In general, host cells are filamentous fungal cells. Specifically, the present invention find A. nidulans, A. niger and T. reesei cells advantageous.

The terms "isolated" or "purified" as used herein refer to a nucleic acid or polypeptide that is removed from at least one component with which it is naturally associated.

As used herein, the terms "active" and "biologically active" refer to a biological activity associated with a particular protein, such as the enzymatic activity associated with a kinase. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art.

The phrase "slowed growth morphology" means the cells exhibit a more slowly growing phenotype than wild type cells. This is evidenced by a more compact colony appearance on solid growth medium. This morphology may be accompanied by hyphal hyper-branching.

II. Target Organisms

In this invention, the source of the polynucleotides that encode cotA is a filamentous fungus. As well as being the source, in a preferred embodiment, the host cell is also a filamentous fungus cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota. The filamentous fungi are characterized by vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides, with vegetative growth by hyphal elongation and carbon catabolism that is obligately aerobic.

In the present invention, the filamentous fungal parent cell may be a cell of a species of, but not limited to, Aspergillus, Humicola and Trichoderma. In one embodiment, the filamentous fungal parent cell is an Aspergillus niger, or an Aspergillus nidulans cell. In a first aspect, the parent cell is an Aspergillus niger cell. In a second aspect, the parent cell is an Aspergillus nidulans cell. In third aspect, the filamentous fungal parent cell is a Trichoderma reesei cell. In a fourth aspect, the filamentous fungal parent cell is Humicola grisea.

III. Methods of Identifying Novel Sequences

It has been discovered that cotA-encoding polynucleotides share significant identity at the 3' terminus. This region encodes the catalytic region of cotA. Thus, it is expected that cotA homologs from other fungal species may be found through searching fungal genomes for homologous sequences or by degenerate PCR cloning of the conserved region. Open reading frames (ORFs) within a fungal genome are analyzed following full or partial sequencing of the target organism (in this case, fungal) genome and are further analyzed using sequence analysis software, and by determining homology to known sequences in databases (public/private). Sequence searching and comparison techniques are well known and readily available via the World Wide Web.

In a one aspect of this invention, cotA homologs are discovered through degenerate PCR cloning. Useful primers include, but are not limited to, 5'-GA T/C AT T/C AA A/G CCNGA T/C AA-3' (SEQ ID NO:7) and 5'-TCNGGNGC G/T/A AT A/G TA A/G TC-3' (SEQ ID NO:8). Other primers will be apparent to those of skill in the art upon review of the sequences listed in FIG. 7. PCR conditions to optimize hybridization of degenerate primers to genomic DNA and subsequent amplification are well within the purview of those of skill in the art. Such conditions may be found in Ausubel and/or Sambrook.

Although genomic sequences can be discovered directly through PCR cloning, in a preferred method, a probe consisting of a partial polynucleotide sequence is generated via PCR cloning. Typically this probe is less than 1000 base pairs, more preferably less than 750 base pairs, even more preferably less than 500 bp and most preferably less than 250 base pairs. In a particularly preferred embodiment, the probe is from about 241 to 269 base pairs in length (FIG. 13 (SEQ ID NO:15) and corresponds approximately to residues 1144-1405 of the N. crassa cot-1 sequence.

IV. cotA Polypeptides and Nucleic Acid Molecules Encoding cotA.

A. cotA Nucleic Acids

The nucleic acid molecules of the present invention include a coding sequence for A. niger cotA presented herein as SEQ. ID. NO:13 or A. nidulans presented herein as SEQ. ID. NO: 14, naturally occurring allelic and splice variants, nucleic acid fragments, and biologically active (functional) derivatives thereof, such as, amino acid sequence variants of the native molecule and sequences which encode fusion proteins.

The nucleic acid molecules of the present invention include a partial native coding sequence for cotA presented herein as SEQ. ID. NO:1, and homologues thereof in other species (for example, SEQ ID NO:3 (cotA from A. nidulans) and SEQ ID NO:5 (cotA from T. reesei)), naturally occurring allelic and splice variants, nucleic acid fragments, and biologically active (functional) derivatives thereof, such as, amino acid sequence variants of the native molecule and sequences which encode fusion proteins. The sequences, both full length and partial sequences, are collectively referred to herein as "cotA-encoding nucleic acid sequences".

A cotA nucleic acid sequence of this invention may be a DNA or RNA sequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized in whole or in part. The DNA may be double-stranded or single-stranded and if single-stranded may be the coding strand or the non-coding (antisense, complementary) strand. The nucleic acid sequence may be cloned, for example, by isolating genomic DNA from an appropriate source, and amplifying and cloning the sequence of interest using a polymerase chain reaction (PCR). Alternatively, nucleic acid sequences may be synthesized, either completely or in part, especially where it is desirable to provide host-preferred sequences for optimal expression. Thus, all or a portion of the desired structural gene (that portion of the gene which encodes a polypeptide or protein) may be synthesized using codons preferred by a selected host, e.g., Aspergillus niger, Aspergillus nidulans or Trichoderma reesei.

Due to the inherent degeneracy of the genetic code, nucleic acid sequences other than the native form that encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and/or express cotA-encoding nucleic acid sequences. Thus, for a given cotA-encoding nucleic acid sequence, it is appreciated that, as a result of the degeneracy of the genetic code, a number of coding sequences can be produced that encode a protein having the same amino acid sequence. For example, the triplet CGT encodes the amino acid arginine. Arginine is alternatively encoded by CGA, CGC, CGG, AGA, and AGG. Therefore it is appreciated that such substitutions in the coding region fall within the nucleic acid sequence variants covered by the present invention. Any and all of these sequence variants can be utilized in the same way as described herein for the native form of a cotA-encoding nucleic acid sequence.

A "variant" cotA-encoding nucleic acid sequence may encode a "variant" cotA amino acid sequence which is altered by one or more amino acids from the native polypeptide sequence, both of which are included within the scope of the invention. Similarly, the term "modified form of", relative to cotA, means a derivative or variant form of the native cotA protein-encoding nucleic acid sequence or the native cotA amino acid sequence.

Similarly, the polynucleotides for use in practicing the invention include sequences which encode native cotA proteins and splice variants thereof, sequences complementary to the native protein coding sequence, and novel fragments of cotA encoding polynucleotides.

In one general embodiment, a cotA-encoding nucleotide sequence has at least 70%, preferably 80%, 85%, 90%, 95%, 98%, or more sequence identity to any one of the cotA coding sequences presented herein as SEQ ID NOs:1, 3 or 5.

In another embodiment, a cotA-encoding nucleotide sequence will hybridize under moderate to high stringency conditions to a nucleotide sequence that encodes a cotA protein. In a related embodiment, a cotA-encoding nucleotide sequence will hybridize under moderate to high stringency conditions to any one of the nucleotide sequences presented as SEQ ID NOs:1, 3 or 5.

It is appreciated that some nucleic acid sequence variants that encode cotA may or may not selectively hybridize to the parent sequence. By way of example, in situations where the coding sequence has been optimized based on the degeneracy of the genetic code, a variant coding sequence may be produced that encodes a cotA protein, but does not hybridize to a native cotA-encoding nucleic acid sequence under moderate to high stringency conditions. This would occur, for example, when the sequence variant includes a different codon for each of the amino acids encoded by the parent nucleotide.

As will be further understood by those of skill in the art, in some cases it may be advantageous to produce nucleotide sequences possessing non-naturally occurring codons. Codons preferred by a particular eukaryotic host (Murray, E. et al., 1989) can be selected, for example, to increase the rate of cotA protein expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from the naturally occurring sequence. Hence, a native cotA-encoding nucleotide sequence may be engineered in order to alter the coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the cotA protein by a cell.

A cotA-encoding nucleotide sequence may be engineered in order to alter the cotA coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of cotA by a cell.

Particularly preferred are nucleic acid substitutions, additions, and deletions that are silent such that they do not alter the properties or activities of the native polynucleotide or polypeptide.

The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., 1986; Zoller et al., 1987], cassette mutagenesis [Wells et al., 1985], restriction selection mutagenesis [Wells et al., 1986] or other known techniques can be performed on the cloned DNA to produce the cotA polypeptide-encoding variant DNA.

However, in some cases it may be advantageous to express variants of cotA which lack the properties or activities of the native cotA polynucleotide or polypeptide. In such cases, mutant or modified forms of the native cotA-encoding nucleic acid sequence may be generated using techniques routinely employed by those of skill in the art. For example, in a preferred embodiment, a fragment of a cotA-encoding polynucleotide is transfected into a fungal host cell. The manufacture of fragments of full length genomic and/or coding sequences is well within the skill of one in the art.

B. cotA Polypeptides

In one embodiment, the invention provides a truncated cotA polypeptide, having a polypeptide sequence comprising the sequence presented in FIG. 2 (SEQ ID NO:2). In another embodiment, a cotA polypeptide of the invention can be the mature cotA polypeptide, part of a fusion protein or a fragment or variant of the cotA polypeptide.

In another embodiment, the invention provides a truncated cotA polypeptide, having a polypeptide sequence comprising the sequence presented in FIG. 4 (SEQ ID NO:4). In another embodiment, a cotA polypeptide of the invention can be the mature cotA polypeptide, part of a fusion protein or a fragment or variant of the cotA polypeptide.

In a third embodiment, the invention provides a truncated cotA polypeptide, having a polypeptide sequence comprising the sequence presented in FIG. 6 (SEQ ID NO:6). In another embodiment, a cotA polypeptide of the invention can be the mature cotA polypeptide, part of a fusion protein or a fragment or variant of the cotA polypeptide.

Ordinarily, a cotA polypeptide of the invention comprises a region having at least 80, 85, 90, 95, 98% or more sequence identity to any one of the cotA polypeptide sequences of FIG. 2, 4 or 6 (SEQ ID NO:2, 4 or 6, respectively), using a sequence alignment program, as detailed herein.

Typically, a "modified form of" a native cotA protein or a "variant" cotA protein has a derivative sequence containing at least one amino acid substitution, deletion or insertion, respectively.

Fragments and variants of any one of the cotA polypeptide sequences of FIG. 2, 4 or 6 (SEQ ID NOs:2, 4 or 6, respectively), are also considered to be a part of the invention. A fragment is a variant polypeptide which has an amino acid sequence that is entirely the same as part but not all of the amino acid sequence of the previously described polypeptides. The fragments can be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or a region, most preferably as a single continuous region. Preferred fragments are biologically active fragments which are those fragments that mediate activities of the polypeptides of the invention, including those with similar activity or improved activity or with a decreased activity. Also included are those fragments that antigenic or immunogenic in an animal, particularly a human. In this aspect, the invention includes (i) fragments of cotA, preferably at least about 20-100 amino acids in length, more preferably about 100-200 amino acids in length, and (ii) a pharmaceutical composition comprising cotA. In various embodiments, the fragment corresponds to the N-terminal domain of cotA or the C-terminal domain of cotA. cotA polypeptides of the invention also include polypeptides that vary from any one of the cotA polypeptide sequences of FIG. 2, 4 or 6 (SEQ ID NO:2, 4 or 6, respectively). These variants may be substitutional, insertional or deletional variants. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as further described below.

A "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

An "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.

A "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.

Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to 5 amino acids.

Substitutions are generally made in accordance with known "conservative substitutions". A "conservative substitution" refers to the substitution of an amino acid in one class by an amino acid in the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature (as determined, e.g., by a standard Dayhoff frequency exchange matrix or BLOSUM matrix). (See generally, Doolittle, R. F., 1986.)

A "non-conservative substitution" refers to the substitution of an amino acid in one class with an amino acid from another class.

cotA polypeptide variants typically exhibit the same qualitative biological activity as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the cotA polypeptide, as needed. For example, glycosylation sites, and more particularly one or more O-linked or N-linked glycosylation sites may be altered or removed. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the cotA polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

Also included within the definition of cotA polypeptides are other related cotA polypeptides. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related polypeptides. Useful probe or primer sequences may be designed to: all or part of the cotA polypeptide sequence, or sequences outside the coding region. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are generally known in the art.

Covalent modifications of cotA polypeptides are also included within the scope of this invention. For example, the invention provides cotA polypeptides that are a mature protein and may comprise additional amino or carboxyl-terminal amino acids, or amino acids within the mature polypeptide (for example, when the mature form of the protein has more than one polypeptide chain). Such sequences can, for example, play a role in the processing of a protein from a precursor to a mature form, allow protein transport, shorten or lengthen protein half-life, or facilitate manipulation of the protein in assays or production. It is contemplated that cellular enzymes are used to remove any additional amino acids from the mature protein.

C. Anti-cotA Antibodies.

The present invention farther provides anti-cotA antibodies. The antibodies may be polyclonal, monoclonal, humanized, bispecific or heteroconjugate antibodies.

Methods of preparing polyclonal antibodies are known to the skilled artisan. The immunizing agent may be a cotA polypeptide or a fusion protein thereof. It may be useful to conjugate the antigen to a protein known to be immunogenic in the mammal being immunized. The immunization protocol may be determined by one skilled in the art based on standard protocols or routine experimentation. Alternatively, the anti-cotA antibodies may be monoclonal antibodies. Monoclonal antibodies may be produced by cells immunized in an animal or using recombinant DNA methods. [See, e.g., Kohler et al., 1975; U.S. Pat. No. 4,816,567]. Antibodies to proteins have many uses well known to those of skill in the art. Here, it is envisioned that antibodies to cotA are useful as a component of staining reagents to determine the expression of cotA in fungal host cells among other uses that will be apparent to those of skill.

V. Expression of Recombinant cotA and cotA Fragments

This invention provides filamentous fungal host cells which have been transduced, transformed or transfected with an expression vector comprising a cotA-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the parental host cell prior to transduction, transformation or transfection and will be apparent to those skilled in the art.

In one approach, a filamentous fungal cell line is transfected with an expression vector having a promoter or biologically active promoter fragment or one or more (e.g., a series) of enhancers which functions in the host cell line, operably linked to a DNA segment encoding cotA, such that cotA is expressed in the cell line. In a preferred embodiment, the DNA sequences encode a partial cotA coding sequence. In another preferred embodiment, the promoter is a regulatable one.

A. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding cotA ("cotA-encoding nucleic acid sequences") may be incorporated into heterologous nucleic acid constructs or vectors, capable of introduction into, and replication in, a filamentous fungal cell. The vectors and methods disclosed herein are suitable for use in host cells for the expression of cotA. Any vector may be used as long as it is replicable and viable in the cells into which it is introduced. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use in filamentous fungal cells are also described in Sambrook et al., 1989, and Ausubel F M et al., 1989, expressly incorporated by reference herein. The appropriate DNA sequence may be inserted into a plasmid or vector (collectively referred to herein as "vectors") by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by standard procedures. Such procedures and related sub-cloning procedures are deemed to be within the scope of knowledge of those skilled in the art.

Appropriate vectors are typically equipped with a selectable marker-encoding nucleic acid sequence, insertion sites, and suitable control elements, such as termination sequences. The vector may comprise regulatory sequences, including, for example, non-coding sequences, such as introns and control elements, i.e., promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in host cells (and/or in a vector or host cell environment in which a modified soluble protein antigen coding sequence is not normally expressed), operably linked to the coding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, many of which are commercially available and/or are described in Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and inducible promoters, examples of which include a CMV promoter, an SV40 early promoter, an RSV promoter, an EF-1.alpha. promoter, a promoter containing the tet responsive element (TRE) in the tet-on or tet-off system as described (Clon Tech and BASF), the beta actin promoter and the metallothienein promoter that can upregulated by addition of certain metal salts. In one embodiment of this invention, glaA promoter is used. This promoter is induced in the presence of maltose. In a preferred embodiment, a promoter that is induced by maltose is used. Such promoters are well known to those of skill in the art.

The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, methotrexate, tetracycline, neomycin (Southern and Berg, J., 1982), mycophenolic acid (Mulligan and Berg, 1980), puromycin, zeomycin, or hygromycin (Sugden et al., 1985). In a preferred embodiment, PyrG is used as a selectable marker.

A selected cotA coding sequence may be inserted into a suitable vector according to well-known recombinant techniques and used to transform a cell line capable of cotA expression. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express cotA, as further detailed above. Therefore it is appreciated that such substitutions in the coding region fall within the sequence variants covered by the present invention. Any and all of these sequence variants can be utilized in the same way as described herein for a parent cotA-encoding nucleic acid sequence.

Once the desired form of a cotA nucleic acid sequence, homologue, variant or fragment thereof, is obtained, it may be modified in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence.

The present invention also includes recombinant nucleic acid constructs comprising one or more of the cotA-encoding nucleic acid sequences as described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.

Heterologous nucleic acid constructs may include the coding sequence for cotA, or a variant, fragment or splice variant thereof: (i) in isolation; (ii) in combination with additional coding sequences; such as fusion protein or signal peptide coding sequences, where the cotA coding sequence is the dominant coding sequence; (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment in which the cotA coding sequence is a heterologous gene.

A heterologous nucleic acid containing the appropriate nucleic acid coding sequence, as described above, together with appropriate promoter and control sequences, may be employed to transform filamentous fungal cells to permit the cells to express cotA.

In one aspect of the present invention, a heterologous nucleic acid construct is employed to transfer a cotA-encoding nucleic acid sequence into a cell in vitro, with established cell lines preferred. Preferably, cell lines that are to be used as production hosts have the nucleic acid sequences of this invention stably integrated. Integration preferably occurs in the cotA locus but ectopic integration is useful as well. It follows that any method effective to generate stable transformants may be used in practicing the invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989), "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

B. Host Cells and Culture Conditions For Regulatable Expression.

Thus, the present invention provides cell lines comprising cells which have been modified, selected and cultured in a manner effective to result in regulatable expression of cotA relative to the corresponding non-transformed parental cell line.

Examples of parental cell lines which may be treated and/or modified for regulatable cotA expression include, but are not limited to filamentous fungal cells. Examples of appropriate primary cell types for use in practicing the invention include, but are not limited to, Aspergillus and Trichoderma.

cotA expressing cells are cultured under conditions typically employed to culture the parental cell line. Generally, cells are cultured in a standard medium containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM or DMEM, typically supplemented with 5-10% serum, such as fetal bovine serum. Culture conditions are also standard, e.g., cultures are incubated at 37.degree. C. in stationary or roller cultures until desired levels of cotA expression are achieved.

Preferred culture conditions for a given cell line may be found in the scientific literature and/or from the source of the cell line such as the American Type Culture Collection (ATCC;). Typically, after cell growth has been established, the cells are exposed to conditions effective to cause or inhibit the expression of cotA and truncated cotA.

In the preferred embodiments, where a cotA coding sequence is under the control of an inducible promoter, the inducing agent, e.g., a carbohydrate, metal salt or antibiotics, is added to the medium at a concentration effective to induce cotA expression.

C. Introduction of a cotA-Encoding Nucleic Acid Sequence into Host Cells.

The invention further provides cells and cell compositions which have been genetically modified to comprise an exogenously provided cotA-encoding nucleic acid sequence. A parental cell or cell line may be genetically modified (i.e., transduced, transformed or transfected) with a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc, as further described above. In a preferred embodiment, a plasmid is used to transfect a filamentous fungal cell.

Various methods may be employed for delivering an expression vector into cells in vitro. Methods of introducing nucleic acids into cells for expression of heterologous nucleic acid sequences are also known to the ordinarily skilled artisan, including, but not limited to electroporation; nuclear microinjection or direct microinjection into single cells; bacterial protoplast fusion with intact cells; use of polycations, e.g., polybrene or polyornithine; membrane fusion with liposomes, lipofectamine or lipofection-mediated transfection; high velocity bombardment with DNA-coated microprojectiles; incubation with calcium phosphate-DNA precipitate; DEAE-Dextran mediated transfection; infection with modified viral nucleic acids; and the like. In addition, heterologous nucleic acid constructs comprising a cotA-encoding nucleic acid sequence can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection.

In a preferred embodiment, the expression vector comprising a truncated cotA and an appropriate promoter is constructed such that the promoter and cotA sequence integrates in the cotA locus. This is accomplished via a single recombination event within the cotA locus. In a more preferred embodiment, the expression vector is constructed such that a double recombination event occurs. The vector comprises a stretch of nucleic acid that is complementary to a stretch of nucleic acid in the cotA locus upstream from the cotA coding sequence. The other site of complementary DNA occurs in the coding region. Upon integration, two crossover events occur so that only the appropriate promoter and the truncated cotA sequence are inserted into the cotA locus instead of the entire expression vector.

Following introduction of a heterologous nucleic acid construct comprising the coding sequence for cotA, the genetically modified cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying expression of a cotA-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

The progeny of cells into which such heterologous nucleic acid constructs have been introduced are generally considered to comprise the cotA-encoding nucleic acid sequence found in the heterologous nucleic acid construct.

VI. Analysis of cotA Nucleic Acids and Proteins

In order to evaluate the expression of cotA by a cell line that has been transformed with a cotA-encoding nucleic acid construct, assays can be carried out at the protein level, the RNA level or by use of functional bioassays particular to growth characteristics of the transfected cell line.

By way of example, the production and/or expression of cotA may be measured in a sample directly, for example, by microscopic examination of transfected cells. Filamentous fungal cells that have been transfected with cotA under the control of an inducible promoter exhibit slowed and more compact growth compared to parental fungal cells when exposed to the compound that induces expression. Nucleic acid-based assays for determining the expression of cotA include, but are not limited to, northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction), or in situ hybridization, using an appropriately labeled probe (based on the nucleic acid coding sequence) and conventional Southern blotting.

Alternatively, protein expression, may be evaluated by immunological methods, such as immunohistochemical staining of cells, tissue sections or immunoassay of tissue culture medium, e.g., by western blot or ELISA. Such immunoassays can be used to qualitatively and quantitatively evaluate expression of cotA. The details of such methods are known to those of skill in the art and many reagents for practicing such methods are commercially available.

A purified form of cotA is typically used to produce either monoclonal or polyclonal antibodies specific to the expressed protein for use in various immunoassays. (See, e.g., Harlow and Lane, 1988). Exemplary assays include ELISA, competitive immunoassays, radioimmunoassays, western blot, indirect immunofluorescent assays and the like. In general, commercially available antibodies and/or kits may be used for the quantitative immunoassay of the expression level of known types of proteins.

VII. Isolation and Purification of Recombinant cotA Protein.

In general, a cotA protein produced in a filamentous fungal cell is not secreted into the medium and therefore must be purified from cell lysates. This can be accomplished by techniques routine employed by those of skill in the art.

Typically, after removal of cell debris, the lysate comprising cotA protein is fractionated to segregate proteins having selected properties, such as binding affinity to particular binding agents, e.g., antibodies or receptors; or which have a selected molecular weight range, or range of isoelectric points.

Once expression of a given cotA protein is achieved, the cotA protein thereby produced is purified from the cells or cell culture. Exemplary procedures suitable for such purification include the following: antibody-affinity column chromatography, ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and gel filtration using, e.g., Sephadex G-75. Various methods of protein purification may be employed and such methods are known in the art and described e.g. in Deutscher, 1990; Scopes, 1982. The purification step(s) selected will depend, e.g., on the nature of the production process used and the particular protein produced.

VIII. Utility of the cotA Polypeptides and Nucleic Acids of this Invention

From the foregoing, it can be appreciated that cells transformed with cotA under the control of an inducible promoter grow more slowly in conditions in which cotA is expressed. By retarding the growth of fungal cell cultures, fermenter cultures of such cells can be maintained for longer periods of time. Because fermenter cultures are maintained for longer periods, expressed protein levels can be maintained for longer periods of time. Thus, elevated concentrations of expressed protein can be achieved. As would be obvious to one of skill, this would lead to lower production costs.

For production of a desired protein in a fungal host cell, an expression vector comprising at least one copy of nucleic acid encoding a desired protein is transformed into the recombinant host cell comprising nucleic acid encoding a protein associated with hyphal growth and cultured under conditions suitable for expression of the protein. Examples of desired proteins include enzymes such as hydrolases including proteases, cellulases, amylases, carbohydrases, and lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases along with proteins of therapeutic value.

Thus, the present invention is particularly useful in enhancing the intracellular and/or extracellular production of proteins. The protein may be homologous or heterologous. Proteins that may produced by the instant invention include, but are not limited to, hormones, enzymes, growth factors, cytokines, antibodies and the like.

Enzymes include, but are not limited to, hydrolases, such as protease, esterase, lipase, phenol oxidase, permease, amylase, pullulanase, cellulase, glucose isomerase, laccase and protein disulfide isomerase.

Hormones include, but are not limited to, follicle-stimulating hormone, luteinizing hormone, corticotropin-releasing factor, somatostatin, gonadotropin hormone, vasopressin, oxytocin, erythropoietin, insulin and the like.

Growth factors are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation. Growth factors include, but are not limited to, platelet-derived growth factor, epidermal growth factor, nerve growth factor, fibroblast growth factors, insulin-like growth factors, transforming growth factors and the like.

Cytokines are a unique family of growth factors. Secreted primarily from leukocytes, cytokines stimulate both the humoral and cellular immune responses, as well as the activation of phagocytic cells. Cytokines include, but are not limited to, colony stimulating factors, the interleukins (IL-1 (.alpha. and .beta.), IL-2 through IL-13) and the interferons (.alpha., .beta. and .gamma.).

Human Interleukin-3 (IL-3) is a 15 kDa protein containing 133 amino acid residues. IL-3 is a species specific colony stimulating factor which stimulates colony formation of megakaryocytes, neutrophils, and macrophages from bone marrow cultures.

Antibodies include, but are not limited to, immunoglobulins from any species from which it is desirable to produce large quantities. It is especially preferred that the antibodies are human antibodies. Immunoglobulins may be from any class, i.e., G, A, M, E or D.

EXAMPLES

The following examples are submitted for illustrative purposes only and should not be interpreted as limiting the invention in any way.

Example 1

Isolation of a Truncated cotA Polynucleotide from Aspergillus niger

Based on an alignment of cot1 from N. crassa, TB3 (a Colletotrichum homologue), KNQ.sub.-- 1/Cbk1p, a related kinase in S. cerevisiae, and Homo sapiens DMK, degenerate oligonucleotides were designed against 2 conserved regions of the coding sequence.

DIKPDN (5' forward primer) (SEQ ID NO:7) 5'-GA T/C AT T/C AA A/G CCNGA T/C AA-3' EPAIYD (3' reverse primer) (SEQ ID NO:8) 5'-TCNGGNGC G/T/A AT A/G TA A/G TC-3'

Using routine PCR conditions and genomic A. niger DNA, a 241 internal fragment was produced. This fragment was sequenced and found to have closest homology to cot-1 of N. crassa. This fragment was used to probe digested A. niger genomic DNA on a Southern blot according to routine methods. A 6.5 kb band from a HindIII digest hybridized with the probe.

A. niger genomic DNA was digested with HindIII, recircularized and ligated. This circularized DNA was subjected to inverse PCR using oligonucleotides designed from the nucleotide sequence of the 241 bp region homologous to cot-1.

(SEQ ID NO:9) INV3' (reverse primer) 5'ACGTCGAGTTCTTCAGC 3' (SEQ ID NO:10) INV5' (forward primer) 5'GCGATCAACCTGACAGT 3'

A 6.5 kb fragment produced from the inverse PCR reaction was inserted into the cloning vector pCR.RTM.2.1. The resulting construct, pPOL, was sequenced. The sequence data allowed orientation of the A. niger cotA within the 6.5 kb fragment. The selected open reading frame of A. niger was aligned with related kinases (See FIG. 7).

As can be seen from FIG. 7, the 6.5 kb fragment contains an open reading frame of approximately 500 amino acids or of 1.5 kb. Alignment of the ORF of the A. niger homologue with cot-1 indicated that about 50 amino acids or 150 base pairs from the C or 3' terminal were missing from the coding region.

Example 2

Expression of Truncated A. niger cotA

1.4 kb of the 5' coding region of cotA under the control of the glaA promoter was inserted into the expression vector pGRT-pyrG1 (Ward, et al, Appl Microbiol and Biotech. 39:738-743 (1993)) to examine the effect of regulated expression of cotA on the growth morphology of A. niger. glaAp is induced by maltose and repressed by xylose. The resulting plasmid, pSMB5, was used to transform an A. niger pyrG-recipient. See FIG. 8 for schematic of transformation. Pyr+ transfmormants were selected on minimal medium with maltose as the sole carbon source and screened for growth morphology on xylose. Transformants that showed restricted growth on xylose but that grew well on maltose, were analyzed by Southern hybridization. In one colony of transformants (SMB540), integration of the plasmid occurred at the cotA locus, in others, ectopic integration took place.

Parental A. niger cotA strains were compared to strains carrying the glaAp-cotA fusion after growth on different C-sources, to regulate expression of glaAp. Morphological changes occurred only during repression of cotA expression, with YEPX more repressing than MM+1% xylose. When cotA+ and glaAp-cotA strains were grown on maltose (non-repressing) then no morphological difference was seen between the strains.

Example 3

Truncated cotA in the Antisense Orientation

To determine what effect disruption of cotA would have on the growth of A. niger, an A. niger strain was transformed with cotA under the control of glaAp as above, except the cotA sequence was in the antisense orientation.

As can be seen in FIG. 9, the morphology of the transformants is very slow growing and compact with very long branches.

Example 4

Point Mutation in the cotA Locus

From the literature, it is known that in N. crassa, a single mutation in the cot-1 locus creates the temperature sensitive hyperbranching phenotype. In cot-1, a histidine naturally occurs at position 352 (see FIG. 7). The cot-1 mutation is caused by a switch to arginine at this position.

Site directed mutagenesis can be used to manufacture the same mutation in the cotA coding sequence of A. niger. Using techniques very similar to those described above, the cotA coding sequence with the point mutation as well as an inducible promoter can be integrated into the cotA locus or ectopically. It is expected that, when induced, the mutation will cause the slow growth morphology described above

Example 5

Isolation of Truncated cot-1 from Trichoderma reesei

Using degenerate PCR, a 264 base pair cot-1 nucleic acid sequence was isolated from genomic T. reesei genomic DNA. The forward primer was 5' GA T/C AT T/C AA A/G CC A/G/C/T GA A/C AA-3' (SEQ ID NO:11) and the reverse primer was 5' TC A/G/C/T GG A/C/G/T GC G/T AT A/G TA A/G TC-3' (SEQ ID NO:12).

The internal cot-1 fragment is shown in FIG. 5 (SEQ ID NO:5) and the translated sequence is shown in FIG. 6 (SEQ ID NO:6).

SEQUENCE LISTING <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 21 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 1784 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 1 tttccatcat ggatcccaac aacaacaacc gactccacct gaatttcggg tacaatgatc 60 gtggtttcaa tgcggcggcg gccaacaacc gcgcctatcc cactaccccc tccgccttcc 120 cccagccgat ctaccaaaac cagggtcccc aggattacat ggacgcccag aacggtgcct 180 acgctcaagg tggttatttc atggccaatc cttaccaagc tcaggcagcc tacggccagc 240 cgcattatgg ccagaacctg cagtcccctc agcccgccta cccatcccgc atgggttaca 300 gcgcgaacga tggcaccaac ggtttgatcc agcagttctc gaaccaggat ctgaattccc 360 ctcgctcggg tttctttgct cgttctgcct cgccagccca gcgaccccga actgccggct 420 cccccgcccc cgggcagcaa cagccaggcc acctggcgcc tcctatgcct cgtagccctc 480 gcacccccgc ggagaacgaa gagttgcaac ggtacccgga acgttactca gagaatgttc 540 acaagcgtgg caaggcagcc aaggagctgg tcagcgtctt cttaatgaga acaatgagcg 600 cgcacgcgat cgcaacatgc ggtgagtatt ccacacaatg ccacggcctc cctcccaacc 660 caacagggaa tttggtatcg ctgactcggg tgcttttcat aggtctgctg agctggacaa 720 gatgatccgt gaacccagta ttcccaagga gaacaagtgc aaggacgcag aggtgcttgc 780 taagaaggaa tcgaatttcc tccggttcct tcgcaccaag gagaccccgc agaacttcca 840 gaccatcaag atcatcggaa agggcgcgtt tggtgaagtg aagctggtac aacggaaggc 900 cgatggcaag atttacgcac tgaagtcgtt gatcaaaacg gagatgttca agcaaggacc 960 agctgctcac gttcgcgcgg aacgtgatat ccttgctgat tccaaggaca acccgtggtt 1020 ggtgaagctg catgcttctt tccaggacac tgcctacttg tatttgctga tggaattctt 1080 gcctggtggt gacttgatga ccatgttgat caagtacgag atcttctccg aggatatcac 1140 tcggttctat atggccgaaa ttgtcatggc gatcgaggct gttcacaagc tcggcttcct 1200 tcaccgggac atcaagcctg ataacatcct tctggatcgc ggtggtcacg tcaagctgac 1260 ggactttggt ctgtccacgg gaggaaagaa gacccacgac aactcctact atcagaatct 1320 gctgaagaac tcgacgtcaa aggacaagaa ccgcaactct ggttacttca acgatgcgat 1380 caacctgaca gtctccaacc gtggccagat caacacctgg agaaagtctc gtcgtgcaat 1440 ggcatactcg acggtcggaa ctccggacta tatcgccccc gagatcttca acggccaagg 1500 atacacctac ctgtgcgatt ggtggtctgt aggtgctatc atgttcgagt gccttgtggg 1560 ttggcccccg ttctgcgcgg aagacaccac cgacacctac cgcaagattg tgaactggag 1620 agaatgcttg tacttccctg aggaactcac cctttcgcgc gattccgagg gtctcatccg 1680 aaggtaagct tgaagccgag aagcgtgagg ccagaaaggc cgcaagcacg aagaacattg 1740 acggagaagt gaagaaggaa gactctgacc cactaggcaa ccag 1784 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 250 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 2 Met Glu Pro Asn Asn Asn Asn Arg Leu His Leu Asn Phe Gly Tyr Asn 1 5 10 15 Asp Arg Gly Phe Asn Ala Ala Ala Ala Asn Asn Arg Ala Tyr Pro Thr 20 25 30 Thr Pro Ser Ala Phe Pro Gln Pro Ile Tyr Gln Asn Gln Gly Pro Gln 35 40 45 Asp Tyr Met Asp Ala Gln Asn Gly Ala Tyr Ala Gln Gly Gly Tyr Phe 50 55 60 Met Ala Asn Pro Tyr Gln Ala Gln Ala Ala Tyr Gly Gln Pro His Tyr 65 70 75 80 Gly Gln Asn Leu Gln Ser Pro Gln Pro Ala Tyr Pro Ser Arg Met Gly 85 90 95 Tyr Ser Ala Asn Asp Gly Thr Asn Gly Leu Ile Gln Gln Phe Ser Asn 100 105 110 Gln Asp Leu Asn Ser Pro Arg Ser Gly Phe Phe Ala Arg Ser Ala Ser 115 120 125 Pro Ala Gln Arg Pro Arg Thr Ala Gly Ser Pro Ala Pro Gly Gln Gln 130 135 140 Gln Pro Gly His Leu Ala Pro Pro Met Pro Arg Ser Pro Arg Thr Pro 145 150 155 160 Ala Glu Asn Glu Glu Leu Gln Arg Tyr Pro Glu Arg Tyr Ser Glu Asn 165 170 175 Val His Lys Arg Gly Lys Ala Ala Lys Glu Leu Val Ser Val Phe Phe 180 185 190 Asn Glu Asn Asn Glu Arg Ala Arg Asp Arg Asn Met Arg Ser Ala Glu 195 200 205 Leu Asp Lys Met Ile Arg Glu Pro Ser Ile Pro Lys Glu Asn Lys Cys 210 215 220 Lys Asp Ala Glu Val Leu Ala Lys Lys Glu Ser Asn Phe Leu Arg Phe 225 230 235 240 Leu Arg Thr Lys Glu Thr Pro Gln Asn Phe 245 250 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 3038 <212> TYPE: DNA <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 3 cgggataggg ctcggaaaag cgagggcttc agagcataag aacatcatca gaaagtggag 60 ctttcgtagc acagtgtcgt gaggtccgtc tgatatggcc ctgaaaagta agcgtagtga 120 gtgggatgct cttctcgctt ttgaacatga ccgtgactct gtctcaatcc acactcaata 180 cctttgtctc cgtgatatgt ttcagatata gaaccttaat gaagagccaa ctttatgaca 240 aatgcatctt cgaggggtgg gtgttgtata gaggcagcgc gggtgggccc cgcggtcttc 300 cgtagcccaa ctcccaaaac agtccagggt aacgtactgg gcaccaccgc actgctttta 360 agctactgct ggtctttaag tctggactct cgataacttg ttgcggcttt gctttttctt 420 tggtgcttat caacccaggt gactttgcga ccacagaatc gttgtcgctt gctcaatcgc 480 ctcctgatcg attatccctc taaggagagc ttgtccagtc gggagcgctc caacactcca 540 ctatagtaac actgttcctt cccctcaagc cgcactcgct cacttgtctc ctgaagccac 600 cgcttcttcc cactaacctt cccctccccc ctttacactt gcacaccccc cccttatatc 660 catcaccttc ctccattcct catctcgccg tccgtccaat tttggtagtc tggagggcac 720 tcttccaaaa tggaccccaa caacaatcgc ccccacctga acttcggcta caatgaacgt 780 gccttcaacc ctgcggccgc aaacaaccgc gcgtatccca ccacgccctc cgcatttcct 840 cagccgatct accagagcca gagcccccag gactacatgg acgctcagaa tggtgtttat 900 ggtcagggat atttcatgcc gaacaactac cctgcgcagg ctgcctatgc ccagccccat 960 tacggccaac ccaatctcca gtctcctcag cccgcctatc agtctcgaat gggatacaat 1020 gtcagcccca acgatggaac aaatggtttg atacagcagt tctcgaatca ggatttaaac 1080 tcgaaccgaa cgggtttctt caatcgctcc gcttcgcctg ctcaaagacc ccgtactgca 1140 ggcaatacag cccccggaca gcagcagcaa cctggacact tggcccctcc agtgcctcgc 1200 agccctcggc tgccccccga gaacgaagaa cttcaacgct acccagagcg cttctctgaa 1260 aatgttcaca aacgtggaaa agctgcgaag gagttggtca acgtattctt tcacgagaat 1320 atcgagcgtg cgcgtgatcg caacatgcgg tgggtttttg ctactgagcg ccgtatttct 1380 ctaaaaagaa ttttgctaac tggagttata actgtacagt tcggcggagc tcgacaagat 1440 gatgcgcgac cccaacattt cacaagatgc aaaggtgaag gaggcggaaa tggttggaaa 1500 gaaagagtcg acattccttc gcttccttcg gacaccagaa actcctgcca acttccaaac 1560 catcaagatt attggaaagg gtgcttttgg tgaagttaag ctggtgcaga ggaagtctga 1620 taacaagatc tatgcgctta agtcgctgat caaatcagag atgtttaaga aagatcagct 1680 cgcccacgtt cgtgctgaac gtgatattct agctgactcg aaggacaacc cttggcttgt 1740 caagctccat gcttcattcc aggatcccgc atacctatac ctcctgatgg agttcttacc 1800 tggaggtgat ttgatgacca tgcttattaa gtacgaaata ttctctgaag atatcacacg 1860 cttctacatg gcggaaattg tgatggcgat tgaggcggtt cacaagctgg gtttccttca 1920 ccggtgagaa taacaatcct ggtctctcgt accatataca gcgtgctaat atacttgtac 1980 tatagagata ttaaacctga caacatcctt ctcgatcgtg gcggtcacgt caagctgacc 2040 gatttcggtc tctcaactgg aggcaagaaa actcacgaca actcatacta tcagaacctg 2100 ttgaagaatt caacatccaa ggataagaac cgaaactctg gatacttcaa cgatgctatc 2160 aacttgacag tatcgaaccg tgggcagatc aacacctgga gaaaatctcg cagggctatg 2220 gcttactcca ctgtcggaac acctgactac attgcacccg aaatttttaa tggtcaagga 2280 tacacctatc tttgcgactg gtggtccgtc ggtgccatca tgtttgaatg tctcgtgggc 2340 tggcctccat tctgcgccga ggatacgacc gacacctatc gcaagattgt gaactggagg 2400 gaatgcctat atttccccga agaattgaca ctgtctcgtg aatcggaggg tctgattcga 2460 aggtatgtta tgtcagcaat ccatttgagc tgcttgtcta accggagatc agcttcctat 2520 gtgacgcaga acaccgcatc ggcaacgaag gtggccaata cggaggtgct acacagatca 2580 aaaatcaccc attcttccgc ggggtagtat gggatcaact gcgcaaaatc cgggcaccgt 2640 tcgaacccag actgacgtca aatatcgacg tatcatattt cccgattgac gagattcctc 2700 aggaggatac cagcgccatt caccgcgccc aggcacgtgc catgccggat gagcagaatg 2760 ctgagatgag cctgcctttt atcggataca catacaaagc attcaacgcc ttccaggcca 2820 gttgagcatg catttaaagt aagaaatata tttgaatgag ccgatgatgg atgccattgg 2880 aaagttttga agcgggcggg cttgcgttga taacttttca atggcgcatc caggtttttg 2940 tgtcggtcgg catagaccct tgttgattgg tattttcatc aagcatatag cgcatacatc 3000 atgtcactgg acacatgagc atctcactac catatgtg 3038 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 637 <212> TYPE: PRT <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 4 Met Asp Pro Asn Asn Asn Arg Pro His Leu Asn Phe Gly Tyr Asn Glu 1 5 10 15 Arg Ala Phe Asn Pro Ala Ala Ala Asn Asn Arg Ala Tyr Pro Thr Thr 20 25 30 Pro Ser Ala Phe Pro Gln Pro Ile Tyr Gln Ser Gln Ser Pro Gln Asp 35 40 45 Tyr Met Asp Ala Gln Asn Gly Val Tyr Gly Gln Gly Tyr Phe Met Pro 50 55 60 Asn Asn Tyr Pro Ala Gln Ala Ala Tyr Ala Gln Pro His Tyr Gly Gln 65 70 75 80 Pro Asn Leu Gln Ser Pro Gln Pro Ala Tyr Gln Ser Arg Met Gly Tyr 85 90 95 Asn Val Ser Pro Asn Asp Gly Thr Asn Gly Leu Ile Gln Gln Phe Ser 100 105 110 Asn Gln Asp Leu Asn Ser Asn Arg Thr Gly Phe Phe Asn Arg Ser Ala 115 120 125 Ser Pro Ala Gln Arg Pro Arg Thr Ala Gly Asn Thr Ala Pro Gly Gln 130 135 140 Gln Gln Gln Pro Gly His Leu Ala Pro Pro Val Pro Arg Ser Pro Arg 145 150 155 160 Leu Pro Pro Glu Asn Glu Glu Leu Gln Arg Tyr Pro Glu Arg Phe Ser 165 170 175 Glu Asn Val His Lys Arg Gly Lys Ala Ala Lys Glu Leu Val Asn Val 180 185 190 Phe Phe His Glu Asn Ile Glu Arg Ala Arg Asp Arg Asn Met Arg Ser 195 200 205 Ala Glu Leu Asp Lys Met Met Arg Asp Pro Asn Ile Ser Gln Asp Ala 210 215 220 Lys Val Lys Glu Ala Glu Met Val Gly Lys Lys Glu Ser Thr Phe Leu 225 230 235 240 Arg Phe Leu Arg Thr Pro Glu Thr Pro Ala Asn Phe Gln Thr Ile Lys 245 250 255 Ile Ile Gly Lys Gly Ala Phe Gly Glu Val Lys Leu Val Gln Arg Lys 260 265 270 Ser Asp Asn Lys Ile Tyr Ala Leu Lys Ser Leu Ile Lys Ser Glu Met 275 280 285 Phe Lys Lys Asp Gln Leu Ala His Val Arg Ala Glu Arg Asp Ile Leu 290 295 300 Ala Asp Ser Lys Asp Asn Pro Trp Leu Val Lys Leu His Ala Ser Phe 305 310 315 320 Gln Asp Pro Ala Tyr Leu Tyr Leu Leu Met Glu Phe Leu Pro Gly Gly 325 330 335 Asp Leu Met Thr Met Leu Ile Lys Tyr Glu Ile Phe Ser Glu Asp Ile 340 345 350 Thr Arg Phe Tyr Met Ala Glu Ile Val Met Ala Ile Glu Ala Val His 355 360 365 Lys Leu Gly Phe Leu His Arg Asp Ile Lys Pro Asp Asn Ile Leu Leu 370 375 380 Asp Arg Gly Gly His Val Lys Leu Thr Asp Phe Gly Leu Ser Thr Gly 385 390 395 400 Gly Lys Lys Thr His Asp Asn Ser Tyr Tyr Gln Asn Leu Leu Lys Asn 405 410 415 Ser Thr Ser Lys Asp Lys Asn Arg Asn Ser Gly Tyr Phe Asn Asp Ala 420 425 430 Ile Asn Leu Thr Val Ser Asn Arg Gly Gln Ile Asn Thr Trp Arg Lys 435 440 445 Ser Arg Arg Ala Met Ala Tyr Ser Thr Val Gly Thr Pro Asp Tyr Ile 450 455 460 Ala Pro Glu Ile Phe Asn Gly Gln Gly Tyr Thr Tyr Leu Cys Asp Trp 465 470 475 480 Trp Ser Val Gly Ala Ile Met Phe Glu Cys Leu Val Gly Trp Pro Pro 485 490 495 Phe Cys Ala Glu Asp Thr Thr Asp Thr Tyr Arg Lys Ile Val Asn Trp 500 505 510 Arg Glu Cys Leu Tyr Phe Pro Glu Glu Leu Thr Leu Ser Arg Glu Ser 515 520 525 Glu Gly Leu Ile Arg Ser Phe Leu Cys Asp Ala Glu His Arg Ile Gly 530 535 540 Asn Glu Gly Gly Gln Tyr Gly Gly Ala Thr Gln Ile Lys Asn His Pro 545 550 555 560 Phe Phe Arg Gly Val Val Trp Asp Gln Leu Arg Lys Ile Arg Ala Pro 565 570 575 Phe Glu Pro Arg Leu Thr Ser Asn Ile Asp Val Ser Tyr Phe Pro Ile 580 585 590 Asp Glu Ile Pro Gln Glu Asp Thr Ser Ala Ile His Arg Ala Gln Ala 595 600 605 Arg Ala Met Pro Asp Glu Gln Asn Ala Glu Met Ser Leu Pro Phe Ile 610 615 620 Gly Tyr Thr Tyr Lys Ala Phe Asn Ala Phe Gln Ala Ser 625 630 635 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 264 <212> TYPE: DNA <213> ORGANISM: Trichoderma reesei <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(264) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 5 ctgctggacc gtggcggccc cgtcaagctg accgactttg gtctctccac gggcttccac 60 cgtctgcacg acaacaacta ctaccagcag ctgctgcagg gccgctccaa ccgcccgcgt 120 gaccgcacct cggttgccat tgatcagatt aacctcacag tcagcaaccg atctcagatt 180 aacgactgga gacgatctcg acggctgatg gcttactcca ccgtcggtac accagactac 240 atcgccccng aaattctcta cctc 264 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 88 <212> TYPE: PRT <213> ORGANISM: Trichoderma reesei <400> SEQUENCE: 6 Leu Leu Asp Arg Gly Gly Pro Val Lys Leu Thr Asp Phe Gly Leu Ser 1 5 10 15 Thr Gly Phe His Arg Leu His Asp Asn Asn Tyr Tyr Gln Gln Leu Leu 20 25 30 Gln Gly Arg Ser Asn Arg Pro Arg Asp Arg Thr Ser Val Ala Ile Asp 35 40 45 Gln Ile Asn Leu Thr Val Ser Asn Arg Ser Gln Ile Asn Asp Trp Arg 50 55 60 Arg Ser Arg Arg Leu Met Ala Tyr Ser Thr Val Gly Thr Pro Asp Tyr 65 70 75 80

Ile Ala Pro Glu Ile Leu Tyr Leu 85 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 7 gayatyaarc cngayaa 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 8 tcnggngcda trtartc 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 9 acgtcgagtt cttcagc 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 10 gcgatcaacc tgacagt 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 11 gayatyaarc cngamaa 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 12 tcnggngcka trtartc 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 13 <211> LENGTH: 2136 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 13 atggatccca acaacaacaa ccgactccac ctgaatttcg ggtacaatga tcgtggtttc 60 aatgcggcgg cggccaacaa ccgcgcctat cccactaccc cctccgcctt cccccagccg 120 atctaccaaa accagggtcc ccaggattac atggacgccc agaacggtgc ctacgctcaa 180 ggtggttatt tcatggccaa tccttaccaa gctcaggcag cctacggcca gccgcattat 240 ggccagaacc tgcagtcccc tcagcccgcc taccaatccc gcatgggtta cagcgcgaac 300 gatggcacca acggtttgat ccagcagttc tcgaaccagg atctgaattc ccctcgctcg 360 ggtttctttg ctcgttctgc ctcgccagcc cagcgactcc gaactgccgg ctcccccgcc 420 cccgggcagc aacagccagg ccacctggcg cctcctatgc ctcgtagccc tcgcaccccc 480 gcggagaacg aagagttgca acggtacccg gaacgttact cagagaatgt tcacaagcgt 540 ggcaaggcag ccaaggagct ggtcagcgtc ttcttcaatg agaacattga gcgcgcacgc 600 gatcgcaaca tgcggtgagt attccacaca atgccacggc ctccctccca acccaacagg 660 gaatttggta tcgctgactc gggtgctttt cataggtctg ctgagctgga caagatgatc 720 cgtgaaccca gtattcccaa ggagaacaag tgcaaggacg cagaggtgct tgctaagaag 780 gaatcgaatt tcctccggtt ccttcgcacc aaggagaccc cgcagaactt ccagaccatc 840 aagatcatcg gaaagggcgc gtttggtgaa gtgaagctgg tacaacggaa gaccgatggc 900 aagatttacg cactgaagtc gttgatcaaa acggagatgt tcaagaagga ccagctggct 960 cacgttcgcg cggaacgtga tatccttgct gattccaagg acaacccgtg gttggtgaag 1020 ctgcatgctt ctttccagga cactgcctac ttgtatttgc tgatggaatt cttgcctggt 1080 ggtgacttga tgaccatgtt gatcaagtac gagatcttct ccgaggatat cactcggttc 1140 tatatggccg aaattgtcat ggcgatcgag gctgttcaca agctcggctt ccttcaccgg 1200 taagtactag atgctcgatg ctgccagcag atgcaaagtt gaagtttcac ggggcggcag 1260 gtgctaattg tttttgtcta tagtgatatc aagcctgata acatccttct ggatcgcggt 1320 ggtcacgtca agctgacgga ctttggtctg tccacgggag gaaagaagac ccacgacaac 1380 tcctactatc agaatctgct gaagaactcg acgtcaaagg acaagaaccg caactctggt 1440 tacttcaacg atgcgatcaa cctgacagtc tccaaccgtg gccagatcaa cacctggaga 1500 aagtctcgtc gtgcaatggc atactcgacg gtcggaactc cggactatat cgcccccgag 1560 atcttcaacg gccaaggata cacctacctg tgcgattggt ggtctgtagg tgctatcatg 1620 ttcgagtgcc ttgtgggttg gcccccgttc tgcgcggaag acaccaccga cacctaccgc 1680 aagattgtga actggagaga atgcttgtac ttccctgagg aactcaccct ttcgcgcgat 1740 tccgagggtc tcatccgaag gtaagctttg tgcacatcat atgcttatgt atcatgctaa 1800 ctcaggatta gcttcctctg cgacgcagaa caccgtatcg gaagcgatgg cggccaattc 1860 ggcggcgcaa cgcagatcaa gaaccacccc ttcttccgcg gcgtcgtttg ggagcaactg 1920 cgcagcatcc gcgcgccgtt cgaaccaaga ctgagctcga acattgacgt gtcgtacttc 1980 ccgatcgatg agattcctca ggaggatacg agtgccatcc accgcgctca ggctcgcgcc 2040 aagccggacg agcaggaggc ggagatgagc cttccattca tcggatacac ctacaaagcg 2100 ttcaacgcct tccagggaaa ttgaagatac agtcga 2136 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14 <211> LENGTH: 3038 <212> TYPE: DNA <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 14 cgggataggg ctcggaaaag cgagggcttc agagcataag aacatcatca gaaagtggag 60 ctttcgtagc acagtgtcgt gaggtccgtc tgatatggcc ctgaaaagta agcgtagtga 120 gtgggatgct cttctcgctt ttgaacatga ccgtgactct gtctcaatcc acactcaata 180 cctttgtctc cgtgatatgt ttcagatata gaaccttaat gaagagccaa ctttatgaca 240 aatgcatctt cgaggggtgg gtgttgtata gaggcagcgc gggtgggccc cgcggtcttc 300 cgtagcccaa ctcccaaaac agtccagggt aacgtactgg gcaccaccgc actgctttta 360 agctactgct ggtctttaag tctggactct cgataacttg ttgcggcttt gctttttctt 420 tggtgcttat caacccaggt gactttgcga ccacagaatc gttgtcgctt gctcaatcgc 480 ctcctgatcg attatccctc taaggagagc ttgtccagtc gggagcgctc caacactcca 540 ctatagtaac actgttcctt cccctcaagc cgcactcgct cacttgtctc ctgaagccac 600 cgcttcttcc cactaacctt cccctccccc ctttacactt gcacaccccc cccttatatc 660 catcaccttc ctccattcct catctcgccg tccgtccaat tttggtagtc tggagggcac 720 tcttccaaaa tggaccccaa caacaatcgc ccccacctga acttcggcta caatgaacgt 780 gccttcaacc ctgcggccgc aaacaaccgc gcgtatccca ccacgccctc cgcatttcct 840 cagccgatct accagagcca gagcccccag gactacatgg acgctcagaa tggtgtttat 900 ggtcagggat atttcatgcc gaacaactac cctgcgcagg ctgcctatgc ccagccccat 960 tacggccaac ccaatctcca gtctcctcag cccgcctatc agtctcgaat gggatacaat 1020 gtcagcccca acgatggaac aaatggtttg atacagcagt tctcgaatca ggatttaaac 1080 tcgaaccgaa cgggtttctt caatcgctcc gcttcgcctg ctcaaagacc ccgtactgca 1140 ggcaatacag cccccggaca gcagcagcaa cctggacact tggcccctcc agtgcctcgc 1200 agccctcggc tgccccccga gaacgaagaa cttcaacgct acccagagcg cttctctgaa 1260 aatgttcaca aacgtggaaa agctgcgaag gagttggtca acgtattctt tcacgagaat 1320 atcgagcgtg cgcgtgatcg caacatgcgg tgggtttttg ctactgagcg ccgtatttct 1380 ctaaaaagaa ttttgctaac tggagttata actgtacagt tcggcggagc tcgacaagat 1440 gatgcgcgac cccaacattt cacaagatgc aaaggtgaag gaggcggaaa tggttggaaa 1500 gaaagagtcg acattccttc gcttccttcg gacaccagaa actcctgcca acttccaaac 1560 catcaagatt attggaaagg gtgcttttgg tgaagttaag ctggtgcaga ggaagtctga 1620 taacaagatc tatgcgctta agtcgctgat caaatcagag atgtttaaga aagatcagct 1680 cgcccacgtt cgtgctgaac gtgatattct agctgactcg aaggacaacc cttggcttgt 1740 caagctccat gcttcattcc aggatcccgc atacctatac ctcctgatgg agttcttacc 1800 tggaggtgat ttgatgacca tgcttattaa gtacgaaata ttctctgaag atatcacacg 1860 cttctacatg gcggaaattg tgatggcgat tgaggcggtt cacaagctgg gtttccttca 1920 ccggtgagaa taacaatcct ggtctctcgt accatataca gcgtgctaat atacttgtac 1980 tatagagata ttaaacctga caacatcctt ctcgatcgtg gcggtcacgt caagctgacc 2040 gatttcggtc tctcaactgg aggcaagaaa actcacgaca actcatacta tcagaacctg 2100 ttgaagaatt caacatccaa ggataagaac cgaaactctg gatacttcaa cgatgctatc 2160 aacttgacag tatcgaaccg tgggcagatc aacacctgga gaaaatctcg cagggctatg 2220 gcttactcca ctgtcggaac acctgactac attgcacccg aaatttttaa tggtcaagga 2280 tacacctatc tttgcgactg gtggtccgtc ggtgccatca tgtttgaatg tctcgtgggc 2340 tggcctccat tctgcgccga ggatacgacc gacacctatc gcaagattgt gaactggagg 2400 gaatgcctat atttccccga agaattgaca ctgtctcgtg aatcggaggg tctgattcga 2460 aggtatgtta tgtcagcaat ccatttgagc tgcttgtcta accggagatc agcttcctat 2520 gtgacgcaga acaccgcatc ggcaacgaag gtggccaata cggaggtgct acacagatca 2580 aaaatcaccc attcttccgc ggggtagtat gggatcaact gcgcaaaatc cgggcaccgt 2640 tcgaacccag actgacgtca aatatcgacg tatcatattt cccgattgac gagattcctc 2700 aggaggatac cagcgccatt caccgcgccc aggcacgtgc catgccggat gagcagaatg 2760 ctgagatgag cctgcctttt atcggataca catacaaagc attcaacgcc ttccaggcca 2820 gttgagcatg catttaaagt aagaaatata tttgaatgag ccgatgatgg atgccattgg 2880 aaagttttga agcgggcggg cttgcgttga taacttttca atggcgcatc caggtttttg 2940 tgtcggtcgg catagaccct tgttgattgg tattttcatc aagcatatag cgcatacatc 3000 atgtcactgg acacatgagc atctcactac catatgtg 3038 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211> LENGTH: 269 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 15 gaattcagat tgacatcaag cctgataaca tccttctgga tcgcggtggt cacgtcaagc 60 tgacggactt tggtctgtcc acgggaggaa agaagaccca cgacaactcc tactatcaga 120 atctgctgaa gaactcgacg tcaaaggaca agaaccgcaa ctctggttac ttcaacgatg 180 cgatcaacct gacagtctcc aaccgtggcc agatcaacac ctggagaaag tctcgtcgtg 240 caatggcata ctcgacggta ggcatccgg 269 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16 <211> LENGTH: 635 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 16 Met Asp Pro Asn Asn Asn Asn Arg Leu His Leu Asn Phe Gly Tyr Asn 1 5 10 15 Asp Arg Gly Phe Asn Ala Ala Ala Ala Asn Asn Arg Ala Tyr Pro Thr 20 25 30 Thr Pro Ser Ala Phe Pro Gln Pro Ile Tyr Gln Asn Gln Gly Pro Gln 35 40 45 Asp Tyr Met Asp Ala Gln Asn Gly Ala Tyr Ala Gln Gly Gly Tyr Phe 50 55 60 Met Ala Asn Pro Tyr Gln Ala Gln Ala Ala Tyr Gly Gln Pro His Tyr 65 70 75 80 Gly Gln Asn Leu Gln Ser Pro Gln Pro Ala Tyr Gln Ser Arg Met Gly 85 90 95 Tyr Ser Ala Asn Asp Gly Thr Asn Gly Leu Ile Gln Gln Phe Ser Asn 100 105 110 Gln Asp Leu Asn Ser Pro Arg Ser Gly Phe Phe Ala Arg Ser Ala Ser 115 120 125 Pro Ala Gln Arg Leu Arg Thr Ala Gly Ser Pro Ala Pro Gly Gln Gln 130 135 140 Gln Pro Gly His Leu Ala Pro Pro Met Pro Arg Ser Pro Arg Thr Pro 145 150 155 160 Ala Glu Asn Glu Glu Leu Gln Arg Tyr Pro Glu Arg Tyr Ser Glu Asn 165 170 175 Val His Lys Arg Gly Lys Ala Ala Lys Glu Leu Val Ser Val Phe Phe 180 185 190 Asn Glu Asn Ile Glu Arg Ala Arg Asp Arg Asn Met Arg Ser Ala Glu 195 200 205 Leu Asp Lys Met Ile Arg Glu Pro Ser Ile Pro Lys Glu Asn Lys Cys 210 215 220 Lys Asp Ala Glu Val Leu Ala Lys Lys Glu Ser Asn Phe Leu Arg Phe 225 230 235 240 Leu Arg Thr Lys Glu Thr Pro Gln Asn Phe Gln Thr Ile Lys Ile Ile 245 250 255 Gly Lys Gly Ala Phe Gly Glu Val Lys Leu Val Gln Arg Lys Thr Asp 260 265 270 Gly Lys Ile Tyr Ala Leu Lys Ser Leu Ile Lys Thr Glu Met Phe Lys 275 280 285 Lys Asp Gln Leu Ala His Val Arg Ala Glu Arg Asp Ile Leu Ala Asp 290 295 300 Ser Lys Asp Asn Pro Trp Leu Val Lys Leu His Ala Ser Phe Gln Asp 305 310 315 320 Thr Ala Tyr Leu Tyr Leu Leu Met Glu Phe Leu Pro Gly Gly Asp Leu 325 330 335 Met Thr Met Leu Ile Lys Tyr Glu Ile Phe Ser Glu Asp Ile Thr Arg 340 345 350 Phe Tyr Met Ala Glu Ile Val Met Ala Ile Glu Ala Val His Lys Leu 355 360 365 Gly Phe Leu His Arg Asp Ile Lys Pro Asp Asn Ile Leu Leu Asp Arg 370 375 380 Gly Gly His Val Lys Leu Thr Asp Phe Gly Leu Ser Thr Gly Gly Lys 385 390 395 400 Lys Thr His Asp Asn Ser Tyr Tyr Gln Asn Leu Leu Lys Asn Ser Thr 405 410 415 Ser Lys Asp Lys Asn Arg Asn Ser Gly Tyr Phe Asn Asp Ala Ile Asn 420 425 430 Leu Thr Val Ser Asn Arg Gly Gln Ile Asn Thr Trp Arg Lys Ser Arg 435 440 445 Arg Ala Met Ala Tyr Ser Thr Val Gly Thr Pro Asp Tyr Ile Ala Pro 450 455 460 Glu Ile Phe Asn Gly Gln Gly Tyr Thr Tyr Leu Cys Asp Trp Trp Ser 465 470 475 480 Val Gly Ala Ile Met Phe Glu Cys Leu Val Gly Trp Pro Pro Phe Cys 485 490 495 Ala Glu Asp Thr Thr Asp Thr Tyr Arg Lys Ile Val Asn Trp Arg Glu 500 505 510 Cys Leu Tyr Phe Pro Glu Glu Leu Thr Leu Ser Arg Asp Ser Glu Gly 515 520 525 Leu Ile Arg Ser Phe Leu Cys Asp Ala Glu His Arg Ile Gly Ser Asp 530 535 540 Gly Gly Gln Phe Gly Gly Ala Thr Gln Ile Lys Asn His Pro Phe Phe 545 550 555 560 Arg Gly Val Val Trp Glu Gln Leu Arg Ser Ile Arg Ala Pro Phe Glu 565 570 575 Pro Arg Leu Ser Ser Asn Ile Asp Val Ser Tyr Phe Pro Ile Asp Glu 580 585 590 Ile Pro Gln Glu Asp Thr Ser Ala Ile His Arg Ala Gln Ala Arg Ala 595 600 605 Lys Pro Asp Glu Gln Glu Ala Glu Met Ser Leu Pro Phe Ile Gly Tyr 610 615 620 Thr Tyr Lys Ala Phe Asn Ala Phe Gln Gly Asn

625 630 635 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 17 <211> LENGTH: 550 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 17 Met Asp Pro Asn Asn Asn Asn Arg Leu His Leu Asn Phe Gly Tyr Asn 1 5 10 15 Asp Arg Gly Phe Asn Ala Ala Ala Ala Asn Asn Arg Ala Tyr Pro Thr 20 25 30 Thr Pro Ser Ala Phe Pro Gln Pro Ile Tyr Gln Asn Gln Gly Pro Gln 35 40 45 Asp Tyr Met Asp Ala Gln Asn Gly Ala Tyr Ala Gln Gly Gly Tyr Phe 50 55 60 Met Ala Asn Pro Tyr Gln Ala Gln Ala Ala Tyr Gly Gln Pro His Tyr 65 70 75 80 Gly Gln Asn Leu Gln Ser Pro Gln Pro Ala Tyr Ser Arg Met Gly Tyr 85 90 95 Ser Ala Asn Asp Gly Thr Asn Gly Leu Ile Gln Gln Phe Ser Asn Gln 100 105 110 Asp Leu Asn Ser Pro Arg Ser Gly Phe Phe Ala Arg Ser Ala Ser Pro 115 120 125 Ala Gln Arg Pro Arg Thr Ala Gly Ser Pro Ala Pro Gly Gln Gln Gln 130 135 140 Pro Gly His Leu Ala Pro Pro Met Pro Arg Ser Pro Arg Thr Pro Ala 145 150 155 160 Glu Asn Glu Glu Leu Gln Arg Tyr Pro Glu Arg Tyr Ser Glu Asn Val 165 170 175 His Lys Arg Gly Lys Ala Ala Lys Glu Leu Val Ser Val Phe Phe Asn 180 185 190 Glu Asn Asn Glu Arg Ala Arg Asp Arg Asn Met Arg Ser Ala Glu Leu 195 200 205 Asp Lys Met Ile Arg Glu Pro Ser Ile Pro Lys Glu Asn Lys Cys Lys 210 215 220 Asp Ala Glu Val Leu Ala Lys Lys Glu Ser Asn Phe Leu Arg Phe Leu 225 230 235 240 Arg Thr Lys Glu Thr Pro Gln Asn Phe Gln Thr Ile Lys Ile Ile Gly 245 250 255 Lys Gly Ala Phe Gly Glu Val Lys Leu Val Gln Arg Lys Ala Asp Gly 260 265 270 Lys Ile Tyr Ala Leu Lys Ser Leu Ile Lys Thr Glu Met Phe Lys Gln 275 280 285 Gly Pro Ala Ala His Val Arg Ala Glu Arg Asp Ile Leu Ala Asp Ser 290 295 300 Lys Asp Asn Pro Trp Leu Val Lys Leu His Ala Ser Phe Gln Asp Thr 305 310 315 320 Ala Tyr Leu Tyr Leu Leu Met Glu Phe Leu Pro Gly Gly Asp Leu Met 325 330 335 Thr Met Leu Ile Lys Tyr Glu Ile Phe Ser Glu Asp Ile Thr Arg Phe 340 345 350 Tyr Met Ala Glu Ile Val Met Ala Ile Glu Ala Val His Lys Leu Gly 355 360 365 Phe Leu His Arg Asp Ile Lys Pro Asp Asn Ile Leu Leu Asp Arg Gly 370 375 380 Gly His Val Lys Leu Thr Asp Phe Gly Leu Ser Thr Gly Gly Lys Lys 385 390 395 400 Thr His Asp Asn Ser Tyr Tyr Gln Asn Leu Leu Lys Asn Ser Thr Ser 405 410 415 Lys Asp Lys Asn Arg Asn Ser Gly Tyr Phe Asn Asp Ala Ile Asn Leu 420 425 430 Thr Val Ser Asn Arg Gly Gln Ile Asn Thr Trp Arg Lys Ser Arg Arg 435 440 445 Ala Met Ala Tyr Ser Thr Val Gly Thr Pro Asp Tyr Ile Ala Pro Glu 450 455 460 Ile Phe Asn Gly Gln Gly Tyr Thr Tyr Leu Cys Asp Trp Trp Ser Val 465 470 475 480 Gly Ala Ile Met Phe Glu Cys Leu Val Gly Trp Pro Pro Phe Cys Ala 485 490 495 Glu Asp Thr Thr Asp Thr Tyr Arg Lys Ile Val Asn Trp Arg Glu Cys 500 505 510 Leu Tyr Phe Pro Glu Glu Leu Thr Leu Ser Arg Asp Ser Glu Gly Leu 515 520 525 Ile Arg Ser Thr Lys Asn Ile Asp Gly Glu Val Lys Lys Glu Asp Ser 530 535 540 Asp Pro Leu Gly Asn Gln 545 550 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 18 <211> LENGTH: 598 <212> TYPE: PRT <213> ORGANISM: Neurospora crassa <400> SEQUENCE: 18 Met Asp Asn Thr Asn Arg Pro His Leu Asn Leu Gly Thr Asn Asp Thr 1 5 10 15 Arg Met Ala Pro Asn Asp Arg Thr Tyr Pro Thr Thr Pro Ser Thr Phe 20 25 30 Pro Gln Pro Val Phe Pro Gly Gln Gln Ala Gly Gly Ser Gln Gln Tyr 35 40 45 Asn Gln Ala Tyr Ala Gln Ser Gly Asn Tyr Tyr Gln Gln Asn His Asn 50 55 60 Asp Pro Asn Thr Gly Leu Ala His Gln Phe Ala His Gln Asn Ile Gly 65 70 75 80 Ser Ala Gly Arg Ala Ser Pro Tyr Gly Ser Arg Gly Pro Ser Pro Ala 85 90 95 Gln Arg Pro Arg Thr Ser Gly Asn Ser Gly Gln Gln Gln Thr Tyr Gly 100 105 110 Asn Tyr Leu Ser Ala Pro Met Pro Ser Asn Thr Gln Thr Glu Phe Ala 115 120 125 Pro Ala Pro Glu Arg Asn Pro Asp Lys Tyr Gly Pro Asn Ala Asn Asn 130 135 140 Asn Gln Lys Lys Cys Ser Gln Leu Ala Ser Asp Phe Phe Lys Asp Ser 145 150 155 160 Val Lys Arg Ala Arg Glu Arg Asn Gln Arg Gln Ser Glu Met Glu Gln 165 170 175 Lys Leu Gly Glu Thr Asn Asp Ala Arg Arg Arg Glu Ser Ile Trp Ser 180 185 190 Thr Ala Gly Arg Lys Glu Gly Gln Tyr Leu Arg Phe Leu Arg Thr Lys 195 200 205 Asp Lys Pro Glu Asn Tyr Gln Thr Ile Lys Ile Ile Gly Lys Gly Ala 210 215 220 Phe Gly Glu Val Lys Leu Val Gln Lys Lys Ala Asp Gly Lys Val Tyr 225 230 235 240 Ala Met Lys Ser Leu Ile Lys Thr Glu Met Phe Lys Lys Asp Gln Leu 245 250 255 Ala His Val Arg Ala Glu Arg Asp Ile Leu Ala Glu Ser Asp Ser Pro 260 265 270 Trp Val Val Lys Leu Tyr Thr Thr Phe Gln Asp Ala Asn Phe Leu Tyr 275 280 285 Met Leu Met Glu Phe Leu Pro Gly Gly Asp Leu Met Thr Met Leu Ile 290 295 300 Lys Tyr Glu Ile Phe Ser Glu Asp Ile Thr Arg Phe Tyr Ile Ala Glu 305 310 315 320 Ile Val Leu Ala Ile Asp Ala Val His Lys Leu Gly Phe Ile His Arg 325 330 335 Asp Ile Lys Pro Asp Asn Ile Leu Leu Asp Arg Gly Gly His Val Lys 340 345 350 Leu Thr Asp Phe Gly Leu Ser Thr Gly Phe His Lys Leu His Asp Asn 355 360 365 Asn Tyr Tyr Thr Gln Leu Leu Gln Gly Lys Ser Asn Lys Pro Arg Asp 370 375 380 Asn Arg Asn Ser Val Ala Ile Asp Gln Ile Asn Leu Thr Val Ser Asn 385 390 395 400 Arg Ala Gln Ile Asn Asp Trp Arg Arg Ser Arg Arg Leu Met Ala Tyr 405 410 415 Ser Thr Val Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Phe Thr Gly 420 425 430 His Gly Tyr Ser Phe Asp Cys Asp Trp Trp Ser Leu Gly Thr Ile Met 435 440 445 Phe Glu Cys Leu Val Gly Trp Pro Pro Phe Cys Ala Glu Asp Ser His 450 455 460 Asp Thr Tyr Arg Lys Ile Val Asn Trp Arg His Ser Leu Tyr Phe Pro 465 470 475 480 Asp Asp Ile Thr Leu Gly Val Asp Ala Glu Asn Leu Ile Arg Ser Leu 485 490 495 Ile Cys Asn Thr Glu Asn Arg Leu Gly Arg Gly Gly Ala His Glu Ile 500 505 510 Lys Ser His Ala Phe Phe Arg Gly Val Glu Phe Asp Ser Leu Arg Arg 515 520 525 Ile Arg Ala Pro Phe Glu Pro Arg Leu Thr Ser Ala Ile Asp Thr Thr 530 535 540 Tyr Phe Pro Thr Asp Glu Ile Asp Gln Thr Asp Asn Ala Thr Leu Leu 545 550 555 560 Lys Ala Gln Gln Ala Ala Arg Gly Ala Ala Ala Pro Ala Gln Gln Glu 565 570 575 Glu Ser Pro Glu Leu Ser Leu Pro Phe Ile Gly Tyr Thr Phe Lys Arg 580 585 590 Phe Asp Asn Asn Phe Arg 595 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19 <211> LENGTH: 665 <212> TYPE: PRT <213> ORGANISM: Colletotrichum trifolii <400> SEQUENCE: 19 Met Asp Asn Asn Asn Asn Arg Leu Tyr Leu Asn Ile Gly Asn Asn Asn 1 5 10 15 Asp Arg Leu Gly Pro Gly Ser Asp Arg Gln Tyr Pro Thr Thr Pro Ser 20 25 30 Thr Phe Pro Gln Pro Val Phe Pro His Gln Gly Gln Gln Gln Gln Gln 35 40 45 Gln Gln Gln Gln Gln Leu His His Gln Gln Gln Pro Gly Met Gln His 50 55 60 Pro Gln Gln Tyr Gln Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln Gln Gln Pro Tyr Gln Thr Gly Tyr Ala Pro Ser Gly Tyr Phe 85 90 95 Asn Pro Asn Gln Gln Ala Ala Gln Tyr Pro Pro Gln Gly His Gly Asp 100 105 110 Tyr Asn Ala Ala Tyr Gln Pro Arg Ser Asn Thr Pro Gly Thr Asn Asp 115 120 125 Pro Asn Val Gly Leu Ala His Gln Phe Ser His Gln Asn Leu Gly Gly 130 135 140 Ala Ala Arg Ala Ser Pro Tyr Gly Ser Arg Gly Pro Ser Pro Gly Gln 145 150 155 160 Arg Pro Arg Thr Ala Gly Ala Ser Gly Gln Pro Pro Ser Gly Tyr Gly 165 170 175 His Tyr Ala Thr Pro Pro Leu Pro Asn Gln Gln Pro Ala Ser Val Asp 180 185 190 Pro Phe Ala Pro Ala Pro Glu Arg Asn Tyr Glu Lys Tyr Gly Pro Asn 195 200 205 Ala Asn Gly Asn Gln Lys Lys Cys Thr Gln Leu Ala Ser Asp Phe Phe 210 215 220 Lys Asp Ser Val Lys Arg Ala Arg Glu Arg Asn Gln Arg Gln Ser Glu 225 230 235 240 Met Glu Ala Lys Leu Ser Glu Pro Asn Gln Ser Gln Ser Arg Arg Glu 245 250 255 Gln Ile Trp Ser Thr Ala Gly Arg Lys Glu Gly Gln Tyr Leu Arg Phe 260 265 270 Leu Arg Thr Lys Asp Lys Pro Glu Asn Tyr Asn Thr Val Lys Ile Ile 275 280 285 Gly Lys Gly Ala Phe Gly Glu Val Lys Leu Val Gln Lys Lys Gly Asp 290 295 300 Gly Lys Val Tyr Ala Met Lys Ser Leu Ile Lys Thr Glu Met Phe Lys 305 310 315 320 Lys Asp Gln Leu Ala His Val Arg Ser Glu Arg Asp Ile Leu Ala Glu 325 330 335 Ser Asp Ser Pro Trp Val Val Lys Leu Tyr Thr Thr Phe Gln Asp Ser 340 345 350 Tyr Phe Leu Tyr Met Leu Met Glu Phe Leu Pro Gly Gly Asp Leu Met 355 360 365 Thr Met Leu Ile Lys Tyr Glu Ile Phe Ser Glu Asp Ile Thr Arg Phe 370 375 380 Tyr Ile Ala Glu Ile Val Leu Ala Ile Glu Ala Val His Lys Leu Gly 385 390 395 400 Phe Ile His Arg Asp Ile Lys Pro Asp Asn Ile Leu Leu Asp Arg Gly 405 410 415 Gly His Val Lys Leu Thr Asp Phe Gly Leu Ser Thr Gly Phe Asn Arg 420 425 430 Leu His Asp Asn Asn Tyr Tyr Gln Gln Leu Leu Gln Gly Arg Ser Asn 435 440 445 Lys Pro Arg Asp Arg Asn Ser Val Ala Ile Asp Gln Ile Asn Leu Thr 450 455 460 Val Ser Asn Arg Ser Gln Ile Asn Asp Trp Arg Arg Ser Arg Arg Leu 465 470 475 480 Met Ala Tyr Ser Thr Val Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile 485 490 495 Phe Thr Gly His Gly Tyr Thr Phe Asp Cys Asp Trp Trp Ser Leu Gly 500 505 510 Thr Ile Met Phe Glu Cys Leu Val Gly Trp Pro Pro Phe Cys Ala Glu 515 520 525 Asp Ser His Asp Thr Tyr Arg Lys Ile Val Asn Trp Arg Gln Thr Leu 530 535 540 Tyr Phe Pro Asp Asp Ile Gln Leu Gly Val Glu Ala Glu Asn Leu Ile 545 550 555 560 Arg Ser Leu Ile Cys Asn Thr Glu Asn Arg Leu Gly Arg Ser Gly Ala 565 570 575 His Glu Ile Lys Ala His Ser Phe Phe Arg Gly Val Glu Phe Asp Ser 580 585 590 Leu Arg Arg Ile Arg Ala Pro Phe Glu Pro Arg Leu Thr Ser Ala Ile 595 600 605 Asp Thr Thr Tyr Phe Pro Thr Asp Glu Ile Asp Gln Thr Asp Asn Ala 610 615 620 Thr Val Leu Lys Ala Gln Ala Ile Gln Gln Ala Arg Ser Gly Ile Pro 625 630 635 640 Gln Val Glu Glu Ser Pro Glu Met Ser Leu Pro Phe Ile Gly Tyr Thr 645 650 655 Phe Lys Arg Phe Asp Asn Asn Phe Arg 660 665 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20

<211> LENGTH: 756 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 20 Met Tyr Asn Ser Ser Thr Asn His His Glu Gly Ala Pro Thr Ser Gly 1 5 10 15 His Gly Tyr Tyr Met Ser Gln Gln Gln Asp Gln Gln His Gln Gln Gln 20 25 30 Gln Gln Tyr Ala Asn Glu Met Asn Pro Tyr Gln Gln Ile Pro Arg Pro 35 40 45 Pro Ala Ala Gly Phe Ser Ser Asn Tyr Met Lys Glu Gln Gly Ser His 50 55 60 Gln Ser Leu Gln Glu His Leu Gln Arg Glu Thr Gly Asn Leu Gly Ser 65 70 75 80 Gly Phe Thr Asp Val Pro Ala Leu Asn Tyr Pro Ala Thr Pro Pro Pro 85 90 95 His Asn Asn Tyr Ala Ala Ser Asn Gln Met Ile Asn Thr Pro Pro Pro 100 105 110 Ser Met Gly Gly Leu Tyr Arg His Asn Asn Asn Ser Gln Ser Met Val 115 120 125 Gln Asn Gly Asn Gly Ser Gly Asn Ala Gln Leu Pro Gln Leu Ser Pro 130 135 140 Gly Gln Tyr Ser Ile Glu Ser Glu Tyr Asn Gln Asn Leu Asn Gly Ser 145 150 155 160 Ser Ser Ser Ser Pro Phe His Gln Pro Gln Thr Leu Arg Ser Asn Gly 165 170 175 Ser Tyr Ser Ser Gly Leu Arg Ser Val Lys Ser Phe Gln Arg Leu Gln 180 185 190 Gln Glu Gln Glu Asn Val Gln Val Gln Gln Gln Leu Ser Gln Ala Gln 195 200 205 Gln Gln Asn Ser Arg Gln Gln Gln Gln Gln Leu Gln Tyr Gln Gln Gln 210 215 220 Gln Gln Gln Gln Gln Gln Gln Gln His Met Gln Ile Gln Gln Gln Gln 225 230 235 240 Gln Gln Gln Gln Gln Gln Gln Gln Ser Gln Ser Pro Val Gln Ser Gly 245 250 255 Phe Asn Asn Gly Thr Ile Ser Asn Tyr Met Tyr Phe Glu Arg Arg Pro 260 265 270 Asp Leu Leu Thr Lys Gly Thr Gln Asp Lys Ala Ala Ala Val Lys Leu 275 280 285 Lys Ile Glu Asn Phe Tyr Gln Ser Ser Val Lys Tyr Ala Ile Glu Arg 290 295 300 Asn Glu Arg Arg Val Glu Leu Glu Thr Glu Leu Thr Ser His Asn Trp 305 310 315 320 Ser Glu Glu Arg Lys Ser Arg Gln Leu Ser Ser Leu Gly Lys Lys Glu 325 330 335 Ser Gln Phe Leu Arg Leu Arg Arg Thr Arg Leu Ser Leu Glu Asp Phe 340 345 350 His Thr Val Lys Val Ile Gly Lys Gly Ala Phe Gly Glu Val Arg Leu 355 360 365 Val Gln Lys Lys Asp Thr Gly Lys Ile Tyr Ala Met Lys Thr Leu Leu 370 375 380 Lys Ser Glu Met Tyr Lys Lys Asp Gln Leu Ala His Val Lys Ala Glu 385 390 395 400 Arg Asp Val Leu Ala Gly Ser Asp Ser Pro Trp Val Val Ser Leu Tyr 405 410 415 Tyr Ser Phe Gln Asp Ala Gln Tyr Leu Tyr Leu Ile Met Glu Phe Leu 420 425 430 Pro Gly Gly Asp Leu Met Thr Met Leu Ile Arg Trp Gln Leu Phe Thr 435 440 445 Glu Asp Val Thr Arg Phe Tyr Met Ala Glu Cys Ile Leu Ala Ile Glu 450 455 460 Thr Ile His Lys Leu Gly Phe Ile His Arg Asp Ile Lys Pro Asp Asn 465 470 475 480 Ile Leu Ile Asp Ile Arg Gly His Ile Lys Leu Ser Asp Phe Gly Leu 485 490 495 Ser Thr Gly Phe His Lys Thr His Asp Ser Asn Tyr Tyr Lys Lys Leu 500 505 510 Leu Gln Gln Asp Glu Ala Thr Asn Gly Ile Ser Lys Pro Gly Thr Tyr 515 520 525 Asn Ala Asn Thr Thr Asp Thr Ala Asn Lys Arg Gln Thr Met Val Val 530 535 540 Asp Ser Ile Ser Leu Thr Met Ser Asn Arg Gln Gln Ile Gln Thr Trp 545 550 555 560 Arg Lys Ser Arg Arg Leu Met Ala Tyr Ser Thr Val Gly Thr Pro Asp 565 570 575 Tyr Ile Ala Pro Glu Ile Phe Leu Tyr Gln Gly Tyr Gly Gln Glu Cys 580 585 590 Asp Trp Trp Ser Leu Gly Ala Ile Met Tyr Glu Cys Leu Ile Gly Trp 595 600 605 Pro Pro Phe Cys Ser Glu Thr Pro Gln Glu Thr Tyr Arg Lys Ile Met 610 615 620 Asn Phe Glu Gln Thr Leu Gln Phe Pro Asp Asp Ile His Ile Ser Tyr 625 630 635 640 Glu Ala Glu Asp Leu Ile Arg Arg Leu Leu Thr His Ala Asp Gln Arg 645 650 655 Leu Gly Arg His Gly Gly Ala Asp Glu Ile Lys Ser His Pro Phe Phe 660 665 670 Arg Gly Val Asp Trp Asn Thr Ile Arg Gln Val Glu Ala Pro Tyr Ile 675 680 685 Pro Lys Leu Ser Ser Ile Thr Asp Thr Arg Phe Phe Pro Thr Asp Glu 690 695 700 Leu Glu Asn Val Pro Asp Ser Pro Ala Met Ala Gln Ala Ala Lys Gln 705 710 715 720 Arg Glu Gln Met Thr Lys Gln Gly Gly Ser Ala Pro Val Lys Glu Asp 725 730 735 Leu Pro Phe Ile Gly Tyr Thr Tyr Ser Arg Phe Asp Tyr Leu Thr Arg 740 745 750 Lys Asn Ala Leu 755 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 21 <211> LENGTH: 639 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 Met Gly Gly His Phe Trp Pro Pro Glu Pro Tyr Thr Val Phe Met Trp 1 5 10 15 Gly Ser Pro Trp Glu Ala Asp Ser Pro Arg Val Lys Leu Arg Gly Arg 20 25 30 Glu Lys Gly Arg Gln Thr Glu Gly Gly Ala Phe Pro Leu Val Ser Ser 35 40 45 Ala Leu Ser Gly Asp Pro Arg Phe Phe Ser Pro Thr Thr Pro Pro Ala 50 55 60 Glu Pro Ile Val Val Arg Leu Lys Glu Val Arg Leu Gln Arg Asp Asp 65 70 75 80 Phe Glu Ile Leu Lys Val Ile Gly Arg Gly Ala Phe Ser Glu Val Ala 85 90 95 Val Val Lys Met Lys Gln Thr Gly Gln Val Tyr Ala Met Lys Ile Met 100 105 110 Asn Lys Trp Asp Met Leu Lys Arg Gly Glu Val Ser Cys Phe Arg Glu 115 120 125 Glu Arg Asp Val Leu Val Asn Gly Asp Arg Arg Trp Ile Thr Gln Leu 130 135 140 His Phe Ala Phe Gln Asp Glu Asn Tyr Leu Tyr Leu Val Met Glu Tyr 145 150 155 160 Tyr Val Gly Gly Asp Leu Leu Thr Leu Leu Ser Lys Phe Gly Glu Arg 165 170 175 Ile Pro Ala Glu Met Ala Arg Phe Tyr Leu Ala Glu Ile Val Met Ala 180 185 190 Ile Asp Ser Val His Arg Leu Gly Tyr Val His Arg Asp Ile Lys Pro 195 200 205 Asp Asn Ile Leu Leu Asp Arg Cys Gly His Ile Arg Leu Ala Asp Phe 210 215 220 Gly Ser Cys Leu Lys Leu Arg Ala Asp Gly Thr Val Arg Ser Leu Val 225 230 235 240 Ala Val Gly Thr Pro Asp Tyr Leu Ser Pro Glu Ile Leu Gln Ala Val 245 250 255 Gly Gly Gly Pro Gly Thr Gly Ser Tyr Gly Pro Glu Cys Asp Trp Trp 260 265 270 Ala Leu Gly Val Phe Ala Tyr Glu Met Phe Tyr Gly Gln Thr Pro Phe 275 280 285 Tyr Ala Asp Ser Thr Ala Glu Thr Tyr Gly Lys Ile Val His Tyr Lys 290 295 300 Glu His Leu Ser Leu Pro Leu Val Asp Glu Gly Val Pro Glu Glu Ala 305 310 315 320 Arg Asp Phe Ile Gln Arg Leu Leu Cys Pro Pro Glu Thr Arg Leu Gly 325 330 335 Arg Gly Gly Ala Gly Asp Phe Arg Thr His Pro Phe Phe Phe Gly Leu 340 345 350 Asp Trp Asp Gly Leu Arg Asp Ser Val Pro Pro Phe Thr Pro Asp Phe 355 360 365 Glu Gly Ala Thr Asp Thr Cys Asn Phe Asp Leu Val Glu Asp Gly Leu 370 375 380 Thr Ala Met Val Ser Gly Gly Gly Glu Thr Leu Ser Asp Ile Arg Glu 385 390 395 400 Gly Ala Pro Leu Gly Val His Leu Pro Phe Val Gly Tyr Ser Tyr Ser 405 410 415 Cys Met Ala Leu Arg Asp Ser Glu Val Pro Gly Pro Thr Pro Met Glu 420 425 430 Val Glu Ala Glu Gln Leu Leu Glu Pro His Val Gln Ala Pro Ser Leu 435 440 445 Glu Pro Ser Val Ser Pro Gln Asp Glu Thr Ala Glu Val Ala Val Pro 450 455 460 Ala Ala Val Pro Ala Ala Glu Ala Glu Ala Glu Val Thr Leu Arg Glu 465 470 475 480 Leu Gln Glu Ala Leu Glu Glu Glu Val Leu Thr Arg Gln Ser Leu Ser 485 490 495 Arg Glu Met Glu Ala Ile Arg Thr Asp Asn Gln Asn Phe Ala Ser Gln 500 505 510 Leu Arg Glu Ala Glu Ala Arg Asn Arg Asp Leu Glu Ala His Val Arg 515 520 525 Gln Leu Gln Glu Arg Met Glu Leu Leu Gln Ala Glu Gly Ala Thr Ala 530 535 540 Val Thr Gly Val Pro Ser Pro Arg Ala Thr Asp Pro Pro Ser His Leu 545 550 555 560 Asp Gly Pro Pro Ala Val Ala Val Gly Gln Cys Pro Leu Val Gly Pro 565 570 575 Gly Pro Met His Arg Arg His Leu Leu Leu Pro Ala Arg Val Pro Arg 580 585 590 Pro Gly Leu Ser Glu Ala Leu Ser Leu Leu Leu Phe Ala Val Val Leu 595 600 605 Ser Arg Ala Ala Ala Leu Gly Cys Ile Gly Leu Val Ala His Ala Gly 610 615 620 Gln Leu Thr Ala Val Trp Arg Arg Pro Gly Ala Ala Arg Ala Pro 625 630 635

* * * * *

Field of search:

Other references:

References: