CN1558913A - Novel genetic products obtained from ashbya gossypii, which are associated with transcription mechanisms, RNA processing and/or translation - Google Patents

Abstract

The invention relates to novel polynucleotides obtained from Ashbya gossypii, to oligonucleotides that hybridise with the latter, to expression cassettes and vectors containing said polynucleotides, to micro-organisms transformed therewith, to polypeptides that have been coded by said polynucleotides and to the use of the novel polypeptides and polynucleotides as targets for modulating the control of transcription and/or translation and/or the control of RNA processing and in particular the improvement of vitamin B2 production in micro-organisms of the genus Ashbya.

Description

Novel gene products from Ashbya gossypii associated with transcriptional, RNA processing and/or translational mechanisms

Technical Field

The present invention relates to novel polynucleotides from Ashbya gossypii (Ashbya gossypii); an oligonucleotide hybridized thereto; expression cassettes and vectors comprising these polynucleotides; a microorganism transformed with the polynucleotide; polypeptides encoded by these polynucleotides; and the use of the novel polypeptides and polynucleotides as targets for modulating the transcription, RNA processing and/or translation operations of microorganisms of the genus Ashbya, and in particular for increasing the production of vitamin B2.

Background

Vitamin B2 (riboflavin, lactoflavin) is an alkali-and light-sensitive vitamin that exhibits yellow-green fluorescence in solution. Deficiency of vitamin B2 can lead to damage to the ectoderm, especially cataracts, keratitis, corneal vascularization, or to disorders of the autonomous genitourinary system. Vitamin B2 is a precursor to FAD and FMN molecules, both of which react with NAD⁺And NADP⁺Biologically important for hydrogen transfer. Vitamin B2 forms both molecules by phosphorylation (FMN) and subsequent adenylation (FAD).

Vitamin B2 is synthesized in plants, yeast and various microorganisms from GTP and ketonic acid-5-phosphate. This reaction pathway first opens the pyrimidine ring of GTP and eliminates one phosphate residue. The remaining phosphate is deaminated, reduced, and eliminated to form 5-amino-6-ribityl amino-2, 4-pyrimidinone. The compound reacts with 3, 4-dihydroxy-2-butanone-4-phosphoric acid to generate bicyclic molecule 6, 7-dimethyl-8-ribitol-based dioxy tetrahydropteridine. The compound is converted to the tricyclic compound riboflavin by a disproportionation reaction that displaces one 4-carbon unit.

Vitamin B2 is present in many vegetables and meats and is present in lower levels in cereal products. Vitamin B2 is required in an amount of about 1.4 to 2 mg per day for an adult. Riboflavin is again the main decomposition product of the coenzymes FMN and FAD in the human body and is excreted as such.

Vitamin B2 is an important nutrient for humans and animals. Researchers have therefore endeavored to make vitamin B2 producible on an industrial scale and suggested that vitamin B2 be synthesized by a microbial pathway. Microorganisms which can be used for this purpose are, for example, Bacillus subtilis, Ashbya ascomycete (Eremothecium ashbyii), Ashbya gossypii and the yeast Candida flareri and Saccharomyces cerevisiae (Saccharomyces cerevisiae). The nutrient medium used for this purpose comprises molasses or vegetable oil as carbon source, inorganic salts, amino acids, animal or vegetable peptones and protein and vitamin additives. In the sterile aerobic submerged culture method, vitamin B2 was produced at a yield of 10g or more per liter of culture solution in several days. The conditions required are good aeration of the culture broth, careful agitation and setting of a temperature below about 30 ℃. The biomass was removed, the concentrate evaporated and dried to give a product enriched in vitamin B2.

For example, microbial production of vitamin B2 is described in WO-A-92/01060, EP-A-0405370 and EP-A-0531708.

Investigations concerning the importance, appearance, production, biosynthesis and use of vitamin B2 can be found in Ullmann's encyclopedia of Industrial chemistry, volume A27, page 521 and the following.

Transcription

Fungal gene expression is controlled primarily at the transcriptional level. Transcription devices are composed of a variety of proteins that can be divided into two groups: RNA polymerase (operational DNA transcriptase) and transcription factors (which control gene transcription by directing RNA polymerase onto specific promoter DNA sequences that recognize these factors). Fungi such as Ashbya gossypii contain a variety of different transcription factors specific for different promoters, growth phases, environmental conditions, substrates, oxygen levels, etc., thereby adapting the organism to different environmental and metabolic conditions.

Promoters are specific DNA sequences that serve as docking sites for RNA polymerase complexes and transcription factors. Many promoter elements have conserved sequence elements that can be identified by homology searches; another possibility is to identify the promoter region of a particular gene using standard techniques such as primer extension. A number of promoter regions are known for eukaryotic cells (Guarente, L (1987), Ann. Rev. biochem., 21; 425-452).

Promoter transcriptional control is affected by a variety of repression or activation mechanisms. Specific regulatory proteins (transcription factors) bind to the promoter, have the ability to block (repressors) or facilitate (activators) RNA holoenzyme binding, and thereby control transcription. In addition, certain enzymes can modify histones that bind to DNA and thereby prevent or enable the first time the proximity of transcription factors to promoters (Loo, S.; Rine, J. (1995); Annu. Rev. cell. Dev. biol., 11, 519-548). The binding of transcription factors is also controlled by their interaction with other molecules such as proteins or other metabolic compounds (Evans, R. (1989), Science, 240, 889-. The ability to respond to a variety of environmental or metabolic signals by controlling gene transcription allows the cell to precisely control when a gene is expressed and how much of the gene product is available in the cell at that time. Thereby preventing unnecessary expenditure of energy or unnecessary use of potentially rare intermediate compounds or cofactors.

RNA processing

RNA is synthesized as heterogeneous fragments, with eukaryotic coding sequences (exons) often interrupted by non-coding sequences (introns). In post-transcriptional RNA processing, introns are excised (spliced) so that the (mRNA) coding sequences can be read on the ribosomes (Sharp, P. (1987), Science; 235, 766-. Since the export of RNA from the cell is also controlled by splicing, the amount of mRNA available on the ribosome can be controlled in this way.

Translation

Translation is the process by which polypeptides are synthesized from amino acids based on the information contained in the RNA molecule. The major components of this process are the ribosomes and specific initiation or elongation factors such as eEF1 and eEF2(Moldave (1985); Ann. Rev. biochem., 54, 1109-. Ribosomes are composed of RNA (rRNA) and specific proteins. It consists of one large and one small subunit, each of which can be characterized by its sedimentation behavior in analytical supercentrifuges. Depending on the physiological state of the cell, ribosome synthesis is controlled by the coordinated production of RNA and protein components.

Each codon of an mRNA molecule encodes a particular amino acid. The conversion of mRNA to amino acids is performed by transfer RNA (tRNA) molecules. These molecules consist of a single strand of RNA (between 60 and 100 bases) with an L-shaped three-dimensional structure with protruding regions or "arms". One of these arms forms a base pair with a specific codon sequence on the mRNA molecule. The other arm specifically interacts with a specific amino acid (encoded by a codon). Other tRNA arms include the variable arm, T Ψ C arm (with thymidylate and pseudouridylate modifications) and D arm (with dihydrouridine modifications). The function of these latter structures is still unknown, but their conservation among tRNA molecules suggests a role in protein synthesis.

To pair a nucleic acid-based tRNA molecule with the correct amino acid requires the action of a family of enzymes called aminoacyl-tRNA synthetases. There are many different enzymes of this type, each specific for a particular tRNA and a particular amino acid. These enzymes bind the 3' -hydroxyl group of the terminal adenylribose unit of the tRNA to the amino acid in a two-step reaction. In the first step, the enzyme is activated by reaction with ATP and amino acids to produce an aminoacyl-tRNA synthetase/aminoacyl-adenylate complex. In the second step, an aminoacyl group is transferred from the enzyme to the tRNA of interest and remains in a high energy state thereon. the binding of the tRNA molecule to the codon recognized by the mRNA molecule causes the high energy amino acid bound to the tRNA to contact the ribosome. Within the ribose body, the amino acid-charged tRNA (aminoacyl-tRNA) occupies a binding site (A site) adjacent to the second site (P site) of the tRNA molecule (peptidyl-tRNA) that carries the amino acid on it to bind to the growing polypeptide chain. The activated amino acid on the aminoacyl-tRNA is sufficiently active to spontaneously form a peptide bond with the nearest amino acid on the growing peptide chain. GTP hydrolysis provides energy for the transfer of the tRNA, now charged with the polypeptide chain, from the A site to the P site of the ribosome, and the process is repeated until the stop codon is reached.

Translation may be controlled at a number of different steps. Including ribosome binding to mRNA, secondary mRNA structure, codon usage or the frequency of a particular tRNA.

There is no description of the use of genes involved in the transcription, RNA processing and/or translation machinery for the production of microorganisms, preferably Ashbya, in particular Ashbya gossypii strains, with improved adaptability to external conditions, such as environmental and metabolic conditions.

Summary of The Invention

It is an object of the present invention to provide novel targets which can be used to influence the transcription and/or translation mechanism and/or RNA processing mechanism of microorganisms of the genus Ashbya, in particular Ashbya gossypii. A particular object is to specifically regulate transcription, RNA processing and/or translation of such microorganisms. It is a further object to improve the production of vitamin B2 by such a microorganism.

We have found that this object is achieved by providing coding nucleic acid sequences which are regulated up or down during the production of vitamin B2 from Ashbya gossypii (based on the results of the MPSS analysis described in detail in the experimental section), in particular as follows:

a) preferably an up-regulated nucleic acid sequence encoding a protein having the function of a 26S proteasome subunit or a TAT binding homolog 7.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and designated internally by the name "Oligo 28".

In yet another preferred embodiment, a DNA clone encoding the complete sequence of the nucleic acid of the present invention is isolated according to the present invention, internally designated "Oligo 28 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 1, or a polynucleotide having the nucleic acid sequence shown in figure 1. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 4 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

The inserts of "Oligo 28" and "Oligo 28 v" have significant homology to the MIPS tag "Yta 7" from Saccharomyces cerevisiae. These inserts have the sequence of SEQ ID NO: 1 or SEQ ID NO: 4. The amino acid sequence derived therefrom has significant sequence homology with the 26S proteasome subunit or TAT binding homolog 7(TBP-7) of Saccharomyces cerevisiae.

b) Preferably an up-regulated nucleic acid sequence encoding a protein having the function of a subunit of a translation initiation factor.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and designated internally by the name "Oligo 45".

In another preferred embodiment a DNA clone encoding the complete sequence of the nucleic acid of the invention is isolated according to the invention under the internal name "Oligo 45 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 6. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 10 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 45" and "Oligo 45 v" with significant homology to the MIPS tag "p 39" or "Tif 34" from saccharomyces cerevisiae have the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 10. The amino acid sequence from it has significant sequence homology with the subunit (P39) of the Saccharomyces cerevisiae translation initiation factor EIF3(IF 32).

c) Preferably a down-regulated nucleic acid sequence encoding a protein having the function of a ribosomal protein.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated under the internal designation "Oligo 85".

In another preferred embodiment of the invention, a DNA clone encoding the complete sequence of the nucleic acid of the invention was isolated according to the invention, with the internal designation "Oligo 85 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 12. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 14 or a fragment thereof. The polynucleotide is preferably isolated from a microorganism of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 85" and "Oligo 85 v" with significant homology to the MIPS tag "Rpl 35 a" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14, or a nucleic acid sequence as set forth in seq id no. The amino acid sequence or amino acid partial sequence from the coding chain has significant sequence homology with saccharomyces cerevisiae ribosomal protein.

d) Preferably an up-regulated nucleic acid sequence encoding a protein having the function of nucleolin.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and designated internally by the name "Oligo 133".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention was isolated according to the present invention, internally designated "Oligo 133 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 17. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 19 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 133" and "Oligo 133 v" having significant homology to the MIPS tag "Nop 13" from saccharomyces cerevisiae, these inserts having the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19. From SEQ ID NO: 17 or the corresponding complementary strand of SEQ ID NO: 19 or the amino acid partial sequence has obvious sequence homology with the saccharomyces cerevisiae nucleolin.

e) Preferably an up-regulated nucleic acid sequence encoding a protein having the function of a translation initiation protein.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 172".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention was isolated according to the present invention, internally designated "Oligo 172 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 21, or a polynucleotide of the nucleic acid sequence shown in figure 21. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 24 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 172 and" Oligo 172v "having significant homology to the MIPS tag" Sua5 "from saccharomyces cerevisiae, these inserts having the amino acid sequence of SEQ ID NO: 21 or SEQ id no: 24. The amino acid sequence or amino acid partial sequence from the coding strand has significant sequence homology with the translation initiation protein from s.cerevisiae.

f) Preferably a down-regulated nucleic acid sequence encoding a protein having the function of a precursor of ribosomal protein S31.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 63".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention was isolated according to the present invention, internally designated "Oligo 63 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 26. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 29 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 63 and" Oligo 63v "having significant homology to the MIPS tag" Rps25a "from saccharomyces cerevisiae, these inserts comprising the amino acid sequence of SEQ ID NO: 26 or SEQ id no: 29, or a variant thereof. From SEQ ID NO: 26 or the corresponding complementary strand of SEQ ID NO: 29 has significant sequence homology with the precursor of S31 ribosomal protein of saccharomyces cerevisiae.

g) Preferably a down-regulated nucleic acid sequence encoding a protein having the function of a nuclear porin.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 132".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention is isolated according to the present invention, internally designated "Oligo 132 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 31, or a polynucleotide having the nucleic acid sequence shown in figure 31. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 36 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 132" and "Oligo 132 v" with significant homology to the MIPS tag "Nic 96" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 36, or a variant thereof. The amino acid sequences from these inserts (corresponding to nucleotides 108 to 764 of SEQ ID NO: 31) have significant sequence homology with the Saccharomyces cerevisiae nucleoporin.

h) Preferably an up-regulated nucleic acid sequence encoding a protein having the function of a component of the ADH-histone acetyltransferase complex.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 174".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention was isolated according to the present invention, internally designated "Oligo 174 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 38, or a polynucleotide of the nucleic acid sequence shown in seq id no. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 40 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 174" and "Oligo 174 v" with significant homology to the MIPS tag "Ahc 1" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 38 or seq id NO: 40, or a variant thereof. From SEQ ID NO: 38 or the corresponding complementary strand of SEQ id no: 40 has significant sequence homology with a component of the saccharomyces cerevisiae ADH-histone acetyltransferase complex.

i) Preferably a down-regulated nucleic acid sequence encoding a protein having the function of an RNA helicase involved in RNA processing.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and designated internally by the name "Oligo 51".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention is isolated according to the present invention, internally designated "Oligo 51 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 42. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 46 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 51" and "Oligo 51 v" having significant homology to the MIPS tag "Rok 1" from saccharomyces cerevisiae, these inserts having the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 46. From SEQ ID NO: 42 or the corresponding complementary strand of SEQ ID NO: 46 has significant sequence homology with the RNA helicase of Saccharomyces cerevisiae involved in RNA processing.

k) Preferably an up-regulated nucleic acid sequence encoding a protein having the function of a non-essential component of RNA pol I.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and designated internally by the name "Oligo 30".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention is isolated according to the present invention, internally designated "Oligo 30 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 48. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 51 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 30" and "Oligo 30 v" with significant homology to the MIPS tag "Rpa 34" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 48 or SEQ ID NO: 51. The amino acid sequence from the coding strand in each case has significant sequence homology with a non-essential component of s.cerevisiae RNA pol I.

l) a nucleic acid sequence, preferably down-regulated, which encodes a protein having the function of an RNA helicase.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 124".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention was isolated according to the present invention, internally designated "Oligo 124 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 53, or a polynucleotide of the nucleic acid sequence shown in 53. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 56 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 124" and "Oligo 124 v" with significant homology to the MIPS tag "Sub 2" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 56. The amino acid sequence or amino acid partial sequence from the coding strand has significant sequence homology with saccharomyces cerevisiae RNA helicase.

m) a nucleic acid sequence, preferably down-regulated, encoding a protein having the function of an mRNA decapping enzyme.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and designated internally by the name "Oligo 139".

In another preferred embodiment of the invention, a DNA clone encoding the complete sequence of the nucleic acid of the invention is isolated according to the invention, internally designated "Oligo 139 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 58. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 60 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 139" and "Oligo 139 v" with significant homology to the MIPS tag "DCP 1" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 60, or a variant thereof. The amino acid sequence or amino acid partial sequence from the coding strand has significant sequence homology with saccharomyces cerevisiae mRNA decapping enzyme.

n) a nucleic acid sequence, preferably down-regulated, encoding a protein having the function of a subunit of the translation initiation factor eIF 3.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 144".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention is isolated according to the present invention, internally designated "Oligo 144 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 63. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 65 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

Inserts of "Oligo 144" and "Oligo 144 v" having significant homology to the MIPS tag "PRT 1" from saccharomyces cerevisiae have the amino acid sequence shown in SEQ ID NO: 63 or SEQ ID NO: 65. The amino acid sequence or amino acid partial sequence from the coding chain has significant sequence homology with the subunit of saccharomyces cerevisiae translation initiation factor eIF 3.

o) a nucleic acid sequence, preferably up-regulated, which encodes a protein having the function of the U3 nucleolar small ribonucleoprotein-related protein involved in processing of pre-ribosomal RNA.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is designated internally by the name "Oligo 168".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention was isolated according to the present invention, internally designated "Oligo 168 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 67, or a polynucleotide having the nucleic acid sequence shown in seq id no. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 70 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

The inserts of "Oligo 168" and "Oligo 168 v" have significant homology to the MIPS tag "rpp 9" from saccharomyces cerevisiae, these inserts having the amino acid sequence shown in SEQ ID NO: 67 or SEQ ID NO: 70 in a sequence listing. The amino acid sequence or amino acid partial sequence from the coding strand has significant sequence homology with U3 nucleolar ribonucleoprotein-related protein of Saccharomyces cerevisiae involved in RNA processing of pre-ribosomes.

p) preferably a down-regulated nucleic acid sequence encoding a protein having the function of ribosomal 60S large subunit protein L7 a.e.B.

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 160".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 72, which polynucleotide is preferably isolated from a microorganism of the genus ashbya, in particular ashbya gossypii. The present invention also relates to polynucleotides complementary to the polynucleotides, and sequences derived from the polynucleotides by degeneracy of the genetic code.

The insert of "Oligo 160" has significant homology to the MIPS tag "Rpl 8 b" from saccharomyces cerevisiae, these inserts have the amino acid sequence of SEQ ID NO: 72, or a nucleic acid sequence as set forth in seq id no. The amino acid sequences from the corresponding complementary strands have significant sequence homology to the ribosomal protein of Saccharomyces cerevisiae (L7 a.e.B; 60S large subunit).

q) We have found that this object is achieved by providing coding nucleic acid sequences which are up-regulated in Ashbya gossypii in the production of vitamin B2 (based on the results of the MPSS analysis described in detail in the experimental section).

In a preferred embodiment of this aspect of the invention, a DNA clone encoding a sequence characteristic of a portion of the nucleic acid sequence of the invention is isolated and is internally designated "Oligo 18".

In another preferred embodiment of the present invention, a DNA clone encoding the complete sequence of the nucleic acid of the present invention is isolated according to the present invention, internally designated "Oligo 18 v".

One aspect of the invention relates to a polypeptide comprising SEQ ID NO: 75 or the nucleic acid sequence set forth in SEQ ID NO: 74, or a polynucleotide complementary thereto. In another aspect the invention relates to a polypeptide comprising SEQ ID NO: 77 or a fragment thereof. These polynucleotides are preferably isolated from microorganisms of the genus Ashbya, in particular Ashbya gossypii. The invention also relates to polynucleotides that are complementary to these polynucleotides, and to sequences derived from these polynucleotides by the degeneracy of the genetic code.

A further aspect of the invention relates to oligonucleotides which hybridize to one of the above polynucleotides, in particular under stringent conditions.

The invention also relates to polynucleotides which can hybridize with one of the oligonucleotides according to the invention and which code for a gene product from a microorganism of the genus Ashbya or for a functional equivalent of this gene product.

The invention further relates to polypeptides or proteins encoded by the above polynucleotides; and peptide fragments having an amino acid sequence comprising SEQ ID NO: 2, 3, 5, 7, 8, 9, 11, 13, 15, 16, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 34, 35, 37, 39, 41, 43, 44, 45, 47, 49, 50, 52, 54, 55, 57, 59, 61, 62, 64, 66, 68, 69, 71, 73, 76, or SEQ ID NO: 78, a nitrogen source; at least 10 consecutive amino acid residues shown in (a); and functional equivalents of the polypeptides or proteins of the invention.

In this respect, functional equivalents differ in amino acid sequence at least one, for example 1 to 30 or 1 to 20 or 1 to 10, sequence positions as a result of additions, insertions, substitutions, deletions or inversions compared to the products specifically disclosed in the invention, but do not lose the originally observed protein function (which can be deduced by sequence alignment with other proteins). Thus, functional equivalents may have substantially the same, higher or lower activity as compared to the native protein.

Other aspects of the invention relate to expression cassettes for the recombinant production of a protein of the invention, comprising one of the above-mentioned nucleic acid sequences operably linked to at least one regulatory nucleic acid sequence; and to recombinant vectors comprising at least one such expression cassette of the invention.

The invention also provides a prokaryotic or eukaryotic host transformed with at least one vector of the above type. A preferred embodiment provides a prokaryotic or eukaryotic host in which the functional expression of at least one gene encoding a polypeptide of the invention as described above is modulated (e.g.suppressed or overexpressed); or the biological activity of a polypeptide as defined above is reduced or increased. Preferred hosts are selected from the group consisting of ascomycetes, in particular the Ashbya genus, preferably the Ashbya gossypii strain.

Regulation of gene expression in the above sense includes suppression of expression, for example by blocking a certain stage of expression (in particular transcription or translation) and specific overexpression of the gene (for example by modifying the regulatory sequences or increasing the copy number of the coding sequence).

Another aspect of the present invention relates to the use of an expression cassette according to the present invention, a vector according to the present invention or a host according to the present invention for the microbial production of vitamin B2 and/or a precursor and/or a derivative thereof.

Another aspect of the present invention relates to the use of an expression cassette according to the invention, a vector according to the invention or a host according to the invention for the recombinant production of a polypeptide according to the invention as described above.

The invention also provides a method for detecting or verifying effector targets that may be used for modulating the microbial production of vitamin B2 and/or its precursors and/or its derivatives. The method involves: treating a microorganism capable of producing vitamin B2 and/or a precursor thereof and/or a derivative thereof microbiologically with an effector capable of interacting with (e.g. non-covalently binding to) a target selected from the above-described polypeptides of the invention or nucleic acid sequences encoding the same, verifying the effect of the effector on the amount of vitamin B2 and/or a precursor thereof and/or a derivative thereof produced by the microorganism, and isolating the target, if appropriate. Verification is preferably made here by direct comparison with the production of vitamin B2 by the microorganism in the absence of the effector and otherwise identical conditions.

Another aspect of the present invention relates to a method for modulating the microbial production (related to the amount and/or rate of production) of vitamin B2 and/or a precursor and/or a derivative thereof, wherein a microorganism capable of microbiologically producing vitamin B2 and/or a precursor and/or a derivative thereof is treated with an effector which can interact with a target selected from the group consisting of a polypeptide of the invention as defined above or a nucleic acid sequence encoding the polypeptide.

Preferred examples of the above effectors which should be mentioned are:

a) an antibody or antigen-binding fragment thereof;

b) a polypeptide ligand which is different from a) and which can interact with the polypeptide of the invention;

c) low molecular weight effectors that can modulate the biological activity of the polypeptides of the invention;

d) antisense nucleic acid sequences that can interact with the nucleic acid sequences of the invention.

The invention also relates to the above-mentioned effectors having specificity for at least one of the above-defined targets of the invention.

Another aspect of the present invention relates to a process for the microbial production of vitamin B2 and/or a precursor and/or a derivative thereof, wherein a host as defined above is cultivated under conditions which favourably yield vitamin B2 and/or a precursor and/or a derivative thereof and the desired product is isolated from the cultivation mixture. In this connection, the host is preferably treated with the above-defined effectors before and/or during the cultivation. Preferred hosts herein are selected from microorganisms of the genus Ashbya, in particular those transformed by the above-described process.

A final aspect of the invention relates to the use of the polynucleotide or polypeptide of the invention as a target for modulating the production of vitamin B2 and/or a precursor and/or a derivative thereof by a microorganism of the genus ashbya.

Drawings

Fig. 1 shows a sequence based on SEQ ID NO: 5 (middle sequence) and a partial sequence from the MIPS tag "Yta 7" of saccharomyces cerevisiae (lower sequence). Consensus sequences are indicated above both sequences. The positions lacking homology are indicated in black boxes.

Fig. 2 shows a sequence based on SEQ ID NO: 11 (middle sequence) and a partial sequence from the MIPS tag "Tif 34" from saccharomyces cerevisiae (lower sequence). Consensus sequences are indicated above both sequences. The positions lacking homology are indicated in black boxes.

FIG. 3 shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 469 to position 825 of SEQ ID NO: 12) (upper sequence) and the partial sequence of the MIPS tag "Rpl 25a from Saccharomyces cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with a "+".

FIG. 4 shows an alignment of the partial sequence of amino acids of the invention (corresponding to the complementary strand from position 114 to position 1 of SEQ ID NO: 17) (upper sequence) and the partial sequence of the MIPS tag "Nop 13" from Saccharomyces cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with a "+".

FIG. 5A shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 2 to position 349 of SEQ ID NO: 21) (upper sequence) and the partial sequence of the MIPS tag "Sua 5" from Saccharomyces cerevisiae (lower sequence). FIG. 5B shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 336 to position 947 of SEQ ID NO: 21) (upper sequence) and the partial sequence of the MIPS tag "Sua 5" from Saccharomyces cerevisiae (lower sequence).

FIG. 6A shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 609 to 562 of SEQ ID NO: 26) (upper sequence) and the partial sequence of the MIPS tag "Rps 25 a" from Saccharomyces cerevisiae (lower sequence). FIG. 6B shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 556 to position 401 of SEQ ID NO: 26) (upper sequence) and the partial sequence of the MIPS tag "Rps 25 a" from Saccharomyces cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with a "+".

Fig. 7 shows a sequence based on SEQ ID NO: 36 (middle sequence) and a partial sequence of the MIPS tag "Nic 96" from saccharomyces cerevisiae (lower sequence). Consensus sequences are indicated above both sequences. The positions lacking homology are indicated in black boxes.

FIG. 8 shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 174 to position 1 of SEQ ID NO: 38) (upper sequence) and the partial sequence of the MIPS tag "Ahc 1" from Saccharomyces cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with a "+".

FIG. 9A shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 1086 to position 1012 of SEQ ID NO: 42) (upper sequence) and the partial sequence of the MIPS tag "Rok 1" from Saccharomyces cerevisiae (lower sequence). FIG. 9B shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 1022 to position 915 of SEQ ID NO: 42) (upper sequence) and the partial sequence of the MIPS tag "Rok 1" from Saccharomyces cerevisiae (lower sequence). FIG. 9C shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 925 to position 689 of SEQ ID NO: 42) (upper sequence) and the partial sequence of the MIPS tag "Rok 1" from Saccharomyces cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with a "+".

FIG. 10A shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 1 to position 102 of SEQ ID NO: 48) (upper sequence) and the partial sequence of the MIPS tag "Rpa 43" from Saccharomyces cerevisiae (lower sequence). FIG. 10B shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 122 to position 400 of SEQ ID NO: 48) (upper sequence) and the partial sequence of the MIPS tag "Rpa 43" from Saccharomyces cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with a "+". Fig. 10C shows SEQ ID NO: 48 and a partial sequence complementary thereto.

FIG. 11A shows an alignment of the amino acid partial sequences of the invention (corresponding to the chain from position 2 to position 148 of SEQ ID NO: 53) (upper sequence) and the partial sequence of the MIPS tag "Sub 2" from Saccharomyces cerevisiae (lower sequence). FIG. 11B shows an alignment of the amino acid partial sequences of the invention (corresponding to the chain from position 150 to position 185 of SEQ ID NO: 53) (upper sequence) and the partial sequence of the MIPS tag "Sub 2" from Saccharomyces cerevisiae (lower sequence). The same sequence position is indicated in the middle of both sequences. Similar sequence positions are marked with a "+".

FIG. 12 shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 2 to position 82 of SEQ ID NO: 58) (upper sequence) and the partial sequence of the MIPS tag "DCP 1" from Saccharomyces cerevisiae (lower sequence). The same sequence position is indicated in the middle of both sequences. Similar sequence positions are marked with a "+".

FIG. 13 shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 21 to position 695 of SEQ ID NO: 63) (upper sequence) and the partial sequence of the MIPS tag "PRT 1" from Saccharomyces cerevisiae (lower sequence). The same sequence position is indicated in the middle of both sequences. Similar sequence positions are marked with a "+".

FIG. 14A shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 1 to position 111 of SEQ ID NO: 67) (upper sequence) and the partial sequence of the MIPS tag "Rrp 9" from Saccharomyces cerevisiae (lower sequence). FIG. 14B shows an alignment of the amino acid partial sequence of the invention (corresponding to the chain from position 144 to position 887 of SEQ ID NO: 67) (upper sequence) and the partial sequence of the MIPS tag "Rrp 9" from Saccharomyces cerevisiae (lower sequence). The same sequence position is indicated in the middle of both sequences. Similar sequence positions are marked with a "+".

FIG. 15 shows an alignment of the amino acid partial sequence of the invention (corresponding to the complementary strand from position 508 to position 176 of SEQ ID NO: 72) (upper sequence) and the partial sequence of the MIPS tag "Rpl 8 b" from Saccharomyces cerevisiae (lower sequence). The same sequence position is indicated in the middle of both sequences. Similar sequence positions are marked with a "+".

FIG. 16 shows the construction strategy for the insertion of an antibiotic resistance cassette (G418 resistance gene under the control of the Ashbya TEF promoter) after the Open Reading Frame (ORF) of "Oligo 18".

Detailed Description

The polypeptides or proteins encoded by the nucleic acid molecules of the invention are referred to herein as transcription, RNA processing and/or translation proteins (e.g., having activity associated with transcription, RNA processing, splicing or translation) or simply as "TT proteins". These TT proteins have functions such as adapting the organism to various growth phases and environments and metabolic conditions such as substrates, oxygen levels, etc.

Since the cloning vectors available for Ashbya gossypii (e.g.as disclosed in Wright and Philipsen (1991) genes (Gene), 109, 99-105) and the genetic manipulation techniques of Ashbya gossypii and related yeast species are known, the nucleic acid molecules of the invention can be used for the genetic manipulation of these organisms, in particular of Ashbya gossypii, thus allowing a better and more efficient production of vitamin B2 and/or its precursors and/or its derivatives. The improvement in yield or production efficiency may be caused by a direct effect of manipulation of the gene of the present invention or an indirect effect of the manipulation.

The present invention is based on the provision of novel molecules, referred to herein as TT nucleic acids and TT proteins, which are involved in (e.g., regulate transcription, RNA processing and/or translation) in, inter alia, Ashbya gossypii. The activity of the TT molecule of the invention in Ashbya gossypii affects the vitamin B2 production of this microorganism. Preferably, the activity of the TT molecule of the invention is modulated such that the metabolic and/or energetic pathway of Ashbya gossypii in which the TT protein of the invention is involved is modulated in the yield, production and/or efficiency of production of vitamin B2, i.e. the yield, production and/or efficiency of production of vitamin B2 in Ashbya gossypii is directly or indirectly modulated.

The nucleic acid sequences provided by the present invention can be isolated, for example, from the genome of an Ashbya gossypii strain, which is freely available from the American type culture Collection (deposit number ATCC 10895).

Improvements in vitamin B2 production

There are several possible mechanisms by which the amount and/or activity of the TT protein of the invention can be varied to directly affect the yield, production and/or efficiency of production of vitamin B2 from the Ashbya gossypii strain.

Thus, by more efficient transcription, RNA processing or translation, which adapts the expression of the desired gene product to external conditions, the formation of valuable desired products can be optimized.

Mutagenesis of one or more of the TT proteins of the invention may also result in altered (increased or decreased) activity of the TT protein, indirectly affecting production of the desired product in the Ashbya gossypii strain. For example, transcription, RNA processing and/or translation processes can be assisted (for example by activators) or blocked (for example by inhibitors) at different points by the TT protein and thus influence gene expression or protein synthesis. The yield of the desired product is thereby also increased or optimized with respect to the ambient conditions.

Polypeptides

The present invention relates to polypeptides comprising the above-described amino acid sequences or characteristic partial sequences thereof and/or encoded by the nucleic acid sequences described herein.

The invention also includes "functional equivalents" of the specifically disclosed novel polypeptides.

"functional equivalents" or analogs of a specifically disclosed polypeptide are for the purposes of the present invention to refer to polypeptides that differ from the specifically disclosed polypeptide but still possess the desired biological activity (e.g., substrate specificity).

"functional equivalents" in the context of the present invention mean, in particular, mutants which have an amino acid other than that specifically disclosed in at least one of the abovementioned sequence positions but nevertheless have one of the abovementioned biological activities. "functional equivalents" thus include mutants which can be obtained by addition, substitution, deletion and/or inversion of one or more amino acids, the modification of which can take place at any sequence position, as long as the resulting mutants can have the property profile according to the invention. Functional equivalents may also be in particular in the form of mutants and unmodified polypeptides which are qualitatively identical with respect to the reaction pattern, i.e.for example differ in the rate of conversion to the same substrate.

"functional equivalents" in the above sense are also precursors of the mentioned polypeptides and functional derivatives and salts of the polypeptides. The term "salt" refers to both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules of the invention. Salts of the carboxyl groups may be prepared in a manner known in the art and include inorganic salts such as sodium, calcium, ammonium, iron and zinc salts and salts with organic bases such as amines such as triethanolamine, arginine, lysine, piperidine and the like. In another aspect, the invention relates to acid addition salts, such as salts with inorganic acids (e.g. hydrochloric or sulfuric acid) and salts with organic acids (e.g. acetic and oxalic acids).

"functional derivatives" of the polypeptides of the invention may also be prepared by known techniques on amino acid side chain functions or at the N-or C-terminus of the polypeptide. Such derivatives include, for example, aliphatic esters of carboxyl groups and amides of carboxyl groups obtainable by reaction with ammonia or with primary or secondary amines; an N-acyl derivative of a free amino group prepared by reaction with an acyl group; or an O-acyl derivative of a free hydroxyl group prepared by reaction with an acyl group.

"functional equivalents" naturally also include polypeptides and naturally occurring variants which can be obtained from other organisms. For example, homologous sequence regions can be found by sequence alignment and equivalent enzymes established according to the specific requirements of the invention.

"functional equivalents" also include fragments having, for example, the desired biological function, preferably single domains or sequence motifs of the polypeptides of the invention.

A "functional equivalent" may also be a fusion protein having one of the above-mentioned polypeptide sequences or a functional equivalent thereof and at least one further heterologous sequence with a different function, which heterologous sequence is functionally linked at the N-or C-terminus of said polypeptide sequence or functional equivalent thereof (i.e.the parts of the fusion protein are functionally minimally impaired with respect to each other). Non-limiting examples of such heterologous sequences are e.g. signal peptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.

According to the present invention, "functional equivalents" include homologues of the proteins specifically disclosed herein. These homologues have at least 60%, preferably at least 75%, in particular at least 85%, for example 90%, 95%, or 99% homology to one of the specifically disclosed sequences, as calculated by the algorithm of Pearson and Lipman, Proc.Natl.Acad.Sci. (USA)85(8), 1988, 2444-2448.

Where glycosylation of a protein may be present, equivalents of the invention include deglycosylated or glycosylated forms of the above-defined protein types, as well as modified forms obtainable by altering the glycosylation pattern.

Mutagenesis may result in homologues of the protein or polypeptide of the invention, for example by point mutation or truncation of the protein. The term "homologue" as used herein relates to variant forms of a protein which may exert an agonist or antagonist effect on the activity of the protein.

Homologs of the proteins of the invention can be identified by screening combinatorial libraries of mutants, for example truncation mutants. A variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides. A number of methods are available for generating libraries of potential homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer and the synthesized gene can then be ligated into a suitable expression vector. All sequences encoding a desired set of potential protein sequences can be provided in a mixture using a degenerate set of genes. Methods for the synthesis of degenerate oligonucleotides are known to the person skilled in the art (for example NaRNAG, S.A. (1983) Tetrahedron 39: 3; Itakura et al (1984) Annu. Rev. biochem. 53: 323; Itakura et al (1984) Science 198: 1056; Ike et al (1983) Nucleic Acids Res.11: 477).

In addition, libraries of fragments of protein codons can also be used to generate diverse populations of protein fragments for screening and subsequent selection of homologs of the proteins of the invention. In one embodiment, a library of fragments of a coding sequence is generated by: double-stranded PCR fragments of the coding sequence are treated with a nuclease under conditions in which only about one cleavage per molecule occurs, the double-stranded DNA is denatured, then the DNA is renatured, thereby forming double-stranded DNA which may contain sense/antisense pairs from different cleavage products, the single-stranded portions are removed from the newly formed duplexes by treatment with S1 nuclease, and the resulting library of fragments is ligated into an expression vector. Expression libraries encoding N-terminal, C-terminal and internal fragments of different sizes of the protein of the invention can be obtained in this way.

Several techniques are known in the art for screening gene products from combinatorial libraries generated by point mutations or truncations and for screening cDNA libraries for gene products having selected properties. These techniques have been adapted to allow rapid screening of gene libraries generated by combinatorial mutagenesis of homologs of the invention. The most commonly used high throughput large gene library screening assay technique involves cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions conducive to isolation of the vector encoding the gene product to be detected in the activity assay for the desired activity. Recursive Ensemble Mutagenesis (REM) technology increases the frequency of functional mutants in a library and can therefore be used in conjunction with screening experiments to identify homologs. (Arkin and Yourvan (1992) PNAS 89: 7811. sup. quadrature. 7815; Delgrave et al (1993) Protein Engineering (Protein Engineering)6 (3): 327. sup. quadrature. 331).

The polypeptides of the invention can be prepared recombinantly (see below) or in their natural form can be isolated from microorganisms, in particular microorganisms of the genus Ashbya, by conventional biochemical techniques. (see Cooper, T.G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or Scopes, R., Protein Purification (Protein Purification), Schpringer, New York, Heidelberg, Berlin).

Nucleic acid sequences

The invention also relates to nucleic acid sequences (single-and double-stranded DNA and RNA sequences, such as cDNA and mRNA, for example) which code for one of the above-mentioned polypeptides and functional equivalents thereof, which nucleic acid sequences can be obtained, for example, using artificial nucleotide analogs.

The present invention relates both to isolated nucleic acid molecules encoding a polypeptide or protein of the invention or a biologically active portion thereof and to nucleic acid fragments that can be used, for example, as hybridization probes or primers for identifying or amplifying the encoding nucleic acids of the invention.

The nucleic acid molecules of the invention may also comprise untranslated sequences from the 3 'and/or 5' end of a coding region of a gene.

An "isolated" nucleic acid molecule is isolated from other nucleic acid molecules present in the natural source of the nucleic acid, and may also be substantially free of other cellular material or culture medium if the nucleic acid molecule is produced by recombinant techniques; if chemically synthesized, are substantially free of chemical precursors or other chemicals.

The nucleic acid molecules of the invention can be isolated by standard techniques of molecular biology and the sequence information provided by the invention. For example, with a specifically disclosed complete sequence or a portion thereof as a hybridization probe, cDNA can be isolated from a suitable cDNA library by standard hybridization techniques (as described in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, second edition, Molecular Cloning: A Laboratory Manual, 2 edition, Cold Spring Harbor Laboratory, U.S.A., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Nucleic acid molecules comprising a disclosed sequence or a portion thereof can also be isolated by polymerase chain reaction using oligonucleotide primers constructed based on the sequence. The nucleic acid molecules amplified by this method can be cloned into suitable vectors and characterized by DNA sequence analysis. Oligonucleotides of the invention corresponding to nucleotide sequences of TT may also be produced by standard synthesis methods, for example using an automated DNA synthesizer.

The invention also includes nucleic acid molecules, or portions thereof, that are complementary to the specifically described nucleotide sequences.

The nucleotide sequences of the present invention allow the generation of probes and primers that can be used to identify and/or clone homologous sequences in other cell types and organisms. Such probes and primers typically comprise a region of nucleotide sequence which can hybridize under stringent conditions to at least about 12, preferably at least about 25, for example about 40, 50 or 75 consecutive nucleotides in a sense strand or corresponding antisense strand of a nucleic acid sequence of the invention.

Other nucleic acid sequences of the invention are derived from SEQ ID NO: 1, 4, 6, 10, 12, 14, 17, 19, 21, 24, 26, 29, 31, 36, 38, 40, 42, 46, 48, 51, 53, 56, 58, 60, 63, 65, 67, 70, 72, 74, 75, or SEQ ID NO: 77 and differs from the sequence from which it is derived by the addition, substitution, insertion or deletion of one or more nucleotides, but still encodes a polypeptide having the desired property profile.

The invention also includes nucleic acid sequences containing so-called silent mutations or modifications, which are based on codon usage of the particular source or host organism compared to the specifically disclosed sequences, as well as naturally occurring variants thereof (e.g., splice variants or allelic variants). The invention also relates to sequences obtainable by conservative nucleotide substitutions (i.e.the relevant amino acid is replaced by an amino acid of the same charge, size, polarity and/or solubility).

The invention also relates to molecules derived from sequence polymorphisms from the specifically disclosed nucleic acids. These genetic polymorphisms may exist in individuals of a population due to natural variation. These natural variations typically result in 1% to 5% changes in the nucleotide sequence of the gene.

The invention also includes nucleic acid sequences that hybridize or are complementary to the coding sequences described above. These polynucleotides can be found by screening genomic or cDNA libraries and, where appropriate, amplified by PCR using suitable primers and isolated, for example, using suitable probes. Another possibility is to transform suitable microorganisms with the polynucleotides or vectors of the invention, propagate the microorganisms and thus amplify the polynucleotides and then isolate them. A further possibility is to synthesize the polynucleotides of the invention by chemical routes.

The property of being "hybridizable" to a polynucleotide means that the polynucleotide or oligonucleotide binds to nearly complementary sequences under stringent conditions, while nonspecific binding between non-complementary sequences does not occur under such conditions. For this purpose, the sequences should have a complementarity of 70% to 100%, preferably 90% to 100%. The property of complementary sequences to be able to bind specifically to one another can be used, for example, for Northern or Southern blot hybridization or for binding of primers in PCR or RT-PCR. Oligonucleotides of 30 base pairs or more in length are generally used for this purpose. Stringent conditions refer to washing at 50-70 deg.C, preferably 60-65 deg.C, for example in Northern blot hybridization, such as elution of non-specifically hybridizing cDNA probes or oligonucleotides with a wash solution containing 0.1XSSC buffer and 0.1% SDS (20 XSSC: 3M sodium chloride, 0.3M sodium citrate, pH 7.0). In this case, as mentioned above, only highly complementary nucleic acids remain bound to each other. Establishment of such stringent conditions is known to those skilled in the art and can be found, for example, in Ausubel et al, Current Protocols in molecular biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

Another aspect of the invention relates to antisense nucleic acids. Comprising a nucleotide sequence complementary to the sense coding nucleic acid. The antisense nucleic acid can be complementary to the entire coding strand or to only a portion thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a non-coding region of the coding strand of the nucleotide sequence. "non-coding region" refers to the portions of the sequence referred to as the 5 '-and 3' -untranslated regions.

The antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. The antisense nucleic acids of the invention can be constructed by chemical synthesis and enzymatic ligation reactions by methods known in the art. Antisense nucleic acids can be chemically synthesized from naturally occurring nucleotides or various modified nucleotides designed to enhance the biological stability of the molecule or to enhance the physical stability of the duplex formed between the antisense and sense nucleic acids. Examples that can be used include phosphorothioate derivatives and acridine substituted nucleotides. Modified nucleosides that can be used to generate antisense nucleic acids are, for example: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,. 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosyl queosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), Wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3- (3-amino-3-carboxypropyl) uracil, (acp3) w, and 2, 6-diaminopurine, and the like. Antisense nucleic acids can also be produced biologically using expression vectors in which the nucleic acid is subcloned in the antisense orientation.

The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they can hybridize to or bind to the cellular mRNA and/or coding DNA, by, for example, inhibiting the expression of transcription and/or translation repressor proteins.

Antisense molecules can be modified to specifically bind to a receptor or antigen expressed on the surface of a selected cell, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be administered to a cell via a vector described herein. In order to achieve a sufficient concentration of antisense molecules in the cell, it is preferred to use vector constructs which are vectors in which the antisense nucleic acid molecule is under the control of a strong bacterial, viral or eukaryotic promoter.

In another embodiment, the antisense nucleic acid molecule of the invention is an α -anomeric nucleic acid molecule. Alpha-anomeric Nucleic acid molecules form unique double-stranded hybrids with complementary RNA, which run parallel to each other, unlike the normal alpha unit (Gaultier et al, (1987) Nucleic Acids Res. 15: 6625-6641). The antisense Nucleic acid molecules may also comprise 2' -O-methyl ribonucleotides (Inoue et al, (1987) Nucleic Acids Res.15: 6131-.

The invention also relates to ribozymes. It is a catalytic RNA molecule with ribonuclease activity that is capable of cleaving a single-stranded nucleic acid, such as an mRNA, having a region complementary thereto. Ribozymes, such as hammerhead ribozymes (see Haselhoff and Gerlach (1988) Nature 334: 585-. Ribozymes specific for the coding nucleic acids of the invention can be formed, for example, on the basis of the cDNAs specifically disclosed in the invention. For example, derivatives of Tetrahymena-L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the coding mRNA of the present invention. (see, for example, US-A-4987071 and US-A-5116742). Alternatively, mRNA can be used to select catalytic RNA having specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel, D., and Szostak, J.W, (1993) Science 261: 1411-1418).

Alternatively, gene expression of the sequences of the invention may be inhibited by targeting nucleotide sequences complementary to regulatory regions of the nucleotide sequences of the invention (e.g., complementary to promoters and/or enhancers of the coding sequences) that cause triple helix formation and thereby prevent transcription of the corresponding gene in the target cell (Helene, C. (1991) Anticancer Drug Res.6(6) 569-584; Helene, C. et al, (1992) Ann.N.Y.Acad.Sci.660: 27-36; and Maher., L.J. (1992) Bioassays14 (12): 807-815).

Expression constructs and vectors:

the invention also relates to expression constructs comprising a nucleic acid sequence encoding a polypeptide of the invention under the genetic control of a regulatory nucleic acid sequence; and a vector comprising at least one such expression construct. Such expression constructs of the invention preferably comprise a promoter located upstream of the 5 'end of the particular coding sequence and a termination sequence located downstream of the 3' end of the particular coding sequence and, where appropriate, further customary regulatory elements, each of which is operably linked to the coding sequence. "operably linked" refers to a sequential arrangement of promoter, coding sequence, terminator and, where appropriate, other regulatory elements in such a way that each of the regulatory elements functions as intended for expression of the coding sequence. Examples of operably linked sequences are leader and enhancer sequences, polyadenylation signals, and the like. Other regulatory elements include selectable markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are described, for example, in Goeddel, gene expression technology: enzymatic methods (Gene Expression Technology: Methodsin Enzymology)185, Academic Press, San Diego, Calif., CA (1990).

In addition to the artificial regulatory sequences, natural regulatory sequences may still be present before the actual structural gene. This natural regulation can be switched off, where appropriate, by genetic modification, so that the expression of the gene can be increased or decreased. However, the gene construct may also have a simpler structure, i.e.no additional regulatory signals are inserted before the structural gene and the natural promoter with the regulatory function is not deleted. Alternatively, the natural regulatory sequence may be mutated so as to no longer have a regulatory effect, thereby enhancing or reducing the expression of the gene. The nucleic acid sequence may have one or more copies in the gene construct.

Examples of promoters that can be used include: cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, lambda-PR or lambda-PL promoters (which are preferably used in gram-negative bacteria); gram-positive bacterial promoters amy and SPO2, yeast promoter ADC1, MF α, AC, P-60, CYC1, GAPDH or plant promoter CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, or ubiquitin or phaseolin promoterAnd a mover. Particular preference is given to using inducible promoters, for example light-inducible promoters, more preferably temperature-inducible promoters such as P_rP_lA promoter. In principle all natural promoters can be used, as well as their regulatory sequences. Furthermore, the use of synthetic promoters may also be advantageous.

The regulatory sequences are intended to achieve specific expression of the nucleic acid sequence. This may mean, for example, that the genes are expressed or overexpressed only after induction or are expressed and/or overexpressed immediately, depending on the host organism.

Furthermore, it is preferred that the regulatory sequences or factors can positively influence and thus increase or decrease expression. Thus, the enhancement of the regulatory elements can advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. However, translation can also be enhanced by, for example, increasing the stability of the mRNA.

An expression cassette can be generated by fusing a suitable promoter with a suitable nucleotide sequence of the invention and a termination signal or polyadenylation signal. Conventional recombinant and cloning techniques can be used for this purpose, see: t.manitis, e.f.fritsch and j.sambrook, molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982); T.J.Silhavy, M.L.Berman and L.W.Enq uist, Gene fusion test (Experiments with Gene fusions), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984); ausubel, F.M. et al, Current Protocols in Molecular Biology, Greene publishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct may advantageously be inserted into a host-specific vector to achieve optimal expression of these genes in the host. Vectors are well known to those skilled in the art and can be found in, for example, "Cloning Vectors" (Pouwels P.H., et al, eds, Elsevier, Amsterdam-New York-Oxford, 1985). Vectors are not only plasmids, but also all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, plasmids, cosmids and linear or circular DNA. These vectors may be autonomously replicating or chromosomally replicating in the host organism.

Examples of suitable expression vectors which may be mentioned are:

conventional fusion expression vectors such as pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5(Pharmacia, Piscataway, NJ) are used to fuse glutathione S-transferase (GST), maltose E-binding protein and protein A, respectively, to a recombinant target protein.

Non-fusion protein expression vectors such as pTrc (Amann et al (1988) Gene 69: 301-315) and pET 11d (student et al, Gene expression technology: Methods in Enzymology 185, Academic Press, san Diego, Calif. (1990) 60-89).

Yeast expression vectors for expression in Saccharomyces cerevisiae are, for example, pYepSec1(Baldari et al (1987) Embo J.6: 229-234), pMF α (Kurjan and Herskowitz (1982) cells (Cell) 30: 933-943), pJRY88(Schultz et al (1987) Gene (Gene) 54: 113-123) and pYES2(Invitrogen Corporation, san Diego, Calif.). For a detailed description of examples of vectors and methods of vector construction suitable for use in other fungi (e.g., filamentous fungi), see, for example: van den hondel, c.a.m.j.j. & Punt, P.J (1991) "development of filamentous fungal gene transfer systems and vectors," Applied Molecular Genetics of fungi, j.f. peberdy et al eds, pp.1-28, Cambridge University Press: cambridge.

Existing baculovirus vectors for expression of proteins in cultured insect cells (e.g., Sf9 cells) include: the pAc series (Smith et al (1983) mol. cell biol.3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology (Virology) 170: 31-39).

Examples of plant expression vectors are described in detail, for example: becker, d., Kemper, e., Schell, j., and Masterson, R. (1992) "new Plant binary vector with selection marker next to the left border", Plant mol.biol.20: 1195-1197; and Bevan, M.W (1984) "binary vectors for agrobacterium used for plant transformation", nucleic. 8711-8721.

Mammalian expression vectors are exemplified by pCDM8(Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al (1987) EMBO J.6: 187-195).

Other expression systems suitable for prokaryotic and eukaryotic cells are described in Sambrook, j., Fritsch, e.f. and manitis, t., molecular cloning: a Laboratory Manual (Molecular cloning: A Laboratory Manual), second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, chapters 16 and 17.

Recombinant microorganism

The vectors of the invention are useful for producing recombinant microorganisms which are transformed, for example, with at least one vector of the invention and which are useful for producing the polypeptides of the invention. Advantageously, the recombinant constructs of the invention described above may be introduced into and expressed in a suitable host system. The person skilled in the art is familiar with methods for cloning and transfection, for example: coprecipitation, protoplast fusion, electroporation, retroviral transfection, and the like, and preferably are used to cause expression of the nucleic acid in a particular expression system. Suitable expression systems are described, for example, in Current Protocols in Molecular Biology (F. Ausubel et al, eds., Wiley Interscience, New York 1997) or in Sambrook et al, Molecular cloning: a Laboratory Manual, second edition (Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

Homologous recombinant microorganisms can also be prepared according to the invention. This entails generating a vector comprising at least one stretch of the gene or coding sequence of the invention, into which at least one amino acid deletion, addition or substitution is introduced, where appropriate, to modify, for example functionally disrupt, the sequence of the invention (knock-out vector). The introduced sequence may also be, for example, a homologue from a related microorganism or from a mammal, yeast or insect. Alternatively, vectors for homologous recombination may be designed such that the endogenous gene is mutated or otherwise modified during homologous recombination and still encodes a functional protein (e.g., regulatory sequences located upstream may be modified to alter expression of the endogenous protein). The modified portion of the TT gene is present in a homologous recombination vector. The construction of suitable homologous recombination vectors is described, for example, in Thomas, k.r. and Capecchi, M.R, (1987) Cell 51: 503.

suitable host organisms are in principle all organisms which are capable of expressing the nucleic acids according to the invention, their allelic variants, their functional equivalents or derivatives. Host organisms are, for example, bacteria, fungi, yeasts, plant or animal cells. Preferred organisms are bacteria, for example of the genus Escherichia (Escherichia) such as E.coli, Streptomyces (Streptomyces), Bacillus (Bacillus) or Pseudomonas (Pseudomonas), eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus (Aspergillus), higher eukaryotic cells from animals or plants such as Sf9 or CHO cells. Preferred organisms are selected from the genus Ashbya, in particular the Ashbya gossypii strain.

Successfully transformed organisms can be selected by marker genes which are likewise present in the vector or expression cassette. Such marker genes are, for example, antibiotic resistance genes and genes which catalyze enzymes which cause a color-forming reaction which leads to staining of transformed cells. Selection can then be performed by automated cell sorting. Microorganisms successfully transformed with the vector and containing the appropriate antibiotic resistance gene (e.g., G418 or hygromycin) can be selected from the appropriate antibiotic-containing medium or nutrient medium. Selection can be performed by affinity chromatography using a marker protein present on the cell surface.

The combination of a host organism with a vector suitable for the organism, for example a plasmid, virus or phage, such as a plasmid with an RNA polymerase/promoter system, lambda or mu phage or other temperate phages or transposons and/or other advantageous regulatory sequences forms an expression system. The term "expression system" refers, for example, to the combination of mammalian cells, such as CHO cells, with vectors suitable for mammalian cells, such as pcDNA3neo vector.

If desired, the gene product can also be expressed in transgenic organisms such as transgenic animals (in particular mice, sheep) or transgenic plants.

Recombinant production of polypeptides

The invention also relates to a process for the recombinant production of a polypeptide according to the invention or a biologically active functional fragment thereof, in which a microorganism capable of producing the polypeptide is cultured, where appropriate the expression of the polypeptide is induced, and the polypeptide is isolated from the culture. If desired, the polypeptide can be produced on an industrial scale by this method.

The recombinant microorganism can be cultured and fermented by a known method. The bacteria can be cultured in, for example, TB or LB medium at pH6 to 9 at 20 to 40 ℃. Suitable culture conditions are described, for example, in t.manitis, e.f.fritsch and j.sambrook, Molecular Cloning: ALabortory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

If the polypeptide is not secreted into the culture medium, the product is obtained from the lysate after disruption of the cells using known protein isolation methods. Alternatively, the cells may be disrupted by high frequency ultrasound, high pressure, for example in a French press, osmolysis (osmolysis), action of denaturants, lytic enzymes or organic solvents, homogenization or a combination of these.

Purification of the polypeptide can be carried out by known chromatography methods such as molecular sieve chromatography (gel filtration), e.g., Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and other commonly used methods such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

The isolation of recombinant proteins is particularly facilitated by the use of vector systems or oligonucleotides which extend the cDNA by a specific nucleotide sequence and thus encode a modified polypeptide or fusion protein which can, for example, simplify purification. Suitable modifications of this type include, for example, so-called tags which act as anchors (e.g.hexa-histidine anchor modification) or epitopes which can be recognized as antigens by Antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can be used to bind the protein to a solid support, such as a polymer matrix, which can be loaded into a chromatography column, for example, or used on a microtiter plate or other support.

These anchors can also be used for protein recognition. Proteins may also be identified using conventional labels, such as fluorescent dyes, enzyme labels or radioactive labels which form a detectable reaction product upon reaction with a substrate, alone or in combination with an anchor.

The invention also relates to a process for the microbial production of vitamin B2 and/or a precursor and/or a derivative thereof.

If the transformation is carried out with a recombinant microorganism, it is preferred that the microorganism is first cultured in a complex medium in the presence of oxygen, for example at a pH of about 6 to 9 at a temperature of about 20 ℃ or more until the cell density is sufficient. In order to be able to better control the response, it is preferred to use inducible promoters. After induction of vitamin B2 production, the culture is continued for 12 hours to 3 days under aerobic conditions.

The following non-limiting examples describe specific embodiments of the present invention.

Description of the general experiments

a) General cloning methods

Cloning techniques employed in the present invention, such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, ligation of DNA fragments, transformation of E.coli cells, culture of bacteria, replication of phages, and sequence analysis of recombinant DNA, were performed as described in Sambrook et al (1989) supra.

b) Polymerase Chain Reaction (PCR)

PCR was performed in the following standard mixture according to standard protocols:

mu.l dNTP mix (200. mu.M), 10. mu.l MgCl free₂Taq polymerase buffer (10X), 8. mu.l MgCl₂(25mM), 1. mu.l (0.1. mu.M) of each primer, 1. mu.l of DNA (to be amplified), 2.5U of Taq polymerase (MBI Fermentas, Vilnius, Lithuania), deionized water to 100. mu.l.

c) Cultivation of Escherichia coli

In LB-amp medium (tryptone 10.0g, sodium chloride 5.0g, yeast extract 5.0g, ampicillin 100g/ml, H₂O to 1000ml) was added, and the recombinant Escherichia coli DH 5. alpha. strain was cultured at 37 ℃. For this purpose, one colony was transferred from the agar plate to 5ml LB-amp with an inoculating loop in each case. After approximately 18 hours of shaking culture at 220rpm, 4ml of the culture was inoculated into 400ml of medium in a2 liter shake flask. Expression of P450 in E.coli was induced by heat shock at 42 ℃ for 3 to 4 hours after OD578 values of between 0.8 and 1.0 were reached.

d) Purification of the desired product from the culture

The desired product can be isolated from the microorganism or culture supernatant using a variety of methods known in the art. If the desired product is not secreted by the cells, the cells can be separated from the culture by slow centrifugation and can be lysed by standard techniques such as mechanical force or sonication.

Cell debris was removed by centrifugation to give a supernatant fraction containing soluble protein for further purification of the desired compound. If the product is secreted by the cells, the cells in the culture are removed by slow centrifugation and the supernatant fraction is retained for further purification.

The supernatant fractions from both purification processes are chromatographed on a suitable resin, with the desired molecule remaining on or passing through the chromatography resin with a selectivity greater than that of the impurity. The chromatography step may be repeated with the same or different chromatography resin if necessary. Those skilled in the art will be able to select the appropriate chromatography resin and use it most efficiently to purify a particular molecule. The purified product may be concentrated by filtration or ultrafiltration and stored at the temperature at which the product is most stable.

Various purification methods are known in the art. These purification techniques are described, for example, in Bailey, j.e. & Ollis, d.f. biochemical Engineering Fundamentals, McGraw-Hill: NewYork (1986).

The identity and purity of the isolated compound can be determined using existing techniques. These techniques include High Performance Liquid Chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic analysis, or microbiological analysis. A summary of these analytical methods is given in: patek et al (1994) appl.environ.microbiol.60: 133-140; malakhova et al (1996) Biotek technology iya 1127-32; and Schmidt et al (1998) Bioprocess Engineer.19: 67 to 70; ullmann's encyclopedia of Industrial Chemistry (1996) Vol.A27, VCH: weinheim, pp.89-90, pp.521-540, pp.540-547, pp.559-566, pp.575-581 and pp.581-587; michal, G (1999) Biochemical Pathways: an Atlas of Biochemistry and molecular Biology, John Wiley and Sons; the use of Fallon, A. et al (1987) HPLC in Biochemistry, Vol.17.

e) General description of MPSS method, clone identification and homology search

The MPSS technique (Massive Parallel signal Sequencing, referenced Brenner et al, Nat. Biotechnol. (2000)18, 630-634) was applied to the filamentous fungus Ashbya gossypii producing vitamin B2. Highly accurate quantitative information about the expression levels of a large number of genes in eukaryotic organisms can be obtained using this technique. This technique involves isolating mRNA from an organism at a particular time X, transcribing it into cDNA with the aid of reverse transcriptase and cloning it into a specific vector with a specific tag sequence. A sufficient number (approximately 1000-fold higher) of vectors with different tag sequences are selected so that statistically every DNA molecule is cloned into a vector that is unique for its tag sequence.

The insert sequence of the vector is then cleaved off together with the tag. The DNA molecules obtained by this method are then incubated with microbeads having the molecular counterparts of the tags. After incubation it can be considered that only one type of DNA molecule is loaded per microbead by a specific tag or counterpart. These microbeads were transferred to a special flow cell and immobilized there, making it possible to perform colony sequencing of all microbeads using fluorescent dye-based modified sequencing and digital color camera. Although this method allows high numbers of assays to be performed, it is limited by the read width of about 16 to 20 base pairs. However, for most organisms, the sequence length is sufficient to establish a clear relationship between the sequence and the possible genes (frequency of 20bp sequence is-1X 10)¹²(ii) a In contrast, the human genome is "only" in size about 3X 10⁹bp)。

The number of identical sequences was counted and the frequencies were compared with each other to analyze the data thus obtained. Sequences that occur frequently reflect high levels of expression, while sequences that occur only once reflect low levels of expression. If mRNA is isolated at two different time points (X and Y), a chronological expression pattern of the individual genes can be constructed.

Example 1: isolation of mRNA from Ashbya gossypii

Ashbya gossypii (nutrient medium: 27.5g/l yeast extract; 0.5g/l magnesium sulfate; 50ml/l soybean oil; pH7) was cultured according to known techniques. Samples of the mycelia of Ashbya gossypii were taken at different times during the fermentation (24h, 48h and 72h) and the corresponding RNA or mRNA was isolated according to the protocol of Sambrook et al (1989).

Example 2: application of MPSS

mRNA isolated from Ashbya gossypii was analyzed using the MPSS assay described above.

The resulting data were analyzed using statistical analysis and classified according to the significance of the expression differences. This necessitates detection of increases and decreases in expression levels. The changes in expression were classified: a) monotonic change, b) after 24h, and c) after 48 h.

A20 bp sequence reflecting the change in expression, which was found by MPSS analysis, was hybridized as a probe with a gene library from Ashbya gossypii, the average size of the insert sequences of the library being approximately 1 kb. The hybridization temperature here is about 30 to 57 ℃.

Example 3: construction of genomic libraries from Ashbya gossypii

To construct a genomic DNA library, chromosomal DNA was first isolated by the methods of Wright and Philippsen (Gene (1991) 109: 99-105) and Mohr (1995, phD article, BioZentrum univorsit _ t Basel, Switzerland).

The DNA was partially digested with Sau 3A. For this purpose, 6. mu.g of genomic DNA were digested with different amounts (0.1 to 1U) of Sau3A enzyme. The DNA fragments were fractionated in a sucrose density gradient. The 1kb region was isolated and subjected to QiaEx extraction. The largest fragment was ligated with the BamHI-cut vector pRS416(Sikorski and Hieter, Genetics (1988) 122; 19-27) (90ng BamHI-cut dephosphorylated vector; 198ng insert DNA; 5ml water; 2. mu.l 10 Xligation buffer; 1U ligase). This ligation mixture was used to transform E.coli laboratory strain XL-1 blue, and the resulting clones were used to identify the insertion.

Example 4: preparation of an ordered Gene library (chip technology)

About 25,000 colonies of the Ashbya gossypii gene library (corresponding to about 3-fold coverage of the genome) were transferred sequentially to a nylon membrane and then examined by Sambrook et al (1989)Colony hybridization processing is described. Synthesis of oligonucleotides from 20bp sequences found by Mpss analysis and use of radioactivity³²And P is marked. In each case 10 labelled oligonucleotides of similar melting point were combined and hybridized together with the nylon membrane. After hybridization and washing, positive clones were identified by autoradiography and analyzed directly by PCR sequencing.

This method identified clones containing the insertion sequence with the internal name "Oligo 28" and having significant homology to the MIPS tag "Yta 7" or TBP7 from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 1, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 45" and having significant homology to the MIPS tag "p 39" or "Tif 34" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 6, and (b) a nucleic acid sequence shown in (b).

This method also identified clones containing an insertion sequence with the internal name "Oligo 85" and having significant homology to the MIPS tag "Rpl 35 a" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 12, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 133" and having significant homology to the MIPS tag "Nop 13" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 17, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 172" and having significant homology to the MIPS tag "Sua 5" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 21, and (b) the nucleic acid sequence shown in figure 21.

This method also identified clones containing the insertion sequence with the internal name "Oligo 63" and having significant homology to the MIPS tag "Rps 25 a" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 26, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 132" and having significant homology to the MIPS tag "Nic 96" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 31, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 174" and having significant homology to the MIPS tag "Ahc 1" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 38, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing an insertion with the internal name "Oligo 51" and having significant homology to the MIPS tag "Rok 1" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 42.

This method also identified clones containing an insertion sequence with the internal name "Oligo 30" and having significant homology to the MIPS tag "Rpa 34" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 48, or a nucleic acid sequence shown in SEQ ID NO.

This method also identified clones containing the insertion sequence with the internal name "Oligo 124" and having significant homology to the MIPS tag "Sub 2" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 53, nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 139" and having significant homology to the MIPS tag "DCP 1" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 58, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 144" and having significant homology to the MIPS tag "PRT 1" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 63, and a nucleic acid sequence shown in seq id no.

This method also identified clones containing an insertion sequence of the internal name "Oligo 168" and having significant homology to the MIPS tag "Rrp 9" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 67, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing an insertion sequence with the internal name "Oligo 160" and having significant homology to the MIPS tag "Rpl 8 b" from saccharomyces cerevisiae. The insertion sequence has the sequence shown in SEQ ID NO: 72, or a nucleic acid sequence shown in seq id no.

This method also identified clones containing the insertion sequence with the internal name "Oligo 18". The insertion sequence has the sequence shown in SEQ ID NO: 75 (complementary sequence of SEQ ID NO: 74). Possible ORFs are located in SEQ ID NO: between position 958 and 1272 of 75.

Example 5: sequence data analysis by BLASTX query

The analysis of the resulting nucleic acid sequences, i.e., the functional amino acid sequence by which the function is deduced, is performed in sequence databases by the BLASTX query method. Almost all amino acid sequence homologs found are related to Saccharomyces cerevisiae (baker's yeast). Since the entire sequence of the organism has been determined, more detailed information about these genes can be consulted:

http：//www.mips.gsf.de/proj/yeast/search/code search.htm。

the following homologies with the amino acid fragment of Saccharomyces cerevisiae were thus found. The corresponding alignment is shown in figures 1 to 15.

a) From SEQ ID NO: 1 (corresponding to nucleotides 3 to 374 and 373 to 1479) has significant sequence homology to the s.cerevisiae 26S proteasome subunit or TAT binding homolog 7(TBP 7). The corresponding alignment is shown in figure 1. SEQ ID NO: 2 and SEQ ID NO: 3 all show the amino acid partial sequences of the invention.

It was therefore concluded that the Ashbya gossypii nucleic acid sequence was found to function as a 26S proteasome subunit or TAT binding homolog 7(TBP 7).

b) From SEQ ID NO: 6 (see, SEQ ID NO: 7; corresponding to nucleotides 5 to 463 of SEQ ID NO: 6) has significant sequence homology with the subunit (P39) of the Saccharomyces cerevisiae translation initiation factor (EIF 3). The corresponding alignment is shown in figure 2. SEQ ID NO: 8 and SEQ ID NO: 9 all show the amino acid partial sequences of the invention.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of translation initiation factor subunit.

c) From SEQ ID NO: 12 has significant sequence homology with the ribosomal proteins of saccharomyces cerevisiae. FIG. 3 depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 469 to 825 of SEQ ID NO: 12) and the partial sequence of the s.cerevisiae protein. SEQ ID NO: 13 shows an N-terminally extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of ribosomal protein.

d) From SEQ ID NO: 17 has significant sequence homology with the saccharomyces cerevisiae nucleolin. FIG. 4 depicts the partial sequence of amino acids from the complementary strand (corresponding to nucleotides 114 to 1 of SEQ ID NO: 17) and the partial sequence of the s.cerevisiae protein. SEQ ID NO: 18 shows an N-terminal extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of nucleolin.

e) From SEQ ID NO: the amino acid sequence of the 21 coding strand has significant sequence homology with the Saccharomyces cerevisiae translation initiation protein. FIG. 5A depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 2 to 349 in SEQ ID NO: 21) and the partial sequence of this s.cerevisiae protein. FIG. 5B depicts another amino acid partial sequence from the coding strand (corresponding to nucleotides 336 to 947 in SEQ ID NO: 21) and the partial sequence of this s.cerevisiae protein. SEQ ID NO: 22 and SEQ ID NO: 23 each show an N-terminally extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of translation initiation protein.

f) From SEQ ID NO: 26 has significant sequence homology with the precursor of S31 ribosomal protein of saccharomyces cerevisiae. FIG. 6A depicts the partial sequence of amino acids from the complementary strand (corresponding to nucleotides 609 to 562 of SEQ ID NO: 26) and the partial sequence of this s.cerevisiae protein. FIG. 6B depicts another amino acid partial sequence from the complementary strand (corresponding to nucleotides 556 to 401 in SEQ ID NO: 26) and the partial sequence of this s.cerevisiae protein. SEQ ID NO: 27 and SEQ ID NO: 28 each shows an N-terminally extended amino acid partial sequence.

Therefore, it was concluded that the Ashbya gossypii nucleic acid sequence was found to function as a precursor of ribosomal protein S31.

g) From SEQ ID NO: 31 (see, SEQ ID NO: 32, corresponding to nucleotides 108 to 764 of SEQ ID NO: 31) have significant sequence homology to the saccharomyces cerevisiae nucleopore protein. Figure 7 depicts the corresponding alignment. Sequence SEQ ID NO: 33 to SEQ ID NO: 35 shows further amino acid partial sequences according to the invention.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of nucleoporin.

h) From SEQ ID NO: 38 has significant sequence homology with the components of the saccharomyces cerevisiae ADH-histone acetyltransferase complex. FIG. 8 depicts the partial sequence of amino acids from the complementary strand (corresponding to nucleotides 174 to 1 of SEQ ID NO: 38) and the partial sequence of the s.cerevisiae protein. SEQ ID NO: 39 shows an N-terminal extended amino acid partial sequence.

It can therefore be concluded that the Ashbya gossypii nucleic acid sequence was found to function as a component of the ADH-histone acetyltransferase complex.

i) From SEQ ID NO: 42 has significant sequence homology with the RNA helicase of Saccharomyces cerevisiae involved in RNA processing. FIG. 9A depicts the partial sequence of amino acids from the complementary strand (corresponding to nucleotides 1086 to 1012 of SEQ ID NO: 42) and the partial sequence of this s.cerevisiae enzyme. FIG. 9B depicts another amino acid partial sequence from the complementary strand (corresponding to nucleotides 1022 to 915 of SEQ ID NO: 42) and a partial sequence of this s.cerevisiae enzyme. FIG. 9C depicts a further amino acid partial sequence from the complementary strand (corresponding to nucleotides 925 to 689 of SEQ ID NO: 42) together with the partial sequence of the s.cerevisiae enzyme. SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 shows the partial sequence of the N-terminal extended amino acid.

Therefore, it was concluded that the Ashbya gossypii nucleic acid sequence was found to function as an RNA helicase involved in RNA processing.

k) From SEQ ID NO: the amino acid sequence of the 48 coding strand has significant sequence homology with a non-essential component of Saccharomyces cerevisiae RNA pol I. FIG. 10A depicts the partial sequence of amino acids from this chain (corresponding to nucleotides 1 to 102 of SEQ ID NO: 48) with the partial sequence of the s.cerevisiae protein. FIG. 10B depicts another amino acid partial sequence from the chain (corresponding to nucleotides 122 to 400 of SEQ ID NO: 48) with a partial sequence of the s.cerevisiae protein. SEQ ID NO: 49 and SEQ ID NO: 50 all show the amino acid partial sequences of the present invention.

It can therefore be concluded that the Ashbya gossypii nucleic acid sequence was found to function as an unnecessary component of RNA pol I.

l) is derived from SEQ ID NO: the amino acid sequence of the 53 coding strand has significant sequence homology with saccharomyces cerevisiae RNA helicase. FIG. 11A depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 2 to 148 of SEQ ID NO: 53) and the partial sequence of the s.cerevisiae enzyme. FIG. 11B depicts another amino acid partial sequence from the coding strand (corresponding to nucleotides 150 to 185 of SEQ ID NO: 53) together with a partial sequence of the s.cerevisiae enzyme. SEQ ID NO: 54 and SEQ ID NO: 55 each shows an N-terminally extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of RNA helicase.

m) is from SEQ ID NO: 58 has significant sequence homology with the saccharomyces cerevisiae mRNA decapping enzyme. FIG. 12 depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 2 to 82 of SEQ ID NO: 58) and the partial sequence of the s.cerevisiae enzyme. SEQ ID NO: 59 shows an N-terminal extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence was found to function as an mRNA decapping enzyme.

n) is from SEQ ID NO: 63 has significant sequence homology with the subunit of the saccharomyces cerevisiae translation initiation factor eIF 3. FIG. 13 depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 21 to 695 of SEQ ID NO: 63) and the partial sequence of the s.cerevisiae protein. SEQ ID NO: 64 shows an N-terminal extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence was found to function as a subunit of the translation initiation factor eIF 3.

o) a sequence derived from SEQ ID NO: 67 has significant sequence homology with the U3 nucleolar ribonucleoprotein-related protein of Saccharomyces cerevisiae involved in RNA processing of the pre-ribosome. FIG. 14A depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 1 to 111 of SEQ ID NO: 67) and the partial sequence of the s.cerevisiae protein. FIG. 14B depicts the partial sequence of amino acids from the coding strand (corresponding to nucleotides 144 to 887 of SEQ ID NO: 67) with the partial sequence of the s.cerevisiae protein. SEQ ID NO: 68 and SEQ ID NO: 69 each show an N-terminally extended amino acid partial sequence.

Therefore, it can be concluded that the Ashbya gossypii nucleic acid sequence has the function of U3 nucleolar ribonucleoprotein-related protein involved in RNA processing of the precore.

p) is from SEQ ID NO: 72 has significant sequence homology with the ribosomal protein of Saccharomyces cerevisiae (L7 a.e.B/60S large subunit). FIG. 15 depicts the partial sequence of amino acids from the complementary strand (corresponding to nucleotides 508 to 176 of SEQ ID NO: 72) and the partial sequence of the s.cerevisiae protein. SEQ ID NO: 73 shows the partial sequence of the N-terminal extended amino acid.

Therefore, it was concluded that the Ashbya gossypii nucleic acid sequence was found to function as a ribosomal protein (L7 a.e.B/60S large subunit).

Example 6: isolation of full-Length DNA

a) Construction of Ashbya gossypii Gene library

High molecular weight cellular total DNA was prepared from 100ml Ashbya gossypii cultures grown for two days in liquid MA2 medium (10 g glucose, 10g peptone, 1 g yeast extract, 0.3g inositol, up to 1000 ml). The mycelium was filtered off and washed twice with distilled water, suspended in 10ml of a solution containing 1M sorbitol, 20mM EDTA and 20mg of zymolase 20T and incubated at 27 ℃ with gentle shaking for 30 to 60 minutes. The protoplast suspension was adjusted to contain 50mM Tris-HCl, pH7.5, 150mM NaCl, 100mM EDTA and 0.5% strength Sodium Dodecyl Sulfate (SDS) and incubated at 65 ℃ for 20 minutes. After two phenol/chloroform (volume ratio 1: 1) extractions, DNA was precipitated with isopropanol, suspended in TE buffer, treated with RNase, precipitated again with isopropanol and suspended in TE buffer.

Genomic DNA selected by size and partially digested with Sau3A was ligated with the dephosphorylated arm of the cosmid vector Super-Cos1(Stratagene) to generate the Ashbya gossypii cosmid gene library. The Super-Cos1 vector was digested with Xbal between the two Cos sitesOpen and dephosphorylate with bovine small intestine alkaline phosphatase (Boehringer), followed by opening of the cloning site with BamHI. This ligation contained 2.5. mu.g of partially digested chromosomal DNA in 20. mu.l, 1. mu.g of the Super-Cos1 vector arm, 40mM Tris-HCl, pH7.5, 10mM MgCl₂1mM dithiothreitol, 0.5mM ATP and 2Weiss units of T4-DNA ligase (Boehringer) were added to the mixture at 15 ℃ overnight. The ligation products were packaged in vitro using the protocol for extracts and Stratagene (Gigapack II Packaging Extract). The packaged product was used to infect E.coli NM554(recA13, araD139,. DELTA. (ara, leu)7696,. DELTA. (lac)17A, galU, galK, hsrR, rps (strr), mcrA, mcrB) and spread on LB plates containing ampicillin (50. mu.g/ml). Transformants containing Ashbya gossypii inserts averaging 30-45kb in length were obtained.

b) Cosmid gene library storage and screening

Will be 4X 10 in total⁴Each fresh single colony was individually inoculated into 100. mu.l of LB medium supplemented with a freezing medium (36mM K) in wells of a 96-well microtiter plate (Falcon, No.3072)₂HPO₄/13.2mM KH₂PO₄1.7mM sodium citrate, 0.4mM MgSO₄、6.8mM(NH₄)₂SO₄4.4% (w/v) glycerol) and ampicillin (50. mu.g/ml), grown overnight with shaking at 37 ℃ and then frozen at-70 ℃. The plates were thawed quickly and replicated in fresh medium using 96-well replica plates sterilized with an alcohol bath followed by evaporation of the alcohol on a hotplate. Before freezing and after thawing (before taking all other measures), the plates were briefly shaken in a microtiter plate shaker (Infors) to ensure homogeneity of the cell suspension. Clones were placed on nylon membranes using an automated mechanical system (Bio-Robotics) capable of transferring small amounts of liquid from 96-well microtiter plates to nylon membranes (GeneScreen Plus, New England Nuclear). After transfer of the cultures from the 96-well microtiter plate (1920 clones), the membrane was placed on the surface of LB agar containing ampicillin (50. mu.g/ml) in a 22X 22 cm petri dish (Nunc) and incubated overnight at 37 ℃. Prior to cell confluence, cells were treated with Herrmann, B.G., Barlow, D.P. and Lehrach, H. (1)987) Cell 48, pp.813-825, including an additional treatment after the first denaturing step, i.e. exposing the filter membrane to steam for 5 minutes above a pad saturated with denaturing solution on a boiling water bath.

By using random hexamer primer labeling (Feinberg, A.P. and Vogelstein, B. (1983), anal. biochem.132, pp.6-13)³²P]dCTP is taken up to obtain a labeled double-stranded probe with high specific activity. These membranes were prehybridized at 42 ℃ in a medium containing 50% (vol/vol) formamide, 600 mM sodium phosphate, pH7.2, 1mM EDTA, 10% dextran sulfate, 1% SDS and 10 XDenhardt's solution, salmon sperm DNA (50. mu.g/ml) and³²p-labeled Probe (0.5-1X 10)⁶cpm/ml) for 6 to 12 hours. Typically, the membrane is washed in 13 to 30mM NaCl, 1.5 to 3 mM sodium citrate, pH6.3, 0.1% SDS at 55 to 65 ℃ for about 1 hour and the membrane is autoradiographed with a Kodak screen at-70 ℃ for 12 to 24 hours. To date, each film has been successfully reused more than 20 times. The probes on the filters were stripped by incubation in 2mM Tris-HCl, pH8.0, 0.2mM EDTA, 0.1% SDS for 2X 20 minutes at 95 ℃.

c) Recovery of Positive colonies from the stored Gene library

Frozen bacterial cultures in wells of microtiter plates were scraped with sterile disposable lancets and streaked onto LB agar plates containing ampicillin (50. mu.g/ml). Single colonies were inoculated into liquid media to produce DNA by alkaline lysis (Birnboim, H.C. and Doly, J. (1979), nucleic acids Res.7, pp.1513-1523).

d) full-Length DNA

Clones containing inserts with the correct complete sequence can be identified as described above. The internal names of these clones are:

"Oligo 28 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 4.

"Oligo 45 v", insert comprising the complete sequence has the sequence shown in SEQ ID NO: 10.

"Oligo 85 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 14, or a nucleic acid sequence as set forth in seq id no. The protein encoded by this sequence preferably comprises SEQ ID NO: 15 and 16.

"Oligo 133 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 19.

"Oligo 172 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 24, or a nucleic acid sequence as set forth in seq id no.

"Oligo 63 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 29, or a nucleic acid sequence as set forth in seq id No. 29.

"Oligo 132 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 36, or a nucleic acid sequence as set forth in seq id no.

"Oligo 174 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 40, or a nucleic acid sequence as set forth in seq id no.

"Oligo 51 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 46, or a nucleic acid sequence as set forth in seq id no.

"Oligo 30 v", insert comprising the complete sequence has the sequence shown in SEQ ID NO: 51, or a nucleic acid sequence as set forth in seq id no.

"Oligo 124 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 56.

"Oligo 139 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 60, or a variant thereof. The protein encoded by this sequence preferably comprises SEQ ID NO: 61 and 62, or a pharmaceutically acceptable salt thereof.

"Oligo 144 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 65, or a nucleic acid sequence as set forth in seq id no.

"Oligo 168 v", an insert comprising the complete sequence has the sequence shown in SEQ ID NO: 70, or a nucleic acid sequence as set forth in seq id no.

"Oligo 18 v", insert comprising the complete sequence has the sequence shown in SEQ ID NO: 77.

Example 7: testing the modulating effect of Oligo 18 on vitamin B2 production

To determine whether integration of DNA in the vicinity of the possible reading frame of Oligo 18 adversely affected riboflavin synthesis, DNA fragments were integrated by homologous recombination into the genome of the Ashbya gossypii strain used (Ashbya TEF promoter + G418 resistance gene-see FIG. 1). Transformation is carried out by known electroporation methods. Positive transformants were identified by PCR using the primer pairs shown in FIG. 1. The vitamin B2 production was investigated in shake flask experiments and in laboratory fermentations for one transformant in which specific integration at the site could be detected. Integration of this DNA fragment was found to increase riboflavin production (approximately 3%). The information in the TEF-G418 construct was unlikely to be the cause. Thus inferring that a position effect is present.

Shake flask test for riboflavin assay

0.5ml of glycerol culture or about one inoculation loop of 5 day old mycelia taken well from SP agar plates were inoculated into 10ml of pre-culture medium (9.5ml [9.5g ] medium +0.5ml soybean oil) in a 100ml double baffle Erlenmeyer flask and shaken at 180rpm (box shaker, amplitude 2.5cm) for 40 hours at 28 ℃.

In a 250ml Erlenmeyer flask 1.1ml of this culture was inoculated into 25.7ml of main medium (21.2ml [21.2g ] medium, 1ml of urea [10g/45ml ] +3.5ml [3.2g ] soybean oil, a final volume of 26.8ml, where 4.4ml compensated for evaporation during non-humidified shaking) or 21.8ml of main medium (17.3ml [17.3g ] medium, 1ml of urea [10g/45ml ] +3.5ml [3.2g ] soybean oil, a final volume of 22.9ml, where 0.5ml compensated for evaporation during shaking in a non-human humidified environment) and shaken at 220rpm (industrial shaker, amplitude 5cm) or at 300rpm (box shaker amplitude, 2.5cm) for 5 days at 28 ℃.

After shaking vigorously a mixture of 0.5ml of the main culture and 4.5ml of a [5g ] 40% strength nicotinamide solution (dilution factor 10) or a mixture of 0.25ml of the main culture and 4.75ml of a [5.27g ] 40% strength nicotinamide solution (dilution factor 20) in a test tube and incubating in a water bath at 70 ℃ for approximately 2X 20 minutes (cell lysis, shaking in between), 40. mu.l were placed in a disposable large cuvette, mixed with 3ml of deionized water and measured in the photometer as soon as possible, since vitamin B2 will decompose very quickly. This requires measurement of the extinction values at 402, 446 and 550nm, calculated as follows:

V＝(W1-W2×C+W3×(C-1))∶(B1-B2×C)

wherein,

b1 ═ 17.36[ constant ]

B2 ═ 31.15 [ constant ]

K ═ colorimetric pool volume ml [ ]Standard value of 3.04ml]

Sample volume of [ P ], [ ml ], [The standard value was 0.04ml]

F is the dilution multipleStandard value of 10Namely: 0.5 ml: 5ml]

Correction factor [ (550 + 405)/(550 + 450) ═ 1.45]

Extinction value of 402nm W1

Extinction value of 446nm when W2 is used

Extinction value of 550nm W3

-＞V＝(W1-1.45W2+0.45W3)∶-27.8075

Vitamin B2 concentration (V × K: P × F)

＝V×3.04∶0.04×10

＝V×760

These values must also be used taking into account the evaporation of the medium during shaking:

g1-weight of flask immediately after inoculation

G2 weight of flask before sampling

KV 1-volume of medium with offset evaporation

[22.4ml+4.4ml＝26.8ml]

KV 2-volume of medium [22.4ml ]

B₂Uncorrected vitamin B2 concentration calculated previously

Vitamin B₂Concentration (corrected) ((KV1- (G1-G2)): KV2) × B₂

＝((26.8-(G1-G2))∶22.4)×B₂

From the above studies, it was concluded that the Ashbya gossypii nucleic acid sequence was found to function as a protein regulating vitamin B2 productivity.

Table 1: sequence overview

SEQID NO：	Oligo	DESCRIPTION OF THE SEQUENCES	Sequence homologs
				1	028	DNA partial sequence	Saccharomyces cerevisiae 26S proteasome subunit or TAT binding homolog 7(TBP7)
2	028	From SEQ ID NO: 1, the amino acid partial sequence of the complementary strand of
			3	028	From SEQ ID NO: 1, the amino acid partial sequence of the complementary strand of
4	028	Full-length DNA sequence
			5	028	Corresponding to SEQ ID NO: 4 from position 245 to position 4222
6	045	DNA partial sequence				Saccharomyces cerevisiae translation initiation factor subunit
			7	045	From SEQ ID NO: 6 in the complementary strand
8	045	From SEQ ID NO: 6 in the complementary strand
			9	045	From SEQ ID NO: 6 in the complementary strand
10	045	Full-length DNA sequence
			11	045	Corresponding to SEQ ID NO: 10 from position 640 to position 1674
12	085	DNA partial sequence					Saccharomyces cerevisiae ribosomal protein
			13	085	From SEQ ID NO: 12 in the coding strand of the sequence
14	085	Full-length DNA sequence
			15	085	Corresponding to SEQ ID NO: 14 amino acid sequence of the coding region from position 92 to 307
16	085	Corresponding to SEQ ID NO: 14 amino acid sequence of the coding region from position 403 to 858
			17	133	DNA partial sequence	Saccharomyces cerevisiae nucleolus protein
18	133	From SEQ ID NO: 17 in the complementary strand
			19	133	Full-length DNA sequence
20	133	Corresponding to SEQ ID NO: 19, 1371 to 2495 th coding region
			21	172	DNA partial sequence		Saccharomyces cerevisiae translation initiation protein
22	172	From SEQ ID NO: 21 in the coding strand of the sequence
			23	172	From SEQ ID NO: 21 in the coding strand of the sequence
24	172	Full-length DNA sequence
			25	172	Corresponding to SEQ ID NO: 24 amino acid sequence of the coding region from position 277 to position 1476

SEQID NO：	Oligo	DESCRIPTION OF THE SEQUENCES	Sequence homologs
				26	063	DNA partial sequence	Saccharomyces cerevisiae ribosomal protein S31
27	063	From SEQ ID NO: 26, the amino acid partial sequence of the complementary strand of
			28	063	From SEQ ID NO: 26, the amino acid partial sequence of the complementary strand of
29	063	Full-length DNA sequence
			30	063	Corresponding to SEQ ID NO: 29 amino acid sequence of the coding region from position 533 to position 856
31	132	DNA partial sequence				Saccharomyces cerevisiae nucleoporin
			32	132	From SEQ ID NO: 31 in the complementary strand of the sequence
33	132	From SEQ ID NO: 31 in the complementary strand of the sequence
			34	132	From SEQ ID NO: 31 in the complementary strand of the sequence
35	132	From SEQ ID NO: 31 in the complementary strand of the sequence
			36	132	Full-length DNA sequence
37	132	Corresponding to SEQ ID NO: 36 amino acid sequence of the coding region from 629 to 3181
			38	174	DNA partial sequence		Saccharomyces cerevisiae ADH-histone acetyltransferase complexes
39	174	From SEQ ID NO: 38 of the complementary strand of the sequence
			40	174	Full-length DNA sequence
41	174	Corresponding to SEQ ID NO: 40 from position 964 to position 2589
			42	051	DNA partial sequence	RNA helicase of saccharomyces cerevisiae participating in RNA processing
43	051	From SEQ ID NO: 42, the amino acid partial sequence of the complementary strand of
			44	051	From SEQ ID NO: 42, the amino acid partial sequence of the complementary strand of
45	051	From SEQ ID NO: 42, the amino acid partial sequence of the complementary strand of
			46	051	Full-length DNA sequence
47	051	Corresponding to SEQ ID NO: 46 from position 502 to 2208
			48	030	DNA partial sequence		Saccharomyces cerevisiae RNA pol I non-essential component
49	030	From SEQ ID NO: 48 in the coding strand of the sequence
			50	030	From SEQ ID NO: 48 in the coding strand of the sequence
51	030	Full-length DNA sequence
			52	030	Corresponding to SEQ ID NO: 51 amino acid sequence of the coding region from position 198 to position 1073

SEQID NO：	Oligo	DESCRIPTION OF THE SEQUENCES	Sequence homologs
				53	124	DNA partial sequence	Saccharomyces cerevisiae RNA helicase
54	124	From SEQ ID NO: 53 coding strand of the sequence of the amino acid part
			55	124	From SEQ ID NO: 53 coding strand of the sequence of the amino acid part
56	124	Full-length DNA sequence
			57	124	Corresponding to SEQ ID NO: 56 amino acid sequence of the coding region from 465 th to 1775 th
58	139	DNA partial sequence				Saccharomyces cerevisiae mRNA decapping enzyme
			59	139	From SEQ ID NO: 58 in the coding strand
60	139	Full-length DNA sequence
			61	139	Corresponding to SEQ ID NO: 60 bits 402 to 638The amino acid sequence of the coding region of (1)
62	139	Corresponding to SEQ ID NO: 60 amino acid sequence of the coding region from position 663 to position 974
			63	144	DNA partial sequence		Subunit of Saccharomyces cerevisiae translation initiation factor eIF3
64	144	From SEQ ID NO: 63, of the coding strand of the coding sequence
			65	144	Full-length DNA sequence
66	144	Corresponding to SEQ ID NO: 65 amino acid sequence of the coding region from position 468 to 2675
			67	168	DNA partial sequence	Saccharomyces cerevisiae U3 nucleolar small ribonucleoprotein related protein involved in processing of RNA of precore
68	168	From SEQ ID NO: 67 amino acid partial sequence of the coding strand
			69	168	From SEQ ID NO: 67 amino acid partial sequence of the coding strand
70	168	Full-length DNA sequence
			71	168	Corresponding to SEQ ID NO: 70 amino acid sequence of the coding region from position 660 to position 2432
72	160	DNA partial sequence				Saccharomyces cerevisiae ribosomal protein (L7 a.e.B; 60S large subunit)
			73	160	From SEQ ID NO: 72 in the complementary strand
74	018	DNA partial sequence					Regulator for vitamin B2 production
			75	018	Full-length DNA sequence
76	018	Corresponding to SEQ ID NO: 75 from 958 to 1272
			77	018	Full-length DNA sequence
78	018	Corresponding to SEQ ID NO: 77 from position 1531 to 1845

Sequence listing

Sequence listing

<110> Pasteur Fungines (BASF Aktiengesellschaft)

<120> novel gene products from Ashbya gossypii associated with transcriptional, RNA processing and/or translational mechanisms

<130> M/42319/PCT

<160> 78

<170> PatentIn version 3.1

<210> 1

<211> 1481

<212> DNA

<213> Ashbya gossypii (Ashbya gossypii)

<220>

<221> misc_feature

<223> Oligo 28

<220>

<221> misc_feaure

<222> (151)..(151)

<223> unknown amino acid

<400> 1

gatctttgaa aacgaatgar agagtgttac ttttagctat tgcggaaggc ttcaccgaag 60

aagagttaca taacacacct ttgtcgttat ttggctttga cctgaatatt gkctctatta 120

gagaaccaty tacttgccca aagacacaac ntacttcaaa actctkgaag agttacctga 180

aaatgaagcc cactaagttt cgagagaaga ggaaaagaaa aaagccactg ccggtcctac 240

ccgtgtgcga tactcccagc aacgatgata tyggttctga tggtgaactm ttaaccgaag 300

acgaaaagty gaggaggaag ttaaaatcat accatcacca agacatgaaa ttgaagaata 360

ctttgaagat aaaagctttc cggtctgatg gatctattta aggggcgtta taaaagattt 420

aggaaaccgc ccattgagga ttccttgctt attcatctat ttgaaccaga agcctatgcc 480

tcaaatcctg aatggcaacc ggcttatgta agagaagatg acatgatttt agaagtttca 540

accggcagga agtactacaa tatggatttg gatattatag aagaaaggtt gtggaacggg 600

tattactctg aaccgaaaca gtatttgaag gatatcgagt taatttaccg cgatgctaat 660

acaactggtg accgagagcg tataataaaa gcgtcagaga tgtttgcaaa cgcacagatg 720

ggcattgaag aaatatatac gcctgagctt atccaggagt gcagggatac acgtcaaaga 780

gaactattaa ggcagcagtt gtatttacgc gaccagcaga aaaagataca cgaggaagaa 840

gctaagctcg agcaggagaa aaaggatccg acttttccaa atgacgataa tcatccagaa 900

gaggccgact taagcgttgg ggctggacag caattgcata gcccatcaca aatgtctcat 960

gaggcttgtg aaagccacgc agaagatgga ctgggggtac cacatggctt tgaaaataac 1020

acatatgcta ttggagataa cacaccagag acgatcgata acgggtctcc taaacccyta 1080

gaggactttt gcggggaaga caagactgtc cctgacgaca aatccgtgcc agaacgattt 1140

gttcaagagg gagaagcttc tgagatgggc tttatcccga aaacaccttc cgccagaccc 1200

gtctccatgc actcaaatcc atcaagcaaa gaatccgtag ttgctcctat gcaaaagact 1260

gggttagaag tgaacagtat cgaggacacg tcagtcgctg ttcaatcaga actgacccgc 1320

gatgaagact tacccatgac tcctgaaccg gcctttatcc tggatgaaac cgttttagcg 1380

aaaataatct cgatgttgaa aacaaaaaca cagggattta cagtcgaaca gctggaaaca 1440

tgctatgctg ccgtactgga gctagtatgg gaggcgagat c 1481

<210> 2

<211> 124

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 28

<220>

<221> misc_feature

<222> (37)..(37)

<223> unknown amino acid

<220>

<221> misc_feature

<222> (43)..(43)

<223> unknown amino acid

<220>

<221> misc_feature

<222> (50)..(50)

<223> unknown amino acid

<220>

<221> misc_feature

<222> (55)..(55)

<223> unknown amino acid

<220>

<221> misc_feature

<222> (103)..(103)

<223> unknown amino acid

<400> 2

Ser Leu Lys Thr Asn Glu Arg Val Leu Leu Leu Ala Ile Ala Glu Gly

5 10 15

Phe Thr Glu Glu Glu Leu His Asn Thr Pro Leu Ser Leu Phe Gly Phe

20 25 30

Asp Leu Asn Ile Xaa Ser Ile Arg Glu Pro Xaa Thr Cys Pro Lys Thr

35 40 45

Gln Xaa Thr Ser Lys Leu Xaa Lys Ser Tyr Leu Lys Met Lys Pro Thr

50 55 60

Lys Phe Arg Glu Lys Arg Lys Arg Lys Lys Pro Leu Pro Val Leu Pro

65 70 75 80

Val Cys Asp Thr Pro Ser Asn Asp Asp Ile Gly Ser Asp Gly Glu Leu

85 90 95

Leu Thr Glu Asp Glu Lys Xaa Arg Arg Lys Leu Lys Ser Tyr His His

100 105 110

Gln Asp Met Lys Leu Lys Asn Thr Leu Lys Ile Lys

115 120

<210> 3

<211> 369

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 28

<400> 3

Lys Leu Ser Gly Leu Met Asp Leu Phe Lys Gly Arg Tyr Lys Arg Phe

1 5 10 15

Arg Lys Pro Pro Ile Glu Asp Ser Leu Leu Ile His Leu Phe Glu Pro

20 25 30

Glu Ala Tyr Ala Ser Asn Pro Glu Trp Gln Pro Ala Tyr Val Arg Glu

35 40 45

Asp Asp Met Ile Leu Glu Val Ser Thr Gly Arg Lys Tyr Tyr Asn Met

50 55 60

Asp Leu Asp Ile Ile Glu Glu Arg Leu Trp Asn Gly Tyr Tyr Ser Glu

65 70 75 80

Pro Lys Gln Tyr Leu Lys Asp Ile Glu Leu Ile Tyr Arg Asp Ala Asn

85 90 95

Thr Thr Gly Asp Arg Glu Arg Ile Ile Lys Ala Ser Glu Met Phe Ala

100 105 110

Asn Ala Gln Met Gly Ile Glu Glu Ile Ser Thr Pro Glu Leu Ile Gln

115 120 125

Glu Cys Arg Asp Thr Arg Gln Arg Glu Leu Leu Arg Gln Gln Leu Tyr

130 135 140

Leu Arg Asp Gln Gln Lys Lys Ile His Glu Glu Glu Ala Lys Leu Glu

145 150 155 160

Gln Glu Lys Lys Asp Pro Thr Phe Pro Asn Asp Asp Asn His Pro Glu

165 170 175

Glu Ala Asp Leu Ser Val Gly Ala Gly Gln Gln Leu His Ser Pro Ser

180 185 190

Gln Met Ser His Glu Ala Cys Glu Ser His Ala Glu Asp Gly Leu Gly

195 200 205

Val Pro His Gly Phe Glu Asn Asn Thr Tyr Ala Ile Gly Asp Asn Thr

210 215 220

Pro Glu Thr Ile Asp Asn Gly Ser Pro Lys Pro Leu Glu Asp Phe Cys

225 230 235 240

Gly Glu Asp Lys Thr Val Pro Asp Asp Lys Ser Val Pro Glu Arg Phe

245 250 255

Val Gln Glu Gly Glu Ala Ser Glu Met Gly Phe Ile Pro Lys Thr Pro

260 265 270

Ser Ala Arg Pro Val Ser Met His Ser Asn Pro Ser Ser Lys Glu Ser

275 280 285

Val Val Ala Pro Met Gln Lys Thr Gly Leu Glu Val Asn Ser Ile Glu

290 295 300

Asp Thr Ser Val Ala Val Gln Ser Glu Leu Thr Arg Asp Glu Asp Leu

305 310 315 320

Pro Met Thr Pro Glu Pro Ala Phe Ile Leu Asp Glu Thr Val Leu Ala

325 330 335

Lys Ile Ile Ser Met Leu Lys Thr Lys Thr Gln Gly Phe Thr Val Glu

340 345 350

Gln Leu Glu Thr Cys Tyr Ala Ala Val Leu Glu Leu Val Trp Glu Ala

355 360 365

Arg

<210> 4

<211> 4670

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (245)..(4222)

<223>

<220>

<221> misc_feature

<223> Oligo 28

<400> 4

tctcgttccg aagctgaggc tggatacgaa tatcaccata tacgaaggcc ccgccacccc 60

gagaaaggca tttttcccct ttgaaatggg ttatgtgacg atgttcatga gttgtgcaga 120

aaagcaggga gggaaagacg agtcattagc atcgaagggc agcattgtgt agtcttagtg 180

tcgtattttt ggatctgcaa taggtttttg atttatctct cggtcttcga tttgatagag 240

aatc atg gcg cgt cat aca agg aga tcg cat cag cac gtg aac gac gaa 289

Met Ala Arg His Thr Arg Arg Ser His Gln His Val Asn Asp Glu

1 5 10 15

gat aca ggt agc gaa ata tac gat aaa aat ggg atc aag cac acg acg 337

Asp Thr Gly Ser Glu Ile Tyr Asp Lys Asn Gly Ile Lys His Thr Thr

20 25 30

ccg agg tcg ctg aag aag atc aat tac gcg gaa atc gaa aac agt tac 385

Pro Arg Ser Leu Lys Lys Ile Asn Tyr Ala Glu Ile Glu Asn Ser Tyr

35 40 45

gat tat atg gag gac tac gat gaa gga gac aag gaa gag gag cct gca 433

Asp Tyr Met Glu Asp Tyr Asp Glu Gly Asp Lys Glu Glu Glu Pro Ala

50 55 60

gaa ctg gag aac aac ggg cgg agc aag gga ctg gta ggc cgc gag cgc 481

Glu Leu Glu Asn Asn Gly Arg Ser Lys Gly Leu Val Gly Arg Glu Arg

65 70 75

ggg cgg gag ccg gag gag gac gag gac gag gag ggg cca gtc cgg ggg 529

Gly Arg Glu Pro Glu Glu Asp Glu Asp Glu Glu Gly Pro Val Arg Gly

80 85 90 95

cgg cgc agc cgg aaa cgc act tat gtg gat gtg gag gag gac gag agc 577

Arg Arg Ser Arg Lys Arg Thr Tyr Val Asp Val Glu Glu Asp Glu Ser

100 105 110

ttc cac gag gag gag gcg gag gac gag gac gag gag gcg ctg gac gac 625

Phe His Glu Glu Glu Ala Glu Asp Glu Asp Glu Glu Ala Leu Asp Asp

115 120 125

gat gag gac gag gac gag gag gag cgg aac tgg cgg cgg cgg cgc gag 673

Asp Glu Asp Glu Asp Glu Glu Glu Arg Asn Trp Arg Arg Arg Arg Glu

130 135 140

cac cag ttt gtt gta gag gac gag gat gat gac gag gac gag gag gac 721

His Gln Phe Val Val Glu Asp Glu Asp Asp Asp Glu Asp Glu Glu Asp

145 150 155

gac tat gga gca cag aaa aaa cgc ggc agg cgg cgg acc cgg cgt ggc 769

Asp Tyr Gly Ala Gln Lys Lys Arg Gly Arg Arg Arg Thr Arg Arg Gly

160 165 170 175

agg tcg cgg cta aca gag cgg cgt tcg ccg cca cgc aag ctg cgg aca 817

Arg Ser Arg Leu Thr Glu Arg Arg Ser Pro Pro Arg Lys Leu Arg Thr

180 185 190

cgc cgg acg cga tcg tct gtg aac atg tat gac agt gag cat gac ggg 865

Arg Arg Thr Arg Ser Ser Val Asn Met Tyr Asp Ser Glu His Asp Gly

195 200 205

act ggc gaa gcg ttg acg ctg gag gac gag att cgg gaa ctg cag gag 913

Thr Gly Glu Ala Leu Thr Leu Glu Asp Glu Ile Arg Glu Leu Gln Glu

210 215 220

gat tcg ccc atc cgc gag aaa cgg tcg ctg cgg gag cgc aca aag ccg 961

Asp Ser Pro Ile Arg Glu Lys Arg Ser Leu Arg Glu Arg Thr Lys Pro

225 230 235

gta aat tac acg ctg ccc cca ccc ctg act gat aac caa atg aac gtt 1009

Val Asn Tyr Thr Leu Pro Pro Pro Leu Thr Asp Asn Gln Met Asn Val

240 245 250 255

gat ggc act gct gca cca gcg tac ggg agc ttc cat tct ccc aga ggc 1057

Asp Gly Thr Ala Ala Pro Ala Tyr Gly Ser Phe His Ser Pro Arg Gly

260 265 270

aag cgt ggg cta cac tcg gga caa agt ttt gga cct atc agg cga ttg 1105

Lys Arg Gly Leu His Ser Gly Gln Ser Phe Gly Pro Ile Arg Arg Leu

275 280 285

ttt cca acg ggc ggc cct ttc ggc gga aac gat gtg aca gct ata ttc 1153

Phe Pro Thr Gly Gly Pro Phe Gly Gly Asn Asp Val Thr Ala Ile Phe

290 295 300

ggc cac aac acc aac ttt tat gca acg gct caa gat ccg act gtg gcc 1201

Gly His Asn Thr Asn Phe Tyr Ala Thr Ala Gln Asp Pro Thr Val Ala

305 310 315

aat aac aag ttt atc gat tcg gac tct tca gat gac gaa att ttg ccg 1249

Asn Asn Lys Phe Ile Asp Ser Asp Ser Ser Asp Asp Glu Ile Leu Pro

320 325 330 335

ctg gga agt acc ccc aaa cca aaa agc tcc gaa gct aag aaa aag aaa 1297

Leu Gly Ser Thr Pro Lys Pro Lys Ser Ser Glu Ala Lys Lys Lys Lys

340 345 350

aag ccg gag att gca gat ctg gat ccg tta gga gta gat atg aat att 1345

Lys Pro Glu Ile Ala Asp Leu Asp Pro Leu Gly Val Asp Met Asn Ile

355 360 365

aat ttt gat gac att ggc ggt ctg gat aac tac ata gac cag ctg aag 1393

Asn Phe Asp Asp Ile Gly Gly Leu Asp Asn Tyr Ile Asp Gln Leu Lys

370 375 380

gag atg gtt gcg ttg ccg tta ctc tat ccg gag ctc tat caa aat ttc 1441

Glu Met Val Ala Leu Pro Leu Leu Tyr Pro Glu Leu Tyr Gln Asn Phe

385 390 395

aac atc aca cct ccg cgt ggt gtg ctc ttc tat ggg cca cca ggt acg 1489

Asn Ile Thr Pro Pro Arg Gly Val Leu Phe Tyr Gly Pro Pro Gly Thr

400 405 410 415

ggt aag acg ctt atg gct aga gcg ttg gca gcg agc tgc tca act gaa 1537

Gly Lys Thr Leu Met Ala Arg Ala Leu Ala Ala Ser Cys Ser Thr Glu

420 425 430

aag agg aag att acg ttc tat atg cgc aag ggg gcc gat atc ctg tcg 1585

Lys Arg Lys Ile Thr Phe Tyr Met Arg Lys Gly Ala Asp Ile Leu Ser

435 440 445

aaa tgg gtt ggc gaa gct gag aga cag ctt cga ctg ctg ttt gag gag 1633

Lys Trp Val Gly Glu Ala Glu Arg Gln Leu Arg Leu Leu Phe Glu Glu

450 455 460

gct aag aag cac cag cct tcg ata att ttc ttt gac gag att gat ggc 1681

Ala Lys Lys His Gln Pro Ser Ile Ile Phe Phe Asp Glu Ile Asp Gly

465 470 475

ctg gcc ccg gtt agg agt tca aag caa gaa caa atc cat gcc agc atc 1729

Leu Ala Pro Val Arg Ser Ser Lys Gln Glu Gln Ile His Ala Ser Ile

480 485 490 495

gta tct acc atg ttg gcg tta atg gat gga atg gac aat agg gga caa 1777

Val Ser Thr Met Leu Ala Leu Met Asp Gly Met Asp Asn Arg Gly Gln

500 505 510

gtg att gta atc ggt gct act aat aga ccg gat gca gta gac cct gct 1825

Val Ile Val Ile Gly Ala Thr Asn Arg Pro Asp Ala Val Asp Pro Ala

515 520 525

tta aga cga cca ggt aga ttt gac aga gag ttt tat ttc cct ctg cct 1873

Leu Arg Arg Pro Gly Arg Phe Asp Arg Glu Phe Tyr Phe Pro Leu Pro

530 535 540

gac ata cga gcc aga gcg aag att ctg gaa att cac acc agg aaa tgg 1921

Asp Ile Arg Ala Arg Ala Lys Ile Leu Glu Ile His Thr Arg Lys Trp

545 550 555

cat cct cca gta tca agt gcg ttt ata gaa aag ctt gct tct ttg acg 1969

His Pro Pro Val Ser Ser Ala Phe Ile Glu Lys Leu Ala Ser Leu Thr

560 565 570 575

aaa gga tac ggt gga gca gat ctt aga gcg cta tgt aca gag gcg gcc 2017

Lys Gly Tyr Gly Gly Ala Asp Leu Arg Ala Leu Cys Thr Glu Ala Ala

580 585 590

tgg aat agc att cag cga aga ttc ccg caa ata tat caa agt gag gtc 2065

Trp Asn Ser Ile Gln Arg Arg Phe Pro Gln Ile Tyr Gln Ser Glu Val

595 600 605

aag ttg gca ata aac cca agg gaa gtg cag gtt aaa gct aaa gac ttt 2113

Lys Leu Ala Ile Asn Pro Arg Glu Val Gln Val Lys Ala Lys Asp Phe

610 615 620

atg att gct atg gaa aaa att aca ccg tct tcc gcg cgg tcg agt ggg 2161

Met Ile Ala Met Glu Lys Ile Thr Pro Ser Ser Ala Arg Ser Ser Gly

625 630 635

aac ttg gcc gaa ccc tta cct cgt acc ata gcg gtt ctg cta aac gat 2209

Asn Leu Ala Glu Pro Leu Pro Arg Thr Ile Ala Val Leu Leu Asn Asp

640 645 650 655

cag ttc gag gaa ata aaa caa aaa ttg aat agc att ttg cct gaa gcg 2257

Gln Phe Glu Glu Ile Lys Gln Lys Leu Asn Ser Ile Leu Pro Glu Ala

660 665 670

tca agt aag tct cat cgc ggt tct tcg cta att aaa gaa tac ctg gag 2305

Ser Ser Lys Ser His Arg Gly Ser Ser Leu Ile Lys Glu Tyr Leu Glu

675 680 685

tac gaa gat gaa gaa gat gaa gaa gat gga gaa gat aat atc gaa ggc 2353

Tyr Glu Asp Glu Glu Asp Glu Glu Asp Gly Glu Asp Asn Ile Glu Gly

690 695 700

aca ggt atc cac tcg tcg aac ggc ttc agc aga cat gaa ttt ttc aag 2401

Thr Gly Ile His Ser Ser Asn Gly Phe Ser Arg His Glu Phe Phe Lys

705 710 715

atg ctt gat cag gct agg acc gtt aaa cca aag ttg tta ata aca ggt 2449

Met Leu Asp Gln Ala Arg Thr Val Lys Pro Lys Leu Leu Ile Thr Gly

720 725 730 735

ccg gct ggc aat ggc caa caa tat att ggt tcc gcg ctt ttg cat cat 2497

Pro Ala Gly Asn Gly Gln Gln Tyr Ile Gly Ser Ala Leu Leu His His

740 745 750

tta gag gat tac aat att cag aat ctt gat tta ggc aca tta ctc tcg 2545

Leu Glu Asp Tyr Asn Ile Gln Asn Leu Asp Leu Gly Thr Leu Leu Ser

755 760 765

gaa agt tta agg aca atg gag tcc gct att gtg cag aca ttt att gag 2593

Glu Ser Leu Arg Thr Met Glu Ser Ala Ile Val Gln Thr Phe Ile Glu

770 775 780

gcg aaa aaa cga caa cca tca gta atc tac atc cct aat gct gat att 2641

Ala Lys Lys Arg Gln Pro Ser Val Ile Tyr Ile Pro Asn Ala Asp Ile

785 790 795

tgg tca aga acg gtc ccc gaa agt gcc ata atg acc ttg gca agc tta 2689

Trp Ser Arg Thr Val Pro Glu Ser Ala Ile Met Thr Leu Ala Ser Leu

800 805 810 815

ttt aga tct ttg aaa acg aat gag aga gtg tta ctt tta gct att gcg 2737

Phe Arg Ser Leu Lys Thr Asn Glu Arg Val Leu Leu Leu Ala Ile Ala

820 825 830

gaa ggc ttc acc gaa gaa gag tta cat aac aca cct ttg tcg tta ttt 2785

Glu Gly Phe Thr Glu Glu Glu Leu His Asn Thr Pro Leu Ser Leu Phe

835 840 845

ggc ttt gac ctg aat att gtc tct att aga gaa cca tct act gcc caa 2833

Gly Phe Asp Leu Asn Ile Val Ser Ile Arg Glu Pro Ser Thr Ala Gln

850 855 860

aga cac aac tac ttc aaa act ctc gaa gag tta ctg aaa atg aag ccc 2881

Arg His Asn Tyr Phe Lys Thr Leu Glu Glu Leu Leu Lys Met Lys Pro

865 870 875

act aag ttt cga gag aag agg aaa aga aaa aag cca ctg ccg gta cta 2929

Thr Lys Phe Arg Glu Lys Arg Lys Arg Lys Lys Pro Leu Pro Val Leu

880 885 890 895

ccc gtg tgc gat act ccc agc aac gat gat atc ggt tct gat ggt gaa 2977

Pro Val Cys Asp Thr Pro Ser Asn Asp Asp Ile Gly Ser Asp Gly Glu

900 905 910

cta tta acc gaa gac gaa aag ttg agg agg aag tta aaa tca tac cat 3025

Leu Leu Thr Glu Asp Glu Lys Leu Arg Arg Lys Leu Lys Ser Tyr His

915 920 925

cac caa gac atg aaa ttg aag aat act ttg aag ata aag ctt tcc ggt 3073

His Gln Asp Met Lys Leu Lys Asn Thr Leu Lys Ile Lys Leu Ser Gly

930 935 940

cta gtg gct cta ttt aag ggg cgt tat aaa aga ttt agg aaa ccg ccc 3121

Leu Val Ala Leu Phe Lys Gly Arg Tyr Lys Arg Phe Arg Lys Pro Pro

945 950 955

att gag gat tcc ttg ctt att cat cta ttt gaa cca gaa gcc tat gcc 3169

Ile Glu Asp Ser Leu Leu Ile His Leu Phe Glu Pro Glu Ala Tyr Ala

960 965 970 975

tca aat cct gaa tgg caa ccg gct tat gta aga gaa gat gac atg att 3217

Ser Asn Pro Glu Trp Gln Pro Ala Tyr Val Arg Glu Asp Asp Met Ile

980 985 990

tta gaa gtt tca acc ggc agg aag tac tac aat atg gat ttg gat att 3265

Leu Glu Val Ser Thr Gly Arg Lys Tyr Tyr Asn Met Asp Leu Asp Ile

995 1000 1005

ata gaa gaa agg ttg tgg aac ggg tat tac tct gaa ccg aaa cag 3310

Ile Glu Glu Arg Leu Trp Asn Gly Tyr Tyr Ser Glu Pro Lys Gln

1010 1015 1020

tat ttg aag gat atc gag tta att tac cgc gat gct aat aca act 3355

Tyr Leu Lys Asp Ile Glu Leu Ile Tyr Arg Asp Ala Asn Thr Thr

1025 1030 1035

ggt gac cga gag cgt ata ata aaa gcg tca gag atg ttt gca aac 3400

Gly Asp Arg Glu Arg Ile Ile Lys Ala Ser Glu Met Phe Ala Asn

1040 1045 1050

gca cag atg ggc att gaa gaa ata tct acg cct gag ctt atc cag 3445

Ala Gln Met Gly Ile Glu Glu Ile Ser Thr Pro Glu Leu Ile Gln

1055 1060 1065

gag tgc agg gat aca cgt caa aga gaa cta tta agg cag cag ttg 3490

Glu Cys Arg Asp Thr Arg Gln Arg Glu Leu Leu Arg Gln Gln Leu

1070 1075 1080

tat tta cgc gac cag cag aaa aag ata cac gag gaa gaa gct aag 3535

Tyr Leu Arg Asp Gln Gln Lys Lys Ile His Glu Glu Glu Ala Lys

1085 1090 1095

ctc gag cag gag aaa aag gat ccg act ttt cca aat gac gat aat 3580

Leu Glu Gln Glu Lys Lys Asp Pro Thr Phe Pro Asn Asp Asp Asn

1100 1105 1110

cat cca gaa gag gcc gac tta agc gtt ggg gct gga cag caa ttg 3625

His Pro Glu Glu Ala Asp Leu Ser Val Gly Ala Gly Gln Gln Leu

1115 1120 1125

cat agc cca tca caa atg tct cat gag gct tgt gaa agc cac gca 3670

His Ser Pro Ser Gln Met Ser His Glu Ala Cys Glu Ser His Ala

1130 1135 1140

gaa gat gga ctg ggg gta cca cat ggc ttt gaa aat aac aca tat 3715

Glu Asp Gly Leu Gly Val Pro His Gly Phe Glu Asn Asn Thr Tyr

1145 1150 1155

gct att gga gat aac aca cca gag acg atc gat aac ggg tct cct 3760

Ala Ile Gly Asp Asn Thr Pro Glu Thr Ile Asp Asn Gly Ser Pro

1160 1165 1170

aaa ccc cta gag gac ttt tgc ggg gaa gac aag act gtc cct gac 3805

Lys Pro Leu Glu Asp Phe Cys Gly Glu Asp Lys Thr Val Pro Asp

1175 1180 1185

gac aaa tcc gtg cca gaa cga ttt gtt caa gag gga gaa gct tct 3850

Asp Lys Ser Val Pro Glu Arg Phe Val Gln Glu Gly Glu Ala Ser

1190 1195 1200

gag atg ggc ttt atc ccg aaa aca cct tcc gcc aga ccc gtc tcc 3895

Glu Met Gly Phe Ile Pro Lys Thr Pro Ser Ala Arg Pro Val Ser

1205 1210 1215

atg cac tca aat cca tca agc aaa gaa tcc gta gtt gct cct atg 3940

Met His Ser Asn Pro Ser Ser Lys Glu Ser Val Val Ala Pro Met

1220 1225 1230

caa aag act ggg tta gaa gtg aac agt atc gag gac acg tca gtc 3985

Gln Lys Thr Gly Leu Glu Val Asn Ser Ile Glu Asp Thr Ser Val

1235 1240 1245

gct gtt caa tca gaa ctg acc cgc gat gaa gac tta ccc atg act 4030

Ala Val Gln Ser Glu Leu Thr Arg Asp Glu Asp Leu Pro Met Thr

1250 1255 1260

cct gaa ccg gcc ttt atc ctg gat gaa acc gtt tta gcg aaa ata 4075

Pro Glu Pro Ala Phe Ile Leu Asp Glu Thr Val Leu Ala Lys Ile

1265 1270 1275

atc tcg atg ttg aaa aca aaa aca cag gga ttt aca gtc gaa cag 4120

Ile Ser Met Leu Lys Thr Lys Thr Gln Gly Phe Thr Val Glu Gln

1280 1285 1290

ctg gaa aca tgc tat gct gcc gta ctg gag cta gta tgg gag gcg 4165

Leu Glu Thr Cys Tyr Ala Ala Val Leu Glu Leu Val Trp Glu Ala

1295 1300 1305

aga tcg tca tgg gat aaa aca agt gtc att aag caa ata gaa aaa 4210

Arg Ser Ser Trp Asp Lys Thr Ser Val Ile Lys Gln Ile Glu Lys

1310 1315 1320

tat ata ggc atg tgagcccgtt agtaggacca agtgtatcta acatttattt 4262

Tyr Ile Gly Met

1325

acacgattaa agcctagagt cgatatatac tagttcaatt gttcttgcat gacgtcctcg 4322

gctgcatcga gtaagccgta gtcatcggca aactgtgcgg tgagttggat acctactccc 4382

ttcattccag caagaggaac agatatgcat ggaagcccag cgagggacat tggcaccgtg 4442

aagacgtcgt tgatgtacga gttgactgga ctcttgttct ccttgctagt gaaggcctcg 4502

agactaggtg ggtggcgcca tgcaggtcag cgagagcagc acatccaccc cagcaggatt 4562

aggtttgttt ccggtgcaga tgttctcaaa ggcaaatatc gagtcaaact ggtttataag 4622

ctcgactctc agtttctgcg ccctcatgta gttggtctta aatgcgcc 4670

<210> 5

<211> 1326

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 28

<400> 5

Met Ala Arg His Thr Arg Arg Ser His Gln His Val Asn Asp Glu Asp

1 5 10 15

Thr Gly Ser Glu Ile Tyr Asp Lys Asn Gly Ile Lys His Thr Thr Pro

20 25 30

Arg Ser Leu Lys Lys Ile Asn Tyr Ala Glu Ile Glu Asn Ser Tyr Asp

35 40 45

Tyr Met Glu Asp Tyr Asp Glu Gly Asp Lys Glu Glu Glu Pro Ala Glu

50 55 60

Leu Glu Asn Asn Gly Arg Ser Lys Gly Leu Val Gly Arg Glu Arg Gly

65 70 75 80

Arg Glu Pro Glu Glu Asp Glu Asp Glu Glu Gly Pro Val Arg Gly Arg

85 90 95

Arg Ser Arg Lys Arg Thr Tyr Val Asp Val Glu Glu Asp Glu Ser Phe

100 105 110

His Glu Glu Glu Ala Glu Asp Glu Asp Glu Glu Ala Leu Asp Asp Asp

115 120 125

Glu Asp Glu Asp Glu Glu Glu Arg Asn Trp Arg Arg Arg Arg Glu His

130 135 140

Gln Phe Val Val Glu Asp Glu Asp Asp Asp Glu Asp Glu Glu Asp Asp

145 150 155 160

Tyr Gly Ala Gln Lys Lys Arg Gly Arg Arg Arg Thr Arg Arg Gly Arg

165 170 175

Ser Arg Leu Thr Glu Arg Arg Ser Pro Pro Arg Lys Leu Arg Thr Arg

180 185 190

Arg Thr Arg Ser Ser Val Asn Met Tyr Asp Ser Glu His Asp Gly Thr

195 200 205

Gly Glu Ala Leu Thr Leu Glu Asp Glu Ile Arg Glu Leu Gln Glu Asp

210 215 220

Ser Pro Ile Arg Glu Lys Arg Ser Leu Arg Glu Arg Thr Lys Pro Val

225 230 235 240

Asn Tyr Thr Leu Pro Pro Pro Leu Thr Asp Asn Gln Met Asn Val Asp

245 250 255

Gly Thr Ala Ala Pro Ala Tyr Gly Ser Phe His Ser Pro Arg Gly Lys

260 265 270

Arg Gly Leu His Ser Gly Gln Ser Phe Gly Pro Ile Arg Arg Leu Phe

275 280 285

Pro Thr Gly Gly Pro Phe Gly Gly Asn Asp Val Thr Ala Ile Phe Gly

290 295 300

His Asn Thr Asn Phe Tyr Ala Thr Ala Gln Asp Pro Thr Val Ala Asn

305 310 315 320

Asn Lys Phe Ile Asp Ser Asp Ser Ser Asp Asp Glu Ile Leu Pro Leu

325 330 335

Gly Ser Thr Pro Lys Pro Lys Ser Ser Glu Ala Lys Lys Lys Lys Lys

340 345 350

Pro Glu Ile Ala Asp Leu Asp Pro Leu Gly Val Asp Met Asn Ile Asn

355 360 365

Phe Asp Asp Ile Gly Gly Leu Asp Asn Tyr Ile Asp Gln Leu Lys Glu

370 375 380

Met Val Ala Leu Pro Leu Leu Tyr Pro Glu Leu Tyr Gln Asn Phe Asn

385 390 395 400

Ile Thr Pro Pro Arg Gly Val Leu Phe Tyr Gly Pro Pro Gly Thr Gly

405 410 415

Lys Thr Leu Met Ala Arg Ala Leu Ala Ala Ser Cys Ser Thr Glu Lys

420 425 430

Arg Lys Ile Thr Phe Tyr Met Arg Lys Gly Ala Asp Ile Leu Ser Lys

435 440 445

Trp Val Gly Glu Ala Glu Arg Gln Leu Arg Leu Leu Phe Glu Glu Ala

450 455 460

Lys Lys His Gln Pro Ser Ile Ile Phe Phe Asp Glu Ile Asp Gly Leu

465 470 475 480

Ala Pro Val Arg Ser Ser Lys Gln Glu Gln Ile His Ala Ser Ile Val

485 490 495

Ser Thr Met Leu Ala Leu Met Asp Gly Met Asp Asn Arg Gly Gln Val

500 505 510

Ile Val Ile Gly Ala Thr Asn Arg Pro Asp Ala Val Asp Pro Ala Leu

515 520 525

Arg Arg Pro Gly Arg Phe Asp Arg Glu Phe Tyr Phe Pro Leu Pro Asp

530 535 540

Ile Arg Ala Arg Ala Lys Ile Leu Glu Ile His Thr Arg Lys Trp His

545 550 555 560

Pro Pro Val Ser Ser Ala Phe Ile Glu Lys Leu Ala Ser Leu Thr Lys

565 570 575

Gly Tyr Gly Gly Ala Asp Leu Arg Ala Leu Cys Thr Glu Ala Ala Trp

580 585 590

Asn Ser Ile Gln Arg Arg Phe Pro Gln Ile Tyr Gln Ser Glu Val Lys

595 600 605

Leu Ala Ile Asn Pro Arg Glu Val Gln Val Lys Ala Lys Asp Phe Met

610 615 620

Ile Ala Met Glu Lys Ile Thr Pro Ser Ser Ala Arg Ser Ser Gly Asn

625 630 635 640

Leu Ala Glu Pro Leu Pro Arg Thr Ile Ala Val Leu Leu Asn Asp Gln

645 650 655

Phe Glu Glu Ile Lys Gln Lys Leu Asn Ser Ile Leu Pro Glu Ala Ser

660 665 670

Ser Lys Ser His Arg Gly Ser Ser Leu Ile Lys Glu Tyr Leu Glu Tyr

675 680 685

Glu Asp Glu Glu Asp Glu Glu Asp Gly Glu Asp Asn Ile Glu Gly Thr

690 695 700

Gly Ile His Ser Ser Asn Gly Phe Ser Arg His Glu Phe Phe Lys Met

705 710 715 720

Leu Asp Gln Ala Arg Thr Val Lys Pro Lys Leu Leu Ile Thr Gly Pro

725 730 735

Ala Gly Asn Gly Gln Gln Tyr Ile Gly Ser Ala Leu Leu His His Leu

740 745 750

Glu Asp Tyr Asn Ile Gln Asn Leu Asp Leu Gly Thr Leu Leu Ser Glu

755 760 765

Ser Leu Arg Thr Met Glu Ser Ala Ile Val Gln Thr Phe Ile Glu Ala

770 775 780

Lys Lys Arg Gln Pro Ser Val Ile Tyr Ile Pro Asn Ala Asp Ile Trp

785 790 795 800

Ser Arg Thr Val Pro Glu Ser Ala Ile Met Thr Leu Ala Ser Leu Phe

805 810 815

Arg Ser Leu Lys Thr Asn Glu Arg Val Leu Leu Leu Ala Ile Ala Glu

820 825 830

Gly Phe Thr Glu Glu Glu Leu His Asn Thr Pro Leu Ser Leu Phe Gly

835 840 845

Phe Asp Leu Asn Ile Val Ser Ile Arg Glu Pro Ser Thr Ala Gln Arg

850 855 860

His Asn Tyr Phe Lys Thr Leu Glu Glu Leu Leu Lys Met Lys Pro Thr

865 870 875 880

Lys Phe Arg Glu Lys Arg Lys Arg Lys Lys Pro Leu Pro Val Leu Pro

885 890 895

Val Cys Asp Thr Pro Ser Asn Asp Asp Ile Gly Ser Asp Gly Glu Leu

900 905 910

Leu Thr Glu Asp Glu Lys Leu Arg Arg Lys Leu Lys Ser Tyr His His

915 920 925

Gln Asp Met Lys Leu Lys Asn Thr Leu Lys Ile Lys Leu Ser Gly Leu

930 935 940

Val Ala Leu Phe Lys Gly Arg Tyr Lys Arg Phe Arg Lys Pro Pro Ile

945 950 955 960

Glu Asp Ser Leu Leu Ile His Leu Phe Glu Pro Glu Ala Tyr Ala Ser

965 970 975

Asn Pro Glu Trp Gln Pro Ala Tyr Val Arg Glu Asp Asp Met Ile Leu

980 985 990

Glu Val Ser Thr Gly Arg Lys Tyr Tyr Asn Met Asp Leu Asp Ile Ile

995 1000 1005

Glu Glu Arg Leu Trp Asn Gly Tyr Tyr Ser Glu Pro Lys Gln Tyr

1010 1015 1020

Leu Lys Asp Ile Glu Leu Ile Tyr Arg Asp Ala Asn Thr Thr Gly

1025 1030 1035

Asp Arg Glu Arg Ile Ile Lys Ala Ser Glu Met Phe Ala Asn Ala

1040 1045 1050

Gln Met Gly Ile Glu Glu Ile Ser Thr Pro Glu Leu Ile Gln Glu

1055 1060 1065

Cys Arg Asp Thr Arg Gln Arg Glu Leu Leu Arg Gln Gln Leu Tyr

1070 1075 1080

Leu Arg Asp Gln Gln Lys Lys Ile His Glu Glu Glu Ala Lys Leu

1085 1090 1095

Glu Gln Glu Lys Lys Asp Pro Thr Phe Pro Asn Asp Asp Asn His

1100 1105 1110

Pro Glu Glu Ala Asp Leu Ser Val Gly Ala Gly Gln Gln Leu His

1115 1120 1125

Ser Pro Ser Gln Met Ser His Glu Ala Cys Glu Ser His Ala Glu

1130 1135 1140

Asp Gly Leu Gly Val Pro His Gly Phe Glu Asn Asn Thr Tyr Ala

1145 1150 1155

Ile Gly Asp Asn Thr Pro Glu Thr Ile Asp Asn Gly Ser Pro Lys

1160 1165 1170

Pro Leu Glu Asp Phe Cys Gly Glu Asp Lys Thr Val Pro Asp Asp

1175 1180 1185

Lys Ser Val Pro Glu Arg Phe Val Gln Glu Gly Glu Ala Ser Glu

1190 1195 1200

Met Gly Phe Ile Pro Lys Thr Pro Ser Ala Arg Pro Val Ser Met

1205 1210 1215

His Ser Asn Pro Ser Ser Lys Glu Ser Val Val Ala Pro Met Gln

1220 1225 1230

Lys Thr Gly Leu Glu Val Asn Ser Ile Glu Asp Thr Ser Val Ala

1235 1240 1245

Val Gln Ser Glu Leu Thr Arg Asp Glu Asp Leu Pro Met Thr Pro

1250 1255 1260

Glu Pro Ala Phe Ile Leu Asp Glu Thr Val Leu Ala Lys Ile Ile

1265 1270 1275

Ser Met Leu Lys Thr Lys Thr Gln Gly Phe Thr Val Glu Gln Leu

1280 1285 1290

Glu Thr Cys Tyr Ala Ala Val Leu Glu Leu Val Trp Glu Ala Arg

1295 1300 1305

Ser Ser Trp Asp Lys Thr Ser Val Ile Lys Gln Ile Glu Lys Tyr

1310 1315 1320

Ile Gly Met

1325

<210> 6

<211> 1049

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 45

<400> 6

gatcgacgtg gaccagcaca cggagtacgc ggtgaccggg agtgcggact tcaktgtgaa 60

ggtctggcgc gtacgcgacg gcagcatcgc gcactcgtgg gacacagcgc acgcccgtgc 120

aggcagcgtg gagttttcgc ccaccggcga ccgtgtgcct tgcaggtgcc tggataacgt 180

gatgaactac gccggcgcca tcgtggtgtt cagcgtgacg cgggatgcga acaaccagat 240

cactggcttc aacagcggcc tttcctgcga gatcctgacg caggagggct gtgcgcccgt 300

gcttgtggcg tcgtggtcgt atgacggcaa gtacatcgtg gccgggcacc aagacggcaa 360

gatcagcaaa tacaacggcg tcacgggcga atgcctggaa atcaaggacc tgcacaagca 420

gcgcgtctcc gacatccaat tctcgcttga ccgcacctac ttctcacgac ctccagagac 480

agctacgcca acctagtcga tgtcgagaca ttcgaggtcc tgaagaccta cgaaactgac 540

tgtccgctga acagcggttg catcaccccg ctaaaggagt tcgtcattct gggcggtggc 600

cagacgcgcg cgatgtcacg accaccagtg cccgcgaggg taagttcgag gccaagttct 660

accacaagct tttccaggtg gaaatcggcc gtgtggacga ccatttcggt cccgtcaact 720

acatcgccgt ttccccgcag ggcacctcgt acgcctccgg cggtgaggac ggtttcgtgc 780

gcctccatca cttcgacaag aggctacttc gacttcaagt tcgacgtcga aaaaagcgcc 840

gacgcccaga agaaggtcga cactgctgac cgctgagcta catacctctg taaactaccc 900

tcttgaacag ctattttcta ctgccctgca tcgtaccagg caaggcctac acacctatac 960

actacctgct ggccttctcg gccttcagcc gctccagccg ctcgcgctcc ttgcgctcgt 1020

ccgccgcctg ctgcttacgc cgcaggatc 1049

<210> 7

<211> 153

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 45

<220>

<221> misc_feature

<222> (17)..(17)

<223> unknown amino acid

<400> 7

Asp Val Asp Gln His Thr Glu Tyr Ala Val Thr Gly Ser Ala Asp Phe

1 5 10 15

Xaa Val Lys Val Trp Arg Val Arg Asp Gly Ser Ile Ala His Ser Trp

20 25 30

Asp Thr Ala His Ala Arg Ala Gly Ser Val Glu Phe Ser Pro Thr Gly

35 40 45

Asp Arg Val Pro Cys Arg Cys Leu Asp Asn Val Met Asn Tyr Ala Gly

50 55 60

Ala Ile Val Val Phe Ser Val Thr Arg Asp Ala Asn Asn Gln Ile Thr

65 70 75 80

Gly Phe Asn Ser Gly Leu Ser Cys Glu Ile Leu Thr Gln Glu Gly Cys

85 90 95

Ala Pro Val Leu Val Ala Ser Trp Ser Tyr Asp Gly Lys Tyr Ile Val

100 105 110

Ala Gly His Gln Asp Gly Lys Ile Ser Lys Tyr Asn Gly Val Thr Gly

115 120 125

Glu Cys Leu Glu Ile Lys Asp Leu His Lys Gln Arg Val Ser Asp Ile

130 135 140

Gln Phe Ser Leu Asp Arg Thr Tyr Phe

145 150

<210> 8

<211> 47

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 45

<400> 8

Leu Thr Thr ser Arg Asp Ser Tyr Ala Asn Leu Val Asp Val Glu Thr

1 5 10 15

Phe Glu Val Leu Lys Thr Tyr Glu Thr Asp Cys Pro Leu Asn Ser Gly

20 25 30

Cys Ile Thr Pro Leu Lys Glu Phe Val Ile Leu Gly Gly Gly Gln

35 40 45

<210> 9

<211> 70

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 45

<400> 9

Gly Arg Trp Pro Asp Ala Arg Asp Val Thr Thr Thr Ser Ala Arg Glu

1 5 10 15

Gly Lys Phe Glu Ala Lys Phe Tyr His Lys Leu Phe Gln Val Glu Ile

20 25 30

Gly Arg Val Asp Asp His Phe Gly Pro Val Asn Tyr Ile Ala Val Ser

35 40 45

Pro Gln Gly Thr Ser Tyr Ala Ser Gly Gly Glu Asp Gly Phe Val Arg

50 55 60

Leu His His Phe Asp Lys

65 70

<210> 10

<211> 4389

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (640)..(1674)

<223>

<400> 10

gattcgctgg agcgcgtgtg caaggaggcc gcgccggggc aggaactgct gctcatcttc 60

ggggacctgg gacacccacg tgccgccggc gggccgcgac ttgttgcgca gcagcctgcg 120

cgaacacggc gtggccgcct ccttcctgga actgcacgcg gcccagcacg ctttcgtgcg 180

cgacgagttc tccaagggcc gcttcgacgg ggccgtcacc tccagctgtc tggggctgat 240

gttcgagcag ttcgaccgcc tgctgaagca caaccttggt ccgcgcgagg cagacgccgg 300

cgagctggag cacgtctgct aaaactacgt aactgccgct tcgatacata acacgccttg 360

ggccctgctg gccctgctgg ccctgctggc cctgctggcc ctgctggccc tgctggccct 420

gctggccctg ccggtacgcc gcatctctgc acttctacct gcacctttgc accgggtcat 480

cgtggtctag caagtggctc gccaatatga aaaattttcg actagttcgc aggccttaga 540

gcactatcac ccaagtgaac ggaccagcca gagcgcatct aagtcacttg gagggtatct 600

gatcacatca cacgcttggc catataccat acttgaaag atg aga ccg atc atg 654

Met Arg Pro Ile Met

1 5

ttg atg gga cac gag cgt tcg ctc acg cag gtg aaa tat aac cgc gag 702

Leu Met Gly His Glu Arg Ser Leu Thr Gln Val Lys Tyr Asn Arg Glu

10 15 20

gga gac cta atc ttc acg tcg ggg aag gat aac gtt gcg tcg gtg tgg 750

Gly Asp Leu Ile Phe Thr Ser Gly Lys Asp Asn Val Ala Ser Val Trp

25 30 35

tat gcg atg aac ggc gag cgt ctg gga acg ctg gag ggt cac aat ggt 798

Tyr Ala Met Asn Gly Glu Arg Leu Gly Thr Leu Glu Gly His Asn Gly

40 45 50

tcg att tgg tcg atc gac gtg gac cag cac acg gag tac gcg gtg acc 846

Ser Ile Trp Ser Ile Asp Val Asp Gln His Thr Glu Tyr Ala Val Thr

55 60 65

ggg agt gcg gac ttc agt gtg aag gtc tgg cgc gta cgc gac ggc agc 894

Gly Ser Ala Asp Phe Ser Val Lys Val Trp Arg Val Arg Asp Gly Ser

70 75 80 85

atc gcg cac tcg tgg gac acg cgc acg ccc gtg cgg cgc gtg gag ttt 942

Ile Ala His Ser Trp Asp Thr Arg Thr Pro Val Arg Arg Val Glu Phe

90 95 100

tcg ccc acc ggc gac cgt gtg ctt gcg gtg ctg gat aac gtg atg aac 990

Ser Pro Thr Gly Asp Arg Val Leu Ala Val Leu Asp Asn Val Met Asn

105 110 115

tac gcc ggc gcc atc gtg gtg ttc agc gtg acg cgg gat gcg aac aac 1038

Tyr Ala Gly Ala Ile Val Val Phe Ser Val Thr Arg Asp Ala Asn Asn

120 125 130

cag atc act ggc ttc aac agc ggc ctt tcc tgc gag atc ctg acg cag 1086

Gln Ile Thr Gly Phe Asn Ser Gly Leu Ser Cys Glu Ile Leu Thr Gln

135 140 145

gag ggc tgt gcg ccc gtg ctt gtg gcg tcg tgg tcg tat gac ggc aag 1134

Glu Gly Cys Ala Pro Val Leu Val Ala Ser Trp Ser Tyr Asp Gly Lys

150 155 160 165

tac atc gtg gcc ggg cac caa gac ggc aag atc agc aaa tac aac ggc 1182

Tyr Ile Val Ala Gly His Gln Asp Gly Lys Ile Ser Lys Tyr Asn Gly

170 175 180

gtc acg ggc gaa tgc ctg gaa atc aag gac ctg cac aag cag cgc gtc 1230

Val Thr Gly Glu Cys Leu Glu Ile Lys Asp Leu His Lys Gln Arg Val

185 190 195

tcc gac atc caa ttc tcg ctt gac cgc acc tacttc ctc acg acc tcc 1278

Ser Asp Ile Gln Phe Ser Leu Asp Arg Thr Tyr Phe Leu Thr Thr Ser

200 205 210

aga gac agc tac gcc aac cta gtc gat gtc gag aca ttc gag gtc ctg 1326

Arg Asp Ser Tyr Ala Asn Leu Val Asp Val Glu Thr Phe Glu Val Leu

215 220 225

aag acc tac gaa act gac tgt ccg ctg aac agc ggt tgc atc acc ccg 1374

Lys Thr Tyr Glu Thr Asp Cys Pro Leu Asn Ser Gly Cys Ile Thr Pro

230 235 240 245

cta aag gag ttc gtc att ctg ggc ggt ggc cag gac gcg cgc gat gtc 1422

Leu Lys Glu Phe Val Ile Leu Gly Gly Gly Gln Asp Ala Arg Asp Val

250 255 260

acg acc acc agt gcc cgc gag ggt aag ttc gag gcc aag ttc tac cac 1470

Thr Thr Thr Ser Ala Arg Glu Gly Lys Phe Glu Ala Lys Phe Tyr His

265 270 275

aag ctt ttc cag gtg gaa atc ggc cgt gtg gac gac cat ttc ggt ccc 1518

Lys Leu Phe Gln Val Glu Ile Gly Arg Val Asp Asp His Phe Gly Pro

280 285 290

gtc aac tac atc gcc gtt tcc ccg cag ggc acc tcg tac gcc tcc ggc 1566

Val Asn Tyr Ile Ala Val Ser Pro Gln Gly Thr Ser Tyr Ala Ser Gly

295 300 305

ggt gag gac ggt ttc gtg cgc ctc cat cac ttc gac aag agc tacttc 1614

Gly Glu Asp Gly Phe Val Arg Leu His His Phe Asp Lys Ser Tyr Phe

310 315 320 325

gac ttc aag ttc gac gtc gaa aaa agc gcc gac gcc cag aag aag gtc 1662

Asp Phe Lys Phe Asp Val Glu Lys Ser Ala Asp Ala Gln Lys Lys Val

330 335 340

gac act gct gac cgctgagcta catacctctg taaactaccc tcttgaacag 1714

Asp Thr Ala Asp

345

ctattttcta cgcccgcatc gtaccaggca agcctacaca cctatacact acctgctggc 1774

cttctcggcc ttcagccgct ccagccgctc gcgctccttg cgctcgtccg ccgcctgctg 1834

cttacgccgc aggatctccg cgtcactctc catccgcttc agggggtccc cagacttttt 1894

ctgccccttg gcgctctcct tctgcttctt caggttcttc tgccgtgcga gctcgcgctg 1954

gtttcctcta gccatcctta gctgaagagt gtcggagggg ccgccacggt ggcagcccgc 2014

cttgcttatc tttgtggcca gccctcccca cgtgaggtat cacgtggcct cctccgactc 2074

ggcggtctga ccttcggaaa aatcggaaaa ttaagccggt ccggaatctc cgccggaccg 2134

gtgttcttcc cctctcgcgg tactcgagaa aaatttactg cagctgttta agttgcgtct 2194

catgcgtgcg gcgttgtgcg aaccgtcagt gaaaacgtgg aaaaaaaatg catcaacgca 2254

ggcgcatact aaatacgaaa tatatatagt gggaaagcgc aggcatcgcg agttgcacac 2314

aacaggtcgt aatgtgtctt aagcaaaaac aagagcgggc gaaataactg agygraatcc 2374

ccctatattc aacaacagtc cgcagtattt cccagatctg aactatttat ctccatggca 2434

cagttattca aacggtgtcg gcaccccaat accaattaac agttccatga ttgggaaccc 2494

aaacctttcc aacgtcccca tgaccaagac gtacgatccc tacgatgttc cacagtcgag 2554

ctttcctgcc tattttaatc cgcgcgcagc gctgaacatg ccccaccgcg agaaggttaa 2614

ccaatggata gaaaatgttc caatacatat tgttcaccga agagtaccct gactcacgac 2674

tgctatagca ttgacgagta catgaactgg gaggaggatg aatttgacat gtctttgttc 2734

cagcgcgggg agaagaccca gattaatatg gcaaccgtcg acgaactatt acaattccag 2794

ctgaaacgga taacctccat ggttttaaga ctatacgagg aaagcccgga ggtcccattg 2854

gacaactctg atgctacatc ttactgaaat actgattcca catcatcatg attaatactt 2914

aatagtttta gtatttatat agtatattat taaatagcca cggggtgcac atcaacgctg 2974

acgccgtaca acggctttcg ggccccgaca acattttagc gcatagtaaa actaagtccc 3034

atacacagcg aataatcgcc cccaagaaag cttcgatcaa aacataagtt actctcgagc 3094

ggcaagatag gctaacagga agaggcagta ctattggacg gggttagcgc tgaaaggtgc 3154

aaatttgggg ccatgagtaa ggaacccgga aagaaggtta cgctgaagga acgcatggta 3214

agcgcctctg ctggctcgct ggtcacgtca ctgtttctca cgccactgga cgtggtccgc 3274

gtgcggcttc aacagcaaga aatgcttcca agttgtacat gtacaggaca gctgtcgaaa 3334

ccggcaggga aagtgttttg gcaggatgag tgctttgcaa acgtcggctg ccgagagcct 3394

gctgcgaggc tgcagggaac tctggagggg ctcaggaaga tagcccaact agagggtctg 3454

ccgactctgt ggcgagggct ggggattacg ctcgtaatgg cggtgccggc taacgtggtg 3514

tacttttccg gctatgaagc actgcgtgat aactcgccct tggcatcccg gctacctgtg 3574

gcaaacccac tagtgtgtgg agcatttgcg cggatattgg ctgcaactac tattgcgccg 3634

ctggagctat tgcgcacacg gctccagagt gtgccccgcg caagagacac agagcgtaca 3694

atatatctga taggcgacct gctgcgagag atgcggcatg aggtttcggt tatgggttac 3754

cgtgcgctat ttaaaggctt ggagatcact ttatggaggg acgtgccctt cagcgcaatc 3814

tattggggaa catacgagtt ctgtaaaacc cagttctggg cccgccatgc cgcaacccat 3874

aatgcatcaa actgggacca tttcatcggc agttttgcct gcggtagcat gggcggtgct 3934

gttgcagcac ttttgacaca tccttttgat gtgggcaaga cccgcatgca gattgcgatt 3994

gccagtccac agcagctaac tgtgggggga aaagctacga aaactgatga ctcaagaggc 4054

atgttctcat ttttgaatgc cattaggaaa tcagaaggta ttagagcgct atataccggc 4114

ctattaccta gggtgatgaa gattgcacca agttgcgcca taatgatctc gacttatgaa 4174

ctgtcgaaga agttcttcac tagttgaact ttgtttcttc ctgtatatac caagtaattc 4234

acactgttga tacacgatac agcatttttc ttaaaaccct gtgatatacg cttgctgtag 4294

tacacgccca aacggtgagg tctataattt tacatgtccg cgagatggta ttggatcggc 4354

agaattttag agctgccgct gtagcccatc cgtcg 4389

<210> 11

<211> 345

<212> PRT

<213> Ashbya gossypii

<400> 11

Met Arg Pro Ile Met Leu Met Gly His Glu Arg Ser Leu Thr Gln Val

1 5 10 15

Lys Tyr Asn Arg Glu Gly Asp Leu Ile Phe Thr Ser Gly Lys Asp Asn

20 25 30

Val Ala Ser Val Trp Tyr Ala Met Asn Gly Glu Arg Leu Gly Thr Leu

35 40 45

Glu Gly His Asn Gly Ser Ile Trp Ser Ile Asp Val Asp Gln His Thr

50 55 60

Glu Tyr Ala Val Thr Gly Ser Ala Asp Phe Ser Val Lys Val Trp Arg

65 70 75 80

Val Arg Asp Gly Ser Ile Ala His Ser Trp Asp Thr Arg Thr Pro Val

85 90 95

Arg Arg Val Glu Phe Ser Pro Thr Gly Asp Arg Val Leu Ala Val Leu

100 105 110

Asp Asn Val Met Asn Tyr Ala Gly Ala Ile Val Val Phe Ser Val Thr

115 120 125

Arg Asp Ala Asn Asn Gln Ile Thr Gly Phe Asn Ser Gly Leu Ser Cys

130 135 140

Glu Ile Leu Thr Gln Glu Gly Cys Ala Pro Val Leu Val Ala Ser Trp

145 150 155 160

Ser Tyr Asp Gly Lys Tyr Ile Val Ala Gly His Gln Asp Gly Lys Ile

165 170 175

Ser Lys Tyr Asn Gly Val Thr Gly Glu Cys Leu Glu Ile Lys Asp Leu

180 185 190

His Lys Gln Arg Val Ser Asp Ile Gln Phe Ser Leu Asp Arg Thr Tyr

195 200 205

Phe Leu Thr Thr Ser Arg Asp Ser Tyr Ala Asn Leu Val Asp Val Glu

210 215 220

Thr Phe Glu Val Leu Lys Thr Tyr Glu Thr Asp Cys Pro Leu Asn Ser

225 230 235 240

Gly Cys Ile Thr Pro Leu Lys Glu Phe Val Ile Leu Gly Gly Gly Gln

245 250 255

Asp Ala Arg Asp Val Thr Thr Thr Ser Ala Arg Glu Gly Lys Phe Glu

260 265 270

Ala Lys Phe Tyr His Lys Leu Phe Gln Val Glu Ile Gly Arg Val Asp

275 280 285

Asp His Phe Gly Pro Val Asn Tyr Ile Ala Val Ser Pro Gln Gly Thr

290 295 300

Ser Tyr Ala Ser Gly Gly Glu Asp Gly Phe Val Arg Leu His His Phe

305 310 315 320

Asp Lys Ser Tyr Phe Asp Phe Lys Phe Asp Val Glu Lys Ser Ala Asp

325 330 335

Ala Gln Lys Lys Val Asp Thr Ala Asp

340 345

<210> 12

<211> 1042

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 45

<220>

<221> misc_feature

<223> Oligo 85

<400> 12

gatctgccga aatcgttaat ttcacctgta ggtcacgtga ttgtgaattg tgtttgactg 60

ttgcatccgt acattcggag gtgtgtgggt tgtatagctg aagattctgc gtcgtccgac 120

agactcgctc gcgcacaccg agcggagcac agtcggtttc cacagcaggc caacatcttc 180

aacagaagag gcagttcatt tggagtggct cttagtgcaa aatacagcaa aatcaatcat 240

aatggtatgt gtcaggcggt gtatagcgtg ggatgatggc atgcagcctt ttcgttgggt 300

atcgaggtga ccgttagagg cttaaatccg tgggacagaa gtgcgcgaca gcagcgcata 360

ccgggttagc cagcgagcgg tagccgaggg gtagatgcgg ccccgcaggt tgccagcaat 420

gtcattccaa gcttgatagg tgcgcggagt actaacatct catctcaggc cggtgttaaa 480

gcttttgaat tgagaaccaa gtccaaggag caactggagc aacagttgat ctccttgaag 540

caggagttgg ctgctttgaa ggtccagaag ctatccagac catccttgcc aaagatcaac 600

accgtcagaa agagcattgc tcgtgtcttg accgtcatca accagaacca gagacaggct 660

gtcagagagt tgtacaaggg caagaagtac cagccaaagg acttgagagc aaagaagacc 720

agagctttga gaagagcttt gacgaagttc gaggctgctc agatcactga gaagcagaga 780

aagaagcaga ttgctttccc acaaagaaag tacgctatta aggcctaaac gtctacggta 840

aaccgttgta tgttaattta gtttactttt aagaacccat ctttagaccc gcgggctcaa 900

gcggcggcgg cctgccagag ggagtagcag gcgaggcgct tttttgatag tgtggaatct 960

atacaggaaa ggtgactact tattgtttac cttgtttctt tgatgctttg aactcctcga 1020

ttctctgttt caactcatga tc 1042

<210> 13

<211> 119

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 85

<400> 13

Ala Gly Val Lys Ala Phe Glu Leu Arg Thr Lys Ser Lys Glu Gln Leu

1 5 10 15

Glu Gln Gln Leu Ile Ser Leu Lys Gln Glu Lau Ala Ala Leu Lys Val

20 25 30

Gln Lys Leu Ser Arg Pro Ser Leu Pro Lys Ile Asn Thr Val Arg Lys

35 40 45

Ser Ile Ala Arg Val Leu Thr Val Ile Asn Gln Asn Gln Arg Gln Ala

50 55 60

Val Arg Glu Leu Tyr Lys Gly Lys Lys Tyr Gln Pro Lys Asp Leu Arg

65 70 75 80

Ala Lys Lys Thr Arg Ala Leu Arg Arg Ala Leu Thr Lys Phe Glu Ala

85 90 95

Ala Gln Ile Thr Glu Lys Gln Arg Lys Lys Gln Ile Ala Phe Pro Gln

100 105 110

Arg Lys Tyr Ala Ile Lys Ala

115

<210> 14

<211> 1072

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (92)..(307)

<223>

<220>

<221> misc_feature

<223> Oligo 85

<220>

<221> CDS

<222> (403)..(858)

<223>

<220>

<221> misc_feature

<222> (279)..(501)

<223> Intron

<400> 14

cggtgttccg tcgcgccttt ggcgctcact ttgcgatctg ccgaaatcgt taatttcacc 60

tgtaggtcac gtgattgtga attgtgtttg a ctg ttg cat ccg tac att cgg 112

Leu Leu His Pro Tyr Ile Arg

1 5

agg tgt gtg ggt tgt ata gct gaa gat tct gcg tcg tcc gac aga ctc 160

Arg Cys Val Gly Cys Ile Ala Glu Asp Ser Ala Ser Ser Asp Arg Leu

10 15 20

gct cgc gca cac cga gcg gag cac agt cgg ttt cca cag cag gcc aac 208

Ala Arg Ala His Arg Ala Glu His Ser Arg Phe Pro Gln Gln Ala Asn

25 30 35

atc ttc aac aga aga ggc agt tca ttt gga gtg gct ctt agt gca aaa 256

Ile Phe Asn Arg Arg Gly Ser Ser Phe Gly Val Ala Leu Ser Ala Lys

40 45 50 55

tac agc aaa atc aat cat aat ggt atg tgt cag gcg gtg tat agc gtg 304

Tyr Ser Lys Ile Asn His Asn Gly Met Cys Gln Ala Val Tyr Ser Val

60 65 70

gga tgatggcatg cagcttttcg ttgggtatcg aggtgaccgt tagaggctta 357

Gly

aatccgtggg acagaagtgc gcgacagcag cgcataccgg gttag cca gcg agc ggt 414

Pro Ala Ser Gly

75

agc cga ggg gta gat gcg gcc ccg cag gtt gcc agc aat gtc att cca 462

Ser Arg Gly Val Asp Ala Ala Pro Gln Val Ala Ser Asn Val Ile Pro

80 85 90

agc ttg ata ggt gcg cgg agt act aac atc tca tct cag gcc ggt gtt 510

Ser Leu Ile Gly Ala Arg Ser Thr Asn Ile Ser Ser Gln Ala Gly Val

95 100 105

aaa gct ttt gaa ttg aga acc aag tcc aag gag caa ctg gag caa cag 558

Lys Ala Phe Glu Leu Arg Thr Lys Ser Lys Glu Gln Leu Glu Gln Gln

110 115 120

ttg atc tcc ttg aag cag gag ttg gct gct ttg aag gtc cag aag cta 606

Leu Ile Ser Leu Lys Gln Glu Leu Ala Ala Leu Lys Val Gln Lys Leu

125 130 135 140

tcc aga cca tcc ttg cca aag atc aac acc gtc aga aag agc att gct 654

Ser Arg Pro Ser Leu Pro Lys Ile Asn Thr Val Arg Lys Ser Ile Ala

145 150 155

cgt gtc ttg acc gtc atc aac cag aac cag aga cag gct gtc aga gag 702

Arg Val Leu Thr Val Ile Asn Gln Asn Gln Arg Gln Ala Val Arg Glu

160 165 170

ttg tac aag ggc aag aag tac cag cca aag gac ttg aga gca aag aag 750

Leu Tyr Lys Gly Lys Lys Tyr Gln Pro Lys Asp Leu Arg Ala Lys Lys

175 180 185

acc aga gct ttg aga aga gct ttg acg aag ttc gag gct gct cag atc 798

Thr Arg Ala Leu Arg Arg Ala Leu Thr Lys Phe Glu Ala Ala Gln Ile

190 195 200

act gag aag cag aga aag aag cag att gct ttc cca caa aga aag tac 846

Thr Glu Lys Gln Arg Lys Lys Gln Ile Ala Phe Pro Gln Arg Lys Tyr

205 210 215 220

gct att aag gcctaaacgtcta cgtaaaccgt tgtatgttaa tttagtttac 898

Ala Ile Lys Ala

ttttaagaac ccatctttag acccgcgggc tcaagcggcg gcggcctgcc agagggagta 958

gcagcgagcg cttttttgat agtgtggaat ctatacagga aaggtgacta cttattgttt 1018

accttgtttc tttgatgctt tgaactcctc gattctctgt ttcaactcat gatc 1072

<210> 15

<211> 72

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 85

<220>

<221> misc_feature

<222> (279)..(501)

<223> Intron

<400> 15

Leu Leu His Pro Tyr Ile Arg Arg Cys Val Gly Cys Ile Ala Glu Asp

1 5 10 15

Ser Ala Ser Ser Asp Arg Leu Ala Arg Ala His Arg Ala Glu His Ser

20 25 30

Arg Phe Pro Gln Gln Ala Asn Ile Phe Asn Arg Arg Gly Ser Ser Phe

35 40 45

Gly Val Ala Leu Ser Ala Lys Tyr Ser Lys Ile Asn His Asn Gly Met

50 55 60

Cys Gln Ala Val Tyr Ser Val Gly

65 70

<210> 16

<211> 152

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 85

<220>

<221> misc_feature

<222> (279)..(501)

<223> Intron

<400> 16

Pro Ala Ser Gly Ser Arg Gly Val Asp Ala Ala Pro Gln Val Ala Ser

1 5 10 15

Asn Val Ile Pro Ser Leu Ile Gly Ala Arg Ser Thr Asn Ile Ser Ser

20 25 30

Gln Ala Gly Val Lys Ala Phe Glu Leu Arg Thr Lys Ser Lys Glu Gln

35 40 45

Leu Glu Gln Gln Leu Ile Ser Leu Lys Gln Glu Leu Ala Ala Leu Lys

50 55 60

Val Gln Lys Leu Ser Arg Pro Ser Leu Pro Lys Ile Asn Thr Val Arg

65 70 75 80

Lys Ser Ile Ala Arg Val Leu Thr Val Ile Asn Gln Asn Gln Arg Gln

85 90 95

Ala Val Arg Glu Leu Tyr Lys Gly Lys Lys Tyr Gln Pro Lys Asp Leu

100 105 110

Arg Ala Lys Lys Thr Arg Ala Leu Arg Arg Ala Leu Thr Lys Phe Glu

115 120 125

Ala Ala Gln Ile Thr Glu Lys Gln Arg Lys Lys Gln Ile Ala Phe Pro

130 135 140

Gln Arg Lys Tyr Ala Ile Lys Ala

145 150

<210> 17

<211> 1011

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 133

<220>

<221> misc_feature

<222> (439)..(439)

<223> unknown nucleotide

<400> 17

cttaccgcgt ctcagcagac gtttctgctt ctttgagagg ggcactggtt gagtcaagtc 60

gatttcaatt tcatcctcgg cctttctctt cttggtgggg tcgcttagcg ctttagcttg 120

catatcaacc tttgctggcc gtggccgatt cttcgtctga catagctcca cgcagtagga 180

atgtatgtag tgaccctatg gattatggtc aagaagtgcg atgcgtgcgg agaactatgc 240

gttacaccat agctcatcgc atctcaaaaa ttttcgaatc accgaaaatt caggtcacgt 300

gatttataat aggccctcac ttgaaaatct cagtccacca gcagacaaac atatgtcggt 360

tgctgaacgg ggcacgacaa aactggcggc atcccgtgta acgctcgcaa gattatttca 420

gaaccaaatt actctttcng kcgkattttt gtatctggta tataccatct acccattaaa 480

taagccgagc ccagtccgtg atttgcgcgg catcaacatt gcatagtatg gcggtactat 540

catgtcactt gccttgtaaa ccgatgatta attagctcga attgctgaag cgggcgaccc 600

ttgctaaacg tactaatcaa cgtctctctt cattataggt aaaacgtcag gggatcggca 660

cgcagatctt gaactaaaat aaaataacga aatgggatga atgtataatt agagcagtca 720

gatagttcca tgagaatatg agaataaccg agaacgaact agagttccct agatttcagg 780

ccgtgggata agggcttcgg tgtacggcgc tttaagttca cctcaaaaat ctcttcaacc 840

tagcaaagaa tccggacctt ctctttggtt cagtggaaga gcggctaggg ctagaattgc 900

tgttcttgct agaaggcctg ccgttgtgag catcaaagct ctgcttcgtg cttggagcgg 960

tagccatact agcagcatcc ttagaatgac gtgactgcgt tacagaagat c 1011

<210> 18

<211> 38

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 133

<400> 18

Lys Ala Leu Ser Asp Pro Thr Lys Lys Arg Lys Ala Glu Asp Glu Ile

1 5 10 15

Glu Ile Asp Leu Thr Gln Pro Val Pro Leu Ser Lys Lys Gln Lys Arg

20 25 30

Leu Leu Arg Arg Gly Lys

35

<210> 19

<211> 3091

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (1371)..(2495)

<223>

<220>

<221> misc_feature

<223> Oligo 133

<400> 19

ttctgaggct catgtgacac cggtaactaa cgttccagta tctgagcaga tattcttcgc 60

cgaaggtgac tcagccagtt gtcgaatcaa gccctgccac tgaatcggag aaggtaatta 120

aggcacctgc tgttgctgct cacgagatat tcccacctca tgtggtcaag ctaacaccgg 180

tgtcgcctaa gaagaagacg tccaccggga gcttgaagaa gcagattatt gcgcctagag 240

aagaggagca gccacagcct gttcacgcag tgacaggtac tgccattgag ctcgaggagg 300

ttccatcgac ggtggaactt gtcgaggtac ctccatctga agtgcccacg actcattccc 360

ctattgtcgt cggaatacca ctatcaaatg acaagattgt cgtcgcccct gtgcagatac 420

aaggagatat cacgacacag acgtctgctc caagtactca gtacactgac acatatgctg 480

ctcccgtccg ccaggatgtg cctcttgcta cgaccgctgg cagatcttct gtaacgcagt 540

cacgtcattc taaggatgct gctagtatgg ctaccgctcc aagcacgaag cagagctttg 600

atgctcacaa cggcaggcct tctagcaaga acagcaattc tagccctagc cgctcttcca 660

ctgaaccaaa gagaaggtcc ggattctttg ctaggttgaa gagatttttg aggtgaactt 720

aaagcgccgt acaccgaagc ccttatccca cggcctgaaa tctagggaac tctagttcgt 780

tctcggttat tctcatattc tcatggaact atctgactgc tctaattata cattcatccc 840

atttcgttat tttattttag ttcaagatct gcgtgccgat cccctgacgt tttacctata 900

atgaagagag acgttgatta gtacgtttag caagggtcgc ccgcttcagc aattcgagct 960

aattaatcat cggtttacaa ggcaagtgac atgatagtac cgccatacta tgcaatgttg 1020

atgccgcgca aatcacggac tgggctcggc ttatttaatg ggtagatggt atataacaga 1080

tacaaaaata cgaccgaaag agtaatttgg ttctgaaata atcttgcgag cgttacacgg 1140

gatgccgcca gttttgtcgt gccccgttca gcaaccgaca tatgtttgtc tgctggtgga 1200

ctgagatttt caagtgaggg cctattataa atcacgtgac ctgaattttc ggtgattcga 1260

aaatttttga gatgcgatga gctatggtgt aacgcatagt tctccgcacg catcgcactt 1320

cttgaccata atccataggg tcactacata cattcctact gcgtggagct atg tca 1376

Met Ser

1

gac gaa gaa tcg gcc acg gcc agc aaa gtt gat atg caa gct aaa gcg 1424

Asp Glu Glu Ser Ala Thr Ala Ser Lys Val Asp Met Gln Ala Lys Ala

5 10 15

cta agc gac ccc acc aag aag aga aag gcc gag gat gaa att gaa atc 1472

Leu Ser Asp Pro Thr Lys Lys Arg Lys Ala Glu Asp Glu Ile Glu Ile

20 25 30

gac ttg act caa cca gtg ccc ctc tca aag aag cag aaa cgt ctg ctg 1520

Asp Leu Thr Gln Pro Val Pro Leu Ser Lys Lys Gln Lys Arg Leu Leu

35 40 45 50

aga cgc ggt aag atc acc ctc gaa cag ctc agc gag aag ttc aac atc 1568

Arg Arg Gly Lys Ile Thr Leu Glu Gln Leu Ser Glu Lys Phe Asn Ile

55 60 65

gat caa aag tcc atc gag gag tac aag aac gag caa aag gaa aca gca 1616

Asp Gln Lys Ser Ile Glu Glu Tyr Lys Asn Glu Gln Lys Glu Thr Ala

70 75 80

gag aac gac gat gcc gaa cca gct gcc gag gag gag cca aag cct gcg 1664

Glu Asn Asp Asp Ala Glu Pro Ala Ala Glu Glu Glu Pro Lys Pro Ala

85 90 95

ccc cgc aaa gag aag aag ttc ggc gtc tgg atc ggt aac atg gct ttc 1712

Pro Arg Lys Glu Lys Lys Phe Gly Val Trp Ile Gly Asn Met Ala Phe

100 105 110

gac acc acg cag gag gag ctt cgc cgc ttt gtt gtt tcc aag acc gct 1760

Asp Thr Thr Gln Glu Glu Leu Arg Arg Phe Val Val Ser Lys Thr Ala

115 120 125 130

ggc atg gag gca ggc gag gtc aca gac gct gac atc gtg cgt gtg aat 1808

Gly Met Glu Ala Gly Glu Val Thr Asp Ala Asp Ile Val Arg Val Asn

135 140 145

atg ccg ctg gcc aag aac gac ggc aaa cag atc aaa aac aag ggc ttt 1856

Met Pro Leu Ala Lys Asn Asp Gly Lys Gln Ile Lys Asn Lys Gly Phe

150 155 160

gcg tac gtg gac ttt gcg act agt gcg cag atg gat gca gta atc ggc 1904

Ala Tyr Val Asp Phe Ala Thr Ser Ala Gln Met Asp Ala Val Ile Gly

165 170 175

ctc agc gaa gca cag ctc aac gga cgc aac ttg ctg atc aag aac gcc 1952

Leu Ser Glu Ala Gln Leu Asn Gly Arg Asn Leu Leu Ile Lys Asn Ala

180 185 190

aag agc tac gac ggc cgc ccg gcg aag aac gac ctc atc tcg atg tcc 2000

Lys Ser Tyr Asp Gly Arg Pro Ala Lys Asn Asp Leu Ile Ser Met Ser

195 200 205 210

aaa aac cct cct tct cgc att ttg ttt gtc ggc aac ctc tcc ttc gac 2048

Lys Asn Pro Pro Ser Arg Ile Leu Phe Val Gly Asn Leu Ser Phe Asp

215 220 225

acc acc gat gag ctg ctg aaa aag cat ttc cag cac tgc ggc gag atc 2096

Thr Thr Asp Glu Leu Leu Lys Lys His Phe Gln His Cys Gly Glu Ile

230 235 240

gtc aaa atc cgc atg gcc acc ttc cag gac tcc ggc aag tgc aag ggt 2144

Val Lys Ile Arg Met Ala Thr Phe Gln Asp Ser Gly Lys Cys Lys Gly

245 250 255

ttc gcc ttc gtt gac ttc cgc gac gag gcc ggt gcc acc gcc gcc ctc 2192

Phe Ala Phe Val Asp Phe Arg Asp Glu Ala Gly Ala Thr Ala Ala Leu

260 265 270

aca gac cgc tcc tgt cgc gcc ata gca ggt cgc ccg ctc cgg atg gag 2240

Thr Asp Arg Ser Cys Arg Ala Ile Ala Gly Arg Pro Leu Arg Met Glu

275 280 285 290

tac ggc gag gac cgc agc aag cgc cac gtc cgc agc cgc ccg gcg ccc 2288

Tyr Gly Glu Asp Arg Ser Lys Arg His Val Arg Ser Arg Pro Ala Pro

295 300 305

tcg ctc gtc gac gcc gag cct cat gcg cct gcc ccc gct gac cca gca 2336

Ser Leu Val Asp Ala Glu Pro His Ala Pro Ala Pro Ala Asp Pro Ala

310 315 320

ccc gcg ccc gcg ccc gcg cct gcc cct agg gcg ccc cgg ccc cgc aag 2384

Pro Ala Pro Ala Pro Ala Pro Ala Pro Arg Ala Pro Arg Pro Arg Lys

325 330 335

ccc acg cgc aat gac cac cac agc cgc cct aag agc agc gtc gcc ctc 2432

Pro Thr Arg Asn Asp His His Ser Arg Pro Lys Ser Ser Val Ala Leu

340 345 350

gcc tcc gcc cag cgc gcc tca gcc gcc atc gtt ccc tct cag ggt aag 2480

Ala Ser Ala Gln Arg Ala Ser Ala Ala Ile Val Pro Ser Gln Gly Lys

355 360 365 370

aag gtc aag ttt gat tagcatgcat ctctacatac cgaatgtaca ttagttattg 2535

Lys Val Lys Phe Asp

375

tgcccgctgc gttgctcggt ctgctaccct acgcccgcat accgtccgtt ctttcgactc 2595

tcgcctcggc gtatgtgatg cagcacactg catgcagcat cgaccccggg gaacatgaac 2655

aaccacgtac tgccaacata ctagccggcc aatgcaaggc aaaaaggacc aagatcgagg 2715

tcagggatgt cacaagatga tatgcccttg tgtgaaagta tttttggtca acctggttgc 2775

tacgttgagt acctaaaaaa ggaaggatcc attaggctcg ataggttcta cggtacaatt 2835

atattctcga cacggcattg gatgcaagac tctgaggaag cggaggtgta actactagga 2895

gagttaagca tggggtataa caatacgagc tctccaatag gctcagggca tgggagttcg 2955

ggggtggaaa aggcatccag gcccagttcg gtgcagtcga cgacgcagat gggagcacgt 3015

cattccgaga gcatgaatct gtccgtgagc tggctctcga cggatgcgtg gagcgcggcg 3075

gagtttgcga cactgg 3091

<210> 20

<211> 375

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 133

<400> 20

Met Ser Asp Glu Glu Ser Ala Thr Ala Ser Lys Val Asp Met Gln Ala

1 5 10 15

Lys Ala Leu Ser Asp Pro Thr Lys Lys Arg Lys Ala Glu Asp Glu Ile

20 25 30

Glu Ile Asp Leu Thr Gln Pro Val Pro Leu Ser Lys Lys Gln Lys Arg

35 40 45

Leu Leu Arg Arg Gly Lys Ile Thr Leu Glu Gln Leu Ser Glu Lys Phe

50 55 60

Asn Ile Asp Gln Lys Ser Ile Glu Glu Tyr Lys Asn Glu Gln Lys Glu

65 70 75 80

Thr Ala Glu Asn Asp Asp Ala Glu Pro Ala Ala Glu Glu Glu Pro Lys

85 90 95

Pro Ala Pro Arg Lys Glu Lys Lys Phe Gly Val Trp Ile Gly Asn Met

100 105 110

Ala Phe Asp Thr Thr Gln Glu Glu Leu Arg Arg Phe Val Val Ser Lys

115 120 125

Thr Ala Gly Met Glu Ala Gly Glu Val Thr Asp Ala Asp Ile Val Arg

130 135 140

Val Asn Met Pro Leu Ala Lys Asn Asp Gly Lys Gln Ile Lys Asn Lys

145 150 155 160

Gly Phe Ala Tyr Val Asp Phe Ala Thr Ser Ala Gln Met Asp Ala Val

165 170 175

Ile Gly Leu Ser Glu Ala Gln Leu Asn Gly Arg Asn Leu Leu Ile Lys

180 185 190

Asn Ala Lys Ser Tyr Asp Gly Arg Pro Ala Lys Asn Asp Leu Ile Ser

195 200 205

Met Ser Lys Asn Pro Pro Ser Arg Ile Leu Phe Val Gly Asn Leu Ser

210 215 220

Phe Asp Thr Thr Asp Glu Leu Leu Lys Lys His Phe Gln His Cys Gly

225 230 235 240

Glu Ile Val Lys Ile Arg Met Ala Thr Phe Gln Asp Ser Gly Lys Cys

245 250 255

Lys Gly Phe Ala Phe Val Asp Phe Arg Asp Glu Ala Gly Ala Thr Ala

260 265 270

Ala Leu Thr Asp Arg Ser Cys Arg Ala Ile Ala Gly Arg Pro Leu Arg

275 280 285

Met Glu Tyr Gly Glu Asp Arg Ser Lys Arg His Val Arg Ser Arg Pro

290 295 300

Ala Pro Ser Leu Val Asp Ala Glu Pro His Ala Pro Ala Pro Ala Asp

305 310 315 320

Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Arg Ala Pro Arg Pro

325 330 335

Arg Lys Pro Thr Arg Asn Asp His His Ser Arg Pro Lys Ser Ser Val

340 345 350

Ala Leu Ala Ser Ala Gln Arg Ala Ser Ala Ala Ile Val Pro Ser Gln

355 360 365

Gly Lys Lys Val Lys Phe Asp

370 375

<210> 21

<211> 1687

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 172

<400> 21

gatcagccat gtgtcatcta ttgcacagct aaaccgtcgc atataccagc aggatagaga 60

gggcgatgtt ctgcggaata ttcctgtagt gtaccacaga gctagttcgt caacctgtgg 120

cctggtccac ctcactatcc ttctccctat caacgaggag accgctctat ctgtactcac 180

gacagcaggc cagccaacct ttgctgttcg catccccgca gatccagtag cgagggcact 240

gattgctctt agcgacaccc ccattgctgc gccgtcggct aacgtctcaa ctaggccttc 300

tccgacggcc gctgagcatg tataccatga cttaaaggga aagataccac ctaattctag 360

atggtggaag ttgccgtgtt ggggttgagt ctactgtcat cgatggcttg gtaaatccgc 420

caatgttgtt acgtcctgga ggatttacgt atgaagaaat catcgaacta ggcggggagc 480

aatggtccca ttgtaaagtt gagaatagga tgactgtagg gagtggggaa aaggttagaa 540

ctcccggtat gaagtacaaa cattactctc ctagggcctc cactgtagca tttgccccaa 600

ttaacgatga tcttccaacg agcgaaagga tgaagattgt gacctccgaa ataatgaaat 660

acatgacaag ccatggcacc gataagcgcc aaaaagtggg gctactgaca agcattatgt 720

tccccaataa cttactggaa tccattacgg acgaagttga tgttgtggtt tattcattgg 780

gctcctctgg gaaagaagtt caatcgaatt tgtttgccat gctgcgaagg ctggacgagg 840

aagatgaggt ggatttgata tttgttgaag gtattagcga taggaatgag ggactggcag 900

tgatgaatag attaagaaaa gccgctgggg ggaacgtagt gtcattttaa tgaaccgggt 960

agtcgcacat ttcatcgtca atagaaacag ccggtgggca atattgatcg atcgttttgt 1020

gtcgcttgtt ggataacttt atctgataaa tacaccctat atgcttgtgt atatgtttag 1080

tatataaatg actttatcat ctctggatga aaaaataaga gaaaacaaca tacgcactct 1140

gtgaattaaa ccatggaaaa gtaatatgcg tcttgatggg tattcttttt ggcgtaatac 1200

tttcgaactt catctgcaat gaaaacactg ctactgatta caatcaaata aatgagatcc 1260

cccaggctca ggcgctctgt cttgaagata ttctggaaaa atggaatgta aatagcgcac 1320

aactgaccta gtaatgagaa accaacagca tagttaaaca tcttattatt gaagatacca 1380

atttcgaaaa tggacttcgt aggcatgcct acacgctaga ggctgctgaa catatcaaaa 1440

aacacgaaac aggtaaacgt cattgtggtg tctctcgcag taacctgtcc atcctcagtc 1500

atttccttga tgaacacgta gatagtacca ccaatgataa aggctgcatt gataaggagt 1560

ctccgcataa cttggggagg tcaagatctt gtctgaacgt tttcttggag gcttcctcat 1620

tacttcgtgg tcaactggct ccacacctag ggattgcgcg ggtggaccgt ccatcaatat 1680

gttgatc 1687

<210> 22

<211> 116

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 172

<400> 22

Ile Ser His Val Ser Ser Ile Ala Gln Leu Asn Arg Arg Ile Tyr Gln

1 5 10 15

Gln Asp Arg Glu Gly Asp Val Leu Arg Asn Ile Pro Val Val Tyr His

20 25 30

Arg Ala Ser Ser Ser Thr Cys Gly Leu Val His Leu Thr Ile Leu Leu

35 40 45

Pro Ile Asn Glu Glu Thr Ala Leu Ser Val Leu Thr Thr Ala Gly Gln

50 55 60

Pro Thr Phe Ala Val Arg Ile Pro Ala Asp Pro Val Ala Arg Ala Leu

65 70 75 80

Ile Ala Leu Ser Asp Thr Pro Ile Ala Ala Pro Ser Ala Asn Val Ser

85 90 95

Thr Arg Pro Ser Pro Thr Ala Ala Glu His Val Tyr His Asp Leu Lys

100 105 110

Gly Lys Ile Pro

115

<210> 23

<211> 204

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 172

<400> 23

Arg Glu Arg Tyr His Leu Ile Leu Asp Gly Gly Ser Cys Arg Val Gly

1 5 10 15

Val Glu Ser Thr Val Ile Asp Gly Leu Val Asn Pro Pro Met Leu Leu

20 25 30

Arg Pro Gly Gly Phe Thr Tyr Glu Glu Ile Ile Glu Leu Gly Gly Glu

35 40 45

Gln Trp Ser His Cys Lys Val Glu Asn Arg Met Thr Val Gly Ser Gly

50 55 60

Glu Lys Val Arg Thr Pro Gly Met Lys Tyr Lys His Tyr Ser Pro Arg

65 70 75 80

Ala Ser Thr Val Ala Phe Ala Pro Ile Asn Asp Asp Leu Pro Thr Ser

85 90 95

Glu Arg Met Lys Ile Val Thr Ser Glu Ile Met Lys Tyr Met Thr Ser

100 105 110

His Gly Thr Asp Lys Arg Gln Lys Val Gly Leu Leu Thr Ser Ile Met

115 120 125

Phe Pro Asn Asn Leu Leu Glu Ser Ile Thr Asp Glu Val Asp Val Val

130 135 140

Val Tyr Ser Leu Gly Ser Ser Gly Lys Glu Val Gln Ser Asn Leu Phe

145 150 155 160

Ala Met Leu Arg Arg Leu Asp Glu Glu Asp Glu Val Asp Leu Ile Phe

165 170 175

Val Glu Gly Ile Ser Asp Arg Asn Glu Gly Leu Ala Val Met Asn Arg

180 185 190

Leu Arg Lys Ala Ala Gly Gly Asn Val Val Ser Phe

195 200

<210> 24

<211> 2641

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (277)..(1476)

<223>

<220>

<221> misc_feature

<223> Oligo 172

<400> 24

caagaccaat ccggtcccat ctctatcgga gaacgttgtt gttcggagca ccataccgtt 60

tgatggggcg gcaaaggcgt gaattacatt ttcgatcacg tgacataact gagaaaaatc 120

gctgcgtctt cgaactgctc gtggcatccg ccgttgtatg tctataatga actgatgccg 180

cagaacacaa gtgtatttgg tggcctcgaa ttttgacagt ttatgcagag gtccttctct 240

tttcttcgga ggtcaggttt tcatagcgca atgtca tat aac act aag gta ctg 294

Tyr Asn Thr Lys Val Leu

1 5

agg gta gac cca agt gcg ata cacttc tcg gca act gcg cat att gat 342

Arg Val Asp Pro Ser Ala Ile His Phe Ser Ala Thr Ala His Ile Asp

10 15 20

ggg tcc ctg ccg cgg att tcg gac cca gag acg gag aaa cac tta ctc 390

Gly Ser Leu Pro Arg Ile Ser Asp Pro Glu Thr Glu Lys His Leu Leu

25 30 35

gag gct gcc aga ctt atc cgg gat gat ggc gag acg gtt gcg ttt ccg 438

Glu Ala Ala Arg Leu Ile Arg Asp Asp Gly Glu Thr Val Ala Phe Pro

40 45 50

aca gaa act gta tat gga ctg ggg ggg tcg tct ctg aat gat act tcg 486

Thr Glu Thr Val Tyr Gly Leu Gly Gly Ser Ser Leu Asn Asp Thr Ser

55 60 65 70

gtg cgc aat atc tac aag gca aag aac aga ccc agt gac aat cca ctg 534

Val Arg Asn Ile Tyr Lys Ala Lys Asn Arg Pro Ser Asp Asn Pro Leu

75 80 85

atc agc cat gtg tca tct att gca cag cta aac cgt cgc ata tac cag 582

Ile Ser His Val Ser Ser Ile Ala Gln Leu Asn Arg Arg Ile Tyr Gln

90 95 100

cag gat aga gag ggc gat gtt ctg cgg aat att cct gta gtg tac cac 630

Gln Asp Arg Glu Gly Asp Val Leu Arg Asn Ile Pro Val Val Tyr His

105 110 115

gag cta gtt cgt caa ctg tgg cct ggt cca ctc act atc ctt ctc cct 678

Glu Leu Val Arg Gln Leu Trp Pro Gly Pro Leu Thr Ile Leu Leu Pro

120 125 130

atc aac gag gag acc gct cta tct gta ctc acg aca gca ggc cag cca 726

Ile Asn Glu Glu Thr Ala Leu Ser Val Leu Thr Thr Ala Gly Gln Pro

135 140 145 150

acc ttt gct gtt cgc atc ccc gca gat cca gta gcg agg gca ctg att 774

Thr Phe Ala Val Arg Ile Pro Ala Asp Pro Val Ala Arg Ala Leu Ile

155 160 165

gct ctt agc gac acc ccc att gct gcg ccg tcg gct aac gtc tca act 822

Ala Leu Ser Asp Thr Pro Ile Ala Ala Pro Ser Ala Asn Val Ser Thr

170 175 180

agg cct tct ccg acg gcc gct gag cat gta tac cat gac tta aag gga 870

Arg Pro Ser Pro Thr Ala Ala Glu His Val Tyr His Asp Leu Lys Gly

185 190 195

aag ata cca cta att cta gat ggt gga agt tgc cgt gtt ggg gtt gag 918

Lys Ile Pro Leu Ile Leu Asp Gly Gly Ser Cys Arg Val Gly Val Glu

200 205 210

tct act gtc atc gat ggc ttg gta aat ccg cca atg ttg tta cgt cct 966

Ser Thr Val Ile Asp Gly Leu Val Asn Pro Pro Met Leu Leu Arg Pro

215 220 225 230

gga gga ttt acg tat gaa gaa atc atc gaa cta ggc ggg gag caa tgg 1014

Gly Gly Phe Thr Tyr Glu Glu Ile Ile Glu Leu Gly Gly Glu Gln Trp

235 240 245

tcc cat tgt aaa gtt gag aat agg atg act gta ggg agt ggg gaa aag 1062

Ser His Cys Lys Val Glu Asn Arg Met Thr Val Gly Ser Gly Glu Lys

250 255 260

gtt aga act ccc ggt atg aag tac aaa cat tac tct cct agg gcc tcc 1110

Val Arg Thr Pro Gly Met Lys Tyr Lys His Tyr Ser Pro Arg Ala Ser

265 270 275

act gta gca ttt gcc cca att aac gat gat ctt cca acg agc gaa agg 1158

Thr Val Ala Phe Ala Pro Ile Asn Asp Asp Leu Pro Thr Ser Glu Arg

280 285 290

atg aag att gtg acc tcc gaa ata atg aaa tac atg aca agc cat ggc 1206

Met Lys Ile Val Thr Ser Glu Ile Met Lys Tyr Met Thr Ser His Gly

295 300 305 310

acc gat aag cgc caa aaa gtg ggg cta ctg aca agc att atg ttc ccc 1254

Thr Asp Lys Arg Gln Lys Val Gly Leu Leu Thr Ser Ile Met Phe Pro

315 320 325

aat aac tta ctg gaa tcc att acg gac gaa gtt gat gtt gtg gtt tat 1302

Asn Asn Leu Leu Glu Ser Ile Thr Asp Glu Val Asp Val Val Val Tyr

330 335 340

tca ttg ggc tcc tct ggg aaa gaa gtt caa tcg aat ttg ttt gcc atg 1350

Ser Leu Gly Ser Ser Gly Lys Glu Val Gln Ser Asn Leu Phe Ala Met

345 350 355

ctg cga agg ctg gac gag gaa gat gag gtg gat ttg ata ttt gtt gaa 1398

Leu Arg Arg Leu Asp Glu Glu Asp Glu Val Asp Leu Ile Phe Val Glu

360 365 370

ggt att agc gat agg aat gag gga ctg gca gtg atg aat aga tta aga 1446

Gly Ile Ser Asp Arg Asn Glu Gly Leu Ala Val Met Asn Arg Leu Arg

375 380 385 390

aaa gcc gct ggg ggg aac gta gtg tca ttt taatgaaccg ggtagtcgca 1496

Lys Ala Ala Gly Gly Asn Val Val Ser Phe

395 400

catttcatcg tcaatagaaa cagccggtgg gcaatattga tcgatcgttt tgtgtcgctt 1556

gttggataac tttatctgat aaatacaccc tatatgcttg tgtatatgtt tagtatataa 1616

atgactttat catctctgga tgaaaaaata agagaaaaca acatactgca ctctgtgaat 1676

taaaccatgg aaaagtaata tgcgtcttga tgggtattct ttttggcgta atactttcga 1736

acttcatctg caatgaaaac actgctactg attacaatca aataaatgag atcccccagg 1796

ctcaggcgct ctgtcttgaa gatattctgg aaaaatggaa tgtaaatagc gcacaactga 1856

cctagtaatg agaaaccaac agcatagtta aacatcttat tattgaagat accaatttcg 1916

aaaatggact tcgtaggcat gcctacacgc tagaggctgc tgaacatatc aaaaaacacg 1976

aaacaggtaa acgtcattgt ggtgtctctc gcaggtaacc tgtccatcct cagtcatttc 2036

cttgatgaac acgtagatag taccaccaat gataaaggct gcattgataa ggagtctccg 2096

cataacttgg ggagtcaaga tcttgtctga acgttttctt ggaggcttcc tcattacttc 2156

gtggtcaact ggctccacac ctagggattg cgcgggtgga ccgtccatca atatgttgat 2216

ccataatatc tgcatggcat ttaacggatt ttgaagctta aatgcagtag ctatggcgac 2276

aagtgacaag gctgcgacag aggtagacaa ttgaaacgtt agaaaactct gaatattgtt 2336

aaaaataccc ttgccttctt caatagcggt taggatggtg ctgaagtcat catcagttaa 2396

taccatatca aaagcttctt tcgcaacatc agtacccata tggcccatgg cgacaccgat 2456

atcagccaat ttcaatgcag gtgcgtcgtt aacaccatct cctgtcatag cgacaatgtc 2516

gcccctcttc tgcaaagcac gcacaatatt cagcttgtgt tcacgtgtag cccgagcaaa 2576

aatattcaca tgatcgatcc gccggccaga tggtccatca atcctttggt caagtctatc 2636

accgg 2641

<210> 25

<211> 400

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 172

<400> 25

Tyr Asn Thr Lys Val Leu Arg Val Asp Pro Ser Ala Ile His Phe Ser

1 5 10 15

Ala Thr Ala His Ile Asp Gly Ser Leu Pro Arg Ile Ser Asp Pro Glu

20 25 30

Thr Glu Lys His Leu Leu Glu Ala Ala Arg Leu Ile Arg Asp Asp Gly

35 40 45

Glu Thr Val Ala Phe Pro Thr Glu Thr Val Tyr Gly Leu Gly Gly Ser

50 55 60

Ser Leu Asn Asp Thr Ser Val Arg Asn Ile Tyr Lys Ala Lys Asn Arg

65 70 75 80

Pro Ser Asp Asn Pro Leu Ile Ser His Val Ser Ser Ile Ala Gln Leu

85 90 95

Asn Arg Arg Ile Tyr Gln Gln Asp Arg Glu Gly Asp Val Leu Arg Asn

100 105 110

Ile Pro Val Val Tyr His Glu Leu Val Arg Gln Leu Trp Pro Gly Pro

115 120 125

Leu Thr Ile Leu Leu Pro Ile Asn Glu Glu Thr Ala Leu Ser Val Leu

130 135 140

Thr Thr Ala Gly Gln Pro Thr Phe Ala Val Arg Ile Pro Ala Asp Pro

145 150 155 160

Val Ala Arg Ala Leu Ile Ala Leu Ser Asp Thr Pro Ile Ala Ala Pro

165 170 175

Ser Ala Asn Val Ser Thr Arg Pro Ser Pro Thr Ala Ala Glu His Val

180 185 190

Tyr His Asp Leu Lys Gly Lys Ile Pro Leu Ile Leu Asp Gly Gly Ser

195 200 205

Cys Arg Val Gly Val Glu Ser Thr Val Ile Asp Gly Leu Val Asn Pro

210 215 220

Pro Met Leu Leu Arg Pro Gly Gly Phe Thr Tyr Glu Glu Ile Ile Glu

225 230 235 240

Leu Gly Gly Glu Gln Trp Ser His Cys Lys Val Glu Asn Arg Met Thr

245 250 255

Val Gly Ser Gly Glu Lys Val Arg Thr Pro Gly Met Lys Tyr Lys His

260 265 270

Tyr Ser Pro Arg Ala Ser Thr Val Ala Phe Ala Pro Ile Asn Asp Asp

275 280 285

Leu Pro Thr Ser Glu Arg Met Lys Ile Val Thr Ser Glu Ile Met Lys

290 295 300

Tyr Met Thr Ser His Gly Thr Asp Lys Arg Gln Lys Val Gly Leu Leu

305 310 315 320

Thr Ser Ile Met Phe Pro Asn Asn Leu Leu Glu Ser Ile Thr Asp Glu

325 330 335

Val Asp Val Val Val Tyr Ser Leu Gly Ser Ser Gly Lys Glu Val Gln

340 345 350

Ser Asn Leu Phe Ala Met Leu Arg Arg Leu Asp Glu Glu Asp Glu Val

355 360 365

Asp Leu Ile Phe Val Glu Gly Ile Ser Asp Arg Asn Glu Gly Leu Ala

370 375 380

Val Met Asn Arg Leu Arg Lys Ala Ala Gly Gly Asn Val Val Ser Phe

385 390 395 400

<210> 26

<211> 609

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 63

<220>

<221> misc_feature

<222> (470)..(470)

<223> unknown nucleotide

<220>

<221> misc_feature

<222> (518)..(518)

<223> unknown nucleotide

<220>

<221> misc_feature

<222> (536)..(536)

<223> unknown nucleotide

<400> 26

gatcataaac gaagaattcc taattaacaa tttgtcctgc atgtacttcc tcagtgagaa 60

atagcagata taatcattag aaagcttccc cgagcacttt agcagcaccg catgccagca 120

taaccccctg gactcagggc agtatgccag gctggcacct cggcacctca tcgcaggcga 180

gacagtccac cactgcgagc accgtagtat ttatactttt ccaggttgaa aaattttcga 240

ccgccccacg ccgcagaggg ctggacgcgc attagggctc acagcggtcg actgccactg 300

ctgccccaac agcgccgcgc atgtaacgtg aaatgatata ttataccttc tgactacaat 360

gtgaaatata caaaggtggc tcataggcgc attgcattta ttcagacgca gtagctctgg 420

tgtagatagc ctgcttggag tgcttggaga ttggcttgat gatgccctcn gtctccaagt 480

gtctcatagc aactctggcc atggaaccgc cgatcttnaa tctgtcgacc aacacngaca 540

cagagacgta tctgtagggt tgggaccctc ctttaagatt ctgtcaagct tgtcctggtc 600

caagatgac 609

<210> 27

<211> 16

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 63

<400> 27

Val Ile Leu Asp Gln Asp Lys Leu Asp Arg Ile Lau Lys Glu Gly Pro

1 5 10 15

<210> 28

<211> 52

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 63

<220>

<221> misc_feature

<222> (13)..(13)

<223> unknown amino acid

<400> 28

Tyr Arg Tyr Val Ser Val Ser Val Leu Val Asp Arg Xaa Lys Ile Gly

1 5 10 15

Gly Ser Met Ala Arg Val Ala Met Arg His Leu Glu Thr Glu Gly Ile

20 25 30

Ile Lys Pro Ile Ser Lys His Ser Lys Gln Ala Ile Tyr Thr Arg Ala

35 40 45

Thr Ala Ser Glu

50

<210> 29

<211> 1850

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (533)..(856)

<223>

<220>

<221> misc_feature

<223> Oligo 63

<400> 29

tagccgcgat ctaccaagcg ctgccggtac ccttacagcc cgcctccaac acgtccgtgt 60

gccgtcttgt ctccgcagcc gctgctcttc ccattagttt ctccgctccg cttcgaaccc 120

cagccctcaa cgggcggcgg cgctagccgc tgcaccacca aagcgaaaat tgtaaatcgt 180

cgcttacgaa agagggtacc ccttctgtga ctgcacatcc gtacatccat ggactccgcc 240

aaatctgagt tcggaccgac gcaatactgg ttccgcaacg ggccctgctc accactgaat 300

cctcccacgc tctattgatt aaagcagaag agctaagtaa gtcgctgaag tctggcaaag 360

tagaaaccag ctaggaacat ttagagtatg tgagaggaca tgtgggacca acgccgttga 420

ggcgcgtagg cgttcaggcg cgcaggaggc taccggcacg cctgcagcca cgcctgccgc 480

cagaagagcg gccagctcgc tgccggacag ctactaacga gcacacagta at atg cca 538

Met Pro

1

cca aaa caa caa tta tct aag gcc cag aag gcc gcc gcc gcc atg gcc 586

Pro Lys Gln Gln Leu Ser Lys Ala Gln Lys Ala Ala Ala Ala Met Ala

5 10 15

ggt ggt aag aag tcc aag aag aag tgg tcc aag aag tcc cac aag gac 634

Gly Gly Lys Lys Ser Lys Lys Lys Trp Ser Lys Lys Ser His Lys Asp

20 25 30

aag gcc cag cac gcc gtc atc ttg gac cag gac aag ctt gac aga atc 682

Lys Ala Gln His Ala Val Ile Leu Asp Gln Asp Lys Leu Asp Arg Ile

35 40 45 50

cta aag gag gtc cca acc tac aga tac gtc tct gtg tcc gtg ttg gtc 730

Leu Lys Glu Val Pro Thr Tyr Arg Tyr Val Ser Val Ser Val Leu Val

55 60 65

gac aga ttg aag atc ggc ggt tcc atg gcc aga gtt gct ttg aga cac 778

Asp Arg Leu Lys Ile Gly Gly Ser Met Ala Arg Val Ala Leu Arg His

70 75 80

ttg gag acc gag ggc atc atc aag cca atc tcc aag cac tcc aag cag 826

Leu Glu Thr Glu Gly Ile Ile Lys Pro Ile Ser Lys His Ser Lys Gln

85 90 95

gct atc tac acc aga gct act gcg tct gaa taaatgcaat gcgcctatga 876

Ala Ile Tyr Thr Arg Ala Thr Ala Ser Glu

100 105

gccacctttg tatatttcac attgtagtca gaaggtataa tatatcattt cacgttacat 936

gcgcggcgct gttggggcag cagtggcagt cgaccgctgt gagccctaat gcgcgtccag 996

ccctctgcgg cgtggggcgg tcgaaaattt ttcaacctgg aaaagtataa atactacggt 1056

gctcgcagtg gtggactgtc tcgcctgcga tgaggtgccg aggtgccagc cggcatactg 1116

ccctgagtcc agggggttat gctggcatgc ggtgctgcta aagtgctcgg ggaagctttc 1176

taatgattat atcgctattt ctcactgagg aagtacatgc aggacaaatt gttaattagg 1236

aattcttcgt ttatgatcgg agagcacctc ccccagcgca gcactgcgca gcccaaccat 1296

gagaccatgg tgtccgacag aaggtgtgct tcgccgttct atgatgaata gcttccacag 1356

ctctcgatcc ggcgatgatt ctcaggaata tgtgccagga tatatcatgc atcaccatct 1416

gagattcgaa caggtaaagt cttctgctgg attggccgta tgcgttcttc tagatgatga 1476

taaaactttt tattcagttc tgcttctgaa catctaggaa gataactcta ttaccaaagc 1536

tcaaaactct gacaggagga acgcataacg gtctgggctg ctagtgccaa agagtaacgt 1596

aaaatctaac gaaagtactt agtgggcagg gccgcctgct cagagcctct tgcgggcctc 1656

ttcctgagtg accttctgct cccgtagttt ccggagcagg aagtcttgca cggcgtccca 1716

gcgttcgcgc tggctcgcgg ggaaccacac gcttcagctt ggaaagaatc cgcaccacgg 1776

ttgtctgcac gtaaaaggga aaagttcagc tttgagccag tagatggccc agtagaagaa 1836

cagcgcgaac cccc 1850

<210> 30

<211> 108

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 63

<400> 30

Met Pro Pro Lys Gln Gln Leu Ser Lys Ala Gln Lys Ala Ala Ala Ala

1 5 10 15

Met Ala Gly Gly Lys Lys Ser Lys Lys Lys Trp Ser Lys Lys Ser His

20 25 30

Lys Asp Lys Ala Gln His Ala Val Ile Leu Asp Gln Asp Lys Leu Asp

35 40 45

Arg Ile Leu Lys Glu Val Pro Thr Tyr Arg Tyr Val Ser Val Ser Val

50 55 60

Leu Val Asp Arg Leu Lys Ile Gly Gly Ser Met Ala Arg Val Ala Leu

65 70 75 80

Arg His Leu Glu Thr Glu Gly Ile Ile Lys Pro Ile Ser Lys His Ser

85 90 95

Lys Gln Ala Ile Tyr Thr Arg Ala Thr Ala Ser Glu

100 105

<210> 31

<211> 933

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 132

<400> 31

gatcctagaa ttgcggtgga atatcttctc ctaatcgctt tagcgcacga agactcacaa 60

attgaacttg ctcacgaagc attgagggag cctggtccta gatactaaag agttcaccat 120

acctcctcgg aaaaatcaac agggatggca ccaggatacc gggcattatc gaggagagac 180

agcctctgct ttacttggcc gacaaagagg actttttgca taagattacg gagcaggcag 240

cgcgcagagc agatgaaaat ggcagagtct acgacagtct gctactgtat cagctggcgg 300

aagagtacga tattgtcatt agcattgtta ataagctgct gagcgaaatg ttgagcaaca 360

ctgacctggc gcaacccctg atgcggcagg acgacaatag cgagactaac cctgttctaa 420

tcgccaagaa gctcattgat gtctatatca agaacttgga aatttcaaag aaggtacaca 480

ggaaaaacaa ggaaacatgc attctgctcc tgaaactcgt ggatataagg agaacatata 540

ttgcaagaca gtggcaaaac accctgcagc agatagagga gctggatctg cttccatctg 600

tcgaagactc ttccccaagg aagaaggcgc aggaattcca caacctggac gactgtatca 660

taaagaatgt ccccaacctg ttgatcatcg ccatgacctg cgtttctaac ctcatcaagc 720

agttgagcaa gggccccttc tccaacgggg ccacgcaagc tcaaggtcga ggctctgaag 780

aaggtcgcca acaactacat gatctacaga ggcatgatcc agtacaagat gcctcgggag 840

gtgtacaaca ccctcataaa catcgaggtg gacctctgac cgctcgtcag accacgatgc 900

agtgtgtacc agaccagtat tatagattag atc 933

<210> 32

<211> 30

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 132

<400> 32

Asp Pro Arg Ile Ala Val Glu Tyr Leu Leu Leu Ile Ala Leu Ala His

1 5 10 15

Glu Asp Ser Gln Ile Glu Leu Ala His Glu Ala Leu Arg Glu

20 25 30

<210> 33

<211> 10

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 132

<400> 33

Leu Val Leu Asp Thr Lys Glu Phe Thr Ile

1 5 10

<210> 34

<211> 219

<212> PRT

<213> Ashbya gossypii

<400> 34

Lys Ser Ser Pro Tyr Leu Leu Gly Lys Ile Asn Arg Asp Gly Thr Arg

1 5 10 15

Ile Pro Gly Ile Ile Glu Glu Arg Gln Pro Leu Leu Tyr Leu Ala Asp

20 25 30

Lys Glu Asp Phe Leu His Lys Ile Thr Glu Gln Ala Ala Arg Arg Ala

35 40 45

Asp Glu Asn Gly Arg Val Tyr Asp Ser Leu Leu Leu Tyr Gln Leu Ala

50 55 60

Glu Glu Tyr Asp Ile Val Ile Ser Ile Val Asn Lys Leu Leu Ser Glu

65 70 75 80

Met Leu Ser Asn Thr Asp Leu Ala Gln Pro Leu Met Arg Gln Asp Asp

85 90 95

Asn Ser Glu Thr Asn Pro Val Leu Ile Ala Lys Lys Leu Ile Asp Val

100 105 110

Tyr Ile Lys Asn Leu Glu Ile Ser Lys Lys Val His Arg Lys Asn Lys

115 120 125

Glu Thr Cys Ile Leu Leu Leu Lys Leu Val Asp Ile Arg Arg Thr Tyr

130 135 140

Ile Ala Arg Gln Trp Gln Asn Thr Leu Gln Gln Ile Glu Glu Leu Asp

145 150 155 160

Leu Leu Pro Ser Val Glu Asp Ser Ser Pro Arg Lys Lys Ala Gln Glu

165 170 175

Phe His Asn Leu Asp Asp Cys Ile Ile Lys Asn Val Pro Asn Leu Leu

180 185 190

Ile Ile Ala Met Thr Cys Val Ser Asn Leu Ile Lys Gln Leu Ser Lys

195 200 205

Gly Pro Phe Ser Asn Gly Ala Thr Gln Ala Gln

210 215

<210> 35

<211> 38

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 132

<400> 35

Lys Val Glu Ala Leu Lys Lys Val Ala Asn Asn Tyr Met Ile Tyr Arg

1 5 10 15

Gly Met Ile Gln Tyr Lys Met Pro Arg Glu Val Tyr Asn Thr Leu Ile

20 25 30

Asn Ile Glu Val Asp Leu

35

<210> 36

<211> 3969

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (629)..(3181)

<223>

<220>

<221> misc_feature

<223> Oligo 132

<400> 36

catgacagct gtgccactta tcccacggaa gactagtggc agacgactac cacgatttag 60

tagctttaaa tccagctatt cgacgagctc aggtccaaga tttactgtgt gagagacgat 120

caattcactg cggcctacaa taatccggat acgagggcta ttccaaacgg tttaactgtc 180

gagctaaatg atgggactac cttggaagag gttgttgtcg agtaccctat tggccataag 240

actagacgtg cagaggcaga accgaagctg cttgagaagt tctacaggca tttgtcaggg 300

cactttgagg gcgacattac taaggtcgag caagtaatga caatgtccac tgacccagag 360

tttgagtcct atagtatcga ccagtatgta gacactttct gtcgttgacg gtatctagat 420

ccaacaaatg tactaagtta cgtaacagtt ttatgatgat gttacataat cttcccagtc 480

gccggagctt cgcgtcaatg atatccgggt aatacttttc tgtcaacaaa attttctcta 540

gggatgaatg aagttttcat ctatcagcaa gtacagtata agcagtttgt tattataaga 600

agtacggcag ctgccttctt cgtctaac atg aat aac tcc atc aat gca tcg 652

Met Asn Asn Ser Ile Asn Ala Ser

1 5

gat tta cgg agc gtc agc ccg cac gaa gcc ccc tta aag ggc gtg tcg 700

Asp Leu Arg Ser Val Ser Pro His Glu Ala Pro Leu Lys Gly Val Ser

10 15 20

aag acc ttt aat gat ttg att gaa acg tcc aag aat ctg cct tct aca 748

Lys Thr Phe Asn Asp Leu Ile Glu Thr Ser Lys Asn Leu Pro Ser Thr

25 30 35 40

tca tcc gag ttg ggt tcc gta cta ttg aat gtc aat gaa ctc aag aag 796

Ser Ser Glu Leu Gly Ser Val Leu Leu Asn Val Asn Glu Leu Lys Lys

45 50 55

cgg gct gca gag tta aga gct aaa aat gtc aag aac aaa tct cct cac 844

Arg Ala Ala Glu Leu Arg Ala Lys Asn Val Lys Asn Lys Ser Pro His

60 65 70

cat acc cgg gca cac tat ttg ttg gca gga agt gga ctg gcg att caa 892

His Thr Arg Ala His Tyr Leu Leu Ala Gly Ser Gly Leu Ala Ile Gln

75 80 85

gat gtt gaa tct tct ttg aaa acc ctt gaa tcg aga cag ctg ttg gag 940

Asp Val Glu Ser Ser Leu Lys Thr Leu Glu Ser Arg Gln Leu Leu Glu

90 95 100

cag aat gtc caa aat aag gtt ccc gat gga gat ctt gac acc tac ctg 988

Gln Asn Val Gln Asn Lys Val Pro Asp Gly Asp Leu Asp Thr Tyr Leu

105 110 115 120

cgc aat aaa aaa gat gaa aac atc ctc tcc tct att gag cag tcc ttg 1036

Arg Asn Lys Lys Asp Glu Asn Ile Leu Ser Ser Ile Glu Gln Ser Leu

125 130 135

tct ttg gcg gca aag gat ttt gat aat ttt gtc aat gcg aac ttc aat 1084

Ser Leu Ala Ala Lys Asp Phe Asp Asn Phe Val Asn Ala Asn Phe Asn

140 145 150

ctg gac tgg aag aag cat aag gag gaa gtg aag agg agc ttt ttg ggt 1132

Leu Asp Trp Lys Lys His Lys Glu Glu Val Lys Arg Ser Phe Leu Gly

155 160 165

ctc gta tgg aag tcg gat cca aac cac aag agt cca act tct gtg tct 1180

Leu Val Trp Lys Ser Asp Pro Asn His Lys Ser Pro Thr Ser Val Ser

170 175 180

gaa cca tca ttt atg acc tgg ccc aag aaa ggt agc gga ata ttg gat 1228

Glu Pro Ser Phe Met Thr Trp Pro Lys Lys Gly Ser Gly Ile Leu Asp

185 190 195 200

ggt gaa tca aag ctg aat atc aac gaa aac tac gtt gtg aga gag aag 1276

Gly Glu Ser Lys Leu Asn Ile Asn Glu Asn Tyr Val Val Arg Glu Lys

205 210 215

ttt gaa aaa tac gcc aga ata atc aat cgg ttc aac aat gcc aga cag 1324

Phe Glu Lys Tyr Ala Arg Ile Ile Asn Arg Phe Asn Asn Ala Arg Gln

220 225 230

ctg cat aat aat ttc cct ttg acg aca gag ttt att act cta ttc caa 1372

Leu His Asn Asn Phe Pro Leu Thr Thr Glu Phe Ile Thr Leu Phe Gln

235 240 245

aat tcc gca gac tac aaa cag agg cag ctg ctc gaa gca tgg aag atc 1420

Asn Ser Ala Asp Tyr Lys Gln Arg Gln Leu Leu Glu Ala Trp Lys Ile

250 255 260

ctt gat aat tac cgc ttg tgt tcg gat tct atg aat att gtg gat att 1468

Leu Asp Asn Tyr Arg Leu Cys Ser Asp Ser Met Asn Ile Val Asp Ile

265 270 275 280

tca aaa ggg tat ctt gaa aac caa ttc atg gac tac gtc gac aat cta 1516

Ser Lys Gly Tyr Leu Glu Asn Gln Phe Met Asp Tyr Val Asp Asn Leu

285 290 295

tac ccc aaa aat gga aat gaa gga ttg cct act aac att aat aag atc 1564

Tyr Pro Lys Asn Gly Asn Glu Gly Leu Pro Thr Asn Ile Asn Lys Ile

300 305 310

aaa tcc ttc att gat tgt aaa tta aaa aat cca aat aac act tgg aag 1612

Lys Ser Phe Ile Asp Cys Lys Leu Lys Asn Pro Asn Asn Thr Trp Lys

315 320 325

ttt ggc aac ttg acg atc gtc aat ggt gtt cct gtg tgg gcc ttg atc 1660

Phe Gly Asn Leu Thr Ile Val Asn Gly Val Pro Val Trp Ala Leu Ile

330 335 340

ttt tat cta tta agg gcc ggt aag ttc cag gaa gct ttg gag gtc gcc 1708

Phe Tyr Leu Leu Arg Ala Gly Lys Phe Gln Glu Ala Leu Glu Val Ala

345 350 355 360

ata aac aat aag cta agt tta aaa aag gtg gag cag tct ttc ctg gta 1756

Ile Asn Asn Lys Leu Ser Leu Lys Lys Val Glu Gln Ser Phe Leu Val

365 370 375

tac ttc aaa gca tat gtc tcc tct aaa gac aag cgg ctg cca cag gag 1804

Tyr Phe Lys Ala Tyr Val Ser Ser Lys Asp Lys Arg Leu Pro Gln Glu

380 385 390

ttc att act aga ttg cac acg gag tac aat caa cac att aaa aac tcc 1852

Phe Ile Thr Arg Leu His Thr Glu Tyr Asn Gln His Ile Lys Asn Ser

395 400 405

ctc gat ggc gac ccc ttc aga ctc gcc gtt tac aag atc att gga aga 1900

Leu Asp Gly Asp Pro Phe Arg Leu Ala Val Tyr Lys Ile Ile Gly Arg

410 415 420

tgt gat ctg acg aga aag aat att tcc gct ata acg ctg agt gtt gaa 1948

Cys Asp Leu Thr Arg Lys Asn Ile Ser Ala Ile Thr Leu Ser Val Glu

425 430 435 440

gac tgg cta tgg gtc cac ttc atg ttg ata aaa gac ggc atc tcc agc 1996

Asp Trp Leu Trp Val His Phe Met Leu Ile Lys Asp Gly Ile Ser Ser

445 450 455

gac gac cca gtt tat gag aga tat agc ctg gtc gac ttc caa aac atc 2044

Asp Asp Pro Val Tyr Glu Arg Tyr Ser Leu Val Asp Phe Gln Asn Ile

460 465 470

atc acg tct tat ggc tct tcc agc ttc aat aac cac tac ttg caa gtc 2092

Ile Thr Ser Tyr Gly Ser Ser Ser Phe Asn Asn His Tyr Leu Gln Val

475 480 485

cta ctt ctc agc ggt caa tat gaa cta gct gtc caa tat gcc tac act 2140

Leu Leu Leu Ser Gly Gln Tyr Glu Leu Ala Val Gln Tyr Ala Tyr Thr

490 495 500

att aac gaa att gac gct gtt cat ttg gcg atc gct ctg gcg gat tat 2188

Ile Asn Glu Ile Asp Ala Val His Leu Ala Ile Ala Leu Ala Asp Tyr

505 510 515 520

aaa tta ttg aag gtg gca gct aat gtg act gac gat gag ttt gtc acg 2236

Lys Leu Leu Lys Val Ala Ala Asn Val Thr Asp Asp Glu Phe Val Thr

525 530 535

tcg ccc act ggc gag aga aag atc aac ttt gca aag att ctg ggg aac 2284

Ser Pro Thr Gly Glu Arg Lys Ile Asn Phe Ala Lys Ile Leu Gly Asn

540 545 550

tat aca aag tcc ttc aag ttc tct gat cct aga gtt gcg gtg gaa tat 2332

Tyr Thr Lys Ser Phe Lys Phe Ser Asp Pro Arg Val Ala Val Glu Tyr

555 560 565

ctt ctc cta atc gct tta gcg cac gaa gac tca caa att gaa ctt gct 2380

Leu Leu Leu Ile Ala Leu Ala His Glu Asp Ser Gln Ile Glu Leu Ala

570 575 580

cac gaa gca ttg agg gag ctg gtc cta gat act aaa gag ttc acc ata 2428

His Glu Ala Leu Arg Glu Leu Val Leu Asp Thr Lys Glu Phe Thr Ile

585 590 595 600

ctc ctc gga aaa atc aac agg gat ggc acc agg ata ccg ggc att atc 2476

Leu Leu Gly Lys Ile Asn Arg Asp Gly Thr Arg Ile Pro Gly Ile Ile

605 610 615

gag gag aga cag cct ctg ctt tac ttg gcc gac aaa gag gac ttt ttg 2524

Glu Glu Arg Gln Pro Leu Leu Tyr Leu Ala Asp Lys Glu Asp Phe Leu

620 625 630

cat aag att acg gag cag gca gcg cgc aga gca gat gaa aat ggc aga 2572

His Lys Ile Thr Glu Gln Ala Ala Arg Arg Ala Asp Glu Asn Gly Arg

635 640 645

gtc tac gac agt ctg cta ctg tat cag ctg gcg gaa gag tac gat att 2620

Val Tyr Asp Ser Leu Leu Leu Tyr Gln Leu Ala Glu Glu Tyr Asp Ile

650 655 660

gtc att agc att gtt aat aag ctg ctg agc gaa atg ttg agc aac act 2668

Val Ile Ser Ile Val Asn Lys Leu Leu Ser Glu Met Leu Ser Asn Thr

665 670 675 680

gac ctg gcg caa ccc ctg atg cgg cag gac gac aat agc gag act aac 2716

Asp Leu Ala Gln Pro Leu Met Arg Gln Asp Asp Asn Ser Glu Thr Asn

685 690 695

cct gtt cta atc gcc aag aag ctc att gat gtc tat atc aag aac ttg 2764

Pro Val Leu Ile Ala Lys Lys Leu Ile Asp Val Tyr Ile Lys Asn Leu

700 705 710

gaa att tca aag aag gta cac agg aaa aac aag gaa aca tgc att ctg 2812

Glu Ile Ser Lys Lys Val His Arg Lys Asn Lys Glu Thr Cys Ile Leu

715 720 725

ctc ctg aaa ctc gtg gat ata agg aga aca tat att gca aga cag tgg 2860

Leu Leu Lys Leu Val Asp Ile Arg Arg Thr Tyr Ile Ala Arg Gln Trp

730 735 740

caa aac acc ctg cag cag ata gag gag ctg gat ctg ctt cca tct gtc 2908

Gln Asn Thr Leu Gln Gln Ile Glu Glu Leu Asp Leu Leu Pro Ser Val

745 750 755 760

gaa gac tct tcc cca agg aag aag gcg cag gaa ttc cac aac ctg gac 2956

Glu Asp Ser Ser Pro Arg Lys Lys Ala Gln Glu Phe His Asn Leu Asp

765 770 775

gac tgt atc ata aag aat gtc ccc aac ctg ttg atc atc gcc atg acc 3004

Asp Cys Ile Ile Lys Asn Val Pro Asn Leu Leu Ile Ile Ala Met Thr

780 785 790

tgc gtt tct aac ctc atc aag cag ttg agc aag ggc ccc ttc tcc aac 3052

Cys Val Ser Asn Leu Ile Lys Gln Leu Ser Lys Gly Pro Phe Ser Asn

795 800 805

ggg gcc acg caa gct caa gtc gag gct ctg aag aag gtc gcc aac aac 3100

Gly Ala Thr Gln Ala Gln Val Glu Ala Leu Lys Lys Val Ala Asn Asn

810 815 820

tac atg atc tac aga ggc atg atc cag tac aag atg cct cgg gag gtg 3148

Tyr Met Ile Tyr Arg Gly Met Ile Gln Tyr Lys Met Pro Arg Glu Val

825 830 835 840

tac aac acc ctc ata aac atc gag gtg gac ctc tgaccgctcg tcagaccacg 3201

Tyr Asn Thr Leu Ile Asn Ile Glu Val Asp Leu

845 850

atgcagtgtg taccagacca gtattataga ttagatccta cgaactttac ctgctattta 3261

tccccgtgca gatacttctg cctgtactga ctgtagtccg gctggtactc gtacgcgttc 3321

ggactactgg atctcttccg ttcgagtttc tgccgacgtc tggcgattcg agcctgccgc 3381

ctcgcttcaa agtccatgct agtgtcgttt tcggattccg aagacgagct cgacgaggca 3441

ctcgacgatg cgtgctcgca ctcacccccg tcctcgtcgt caaagttctg ctgcgggtgg 3501

aaaatgcagc atatctttgt cttcttctta ttcatatgct cgttatctac cacattctca 3561

tcccatctga ctttcgactt ctgctctttc ttggctaact ttggcacctg ttccgtttga 3621

tttgcccgga gctgaaggat ctgcgatgtc tccgaaacag ttacagtgtg tgacctgtta 3681

gcaagtgact cctggttgtt gcttgacatc ggaacagctc ttcccacttc cagaccaatc 3741

taatagtctt aacgttgttt gctcctgatt gtgtaccgct tacatacctt ccagctatta 3801

tacagaataa ccccttcgta actggtgata tcagcttgta tgggtggcaa attcaagggc 3861

cttttggcgt attatggttt acaacagcca taatctgaac agcggttaca gagaactgat 3921

cagatacact agccccttat tcgacgtgga tgggatagat tacagaga 3969

<210> 37

<211> 851

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 132

<400> 37

Met Asn Asn Ser Ile Asn Ala Ser Asp Leu Arg Ser Val Ser Pro His

1 5 10 15

Glu Ala Pro Leu Lys Gly Val Ser Lys Thr Phe Asn Asp Leu Ile Glu

20 25 30

Thr Ser Lys Asn Leu Pro Ser Thr Ser Ser Glu Leu Gly Ser Val Leu

35 40 45

Leu Asn Val Asn Glu Leu Lys Lys Arg Ala Ala Glu Leu Arg Ala Lys

50 55 60

Asn Val Lys Asn Lys Ser Pro His His Thr Arg Ala His Tyr Leu Leu

65 70 75 80

Ala Gly Ser Gly Leu Ala Ile Gln Asp Val Glu Ser Ser Leu Lys Thr

85 90 95

Leu Glu Ser Arg Gln Leu Leu Glu Gln Asn Val Gln Asn Lys Val Pro

100 105 110

Asp Gly Asp Leu Asp Thr Tyr Leu Arg Asn Lys Lys Asp Glu Asn Ile

115 120 125

Leu Ser Ser Ile Glu Gln Ser Leu Ser Leu Ala Ala Lys Asp Phe Asp

130 135 140

Asn Phe Val Asn Ala Asn Phe Asn Leu Asp Trp Lys Lys His Lys Glu

145 150 155 160

Glu Val Lys Arg Ser Phe Leu Gly Leu Val Trp Lys Ser Asp Pro Asn

165 170 175

His Lys Ser Pro Thr Ser Val Ser Glu Pro Ser Phe Met Thr Trp Pro

180 185 190

Lys Lys Gly Ser Gly Ile Leu Asp Gly Glu Ser Lys Leu Asn Ile Asn

195 200 205

Glu Asn Tyr Val Val Arg Glu Lys Phe Glu Lys Tyr Ala Arg Ile Ile

210 215 220

Asn Arg Phe Asn Asn Ala Arg Gln Leu His Asn Asn Phe Pro Leu Thr

225 230 235 240

Thr Glu Phe Ile Thr Leu Phe Gln Asn Ser Ala Asp Tyr Lys Gln Arg

245 250 255

Gln Leu Leu Glu Ala Trp Lys Ile Leu Asp Asn Tyr Arg Leu Cys Ser

260 265 270

Asp Ser Met Asn Ile Val Asp Ile Ser Lys Gly Tyr Leu Glu Asn Gln

275 280 285

Phe Met Asp Tyr Val Asp Asn Leu Tyr Pro Lys Asn Gly Asn Glu Gly

290 295 300

Leu Pro Thr Asn Ile Asn Lys Ile Lys Ser Phe Ile Asp Cys Lys Leu

305 310 315 320

Lys Asn Pro Asn Asn Thr Trp Lys Phe Gly Asn Leu Thr Ile Val Asn

325 330 335

Gly Val Pro Val Trp Ala Leu Ile Phe Tyr Leu Leu Arg Ala Gly Lys

340 345 350

Phe Gln Glu Ala Leu Glu Val Ala Ile Asn Asn Lys Leu Ser Leu Lys

355 360 365

Lys Val Glu Gln Ser Phe Leu Val Tyr Phe Lys Ala Tyr Val Ser Ser

370 375 380

Lys Asp Lys Arg Leu Pro Gln Glu Phe Ile Thr Arg Leu His Thr Glu

385 390 395 400

Tyr Asn Gln His Ile Lys Asn Ser Leu Asp Gly Asp Pro Phe Arg Leu

405 410 415

Ala Val Tyr Lys Ile Ile Gly Arg Cys Asp Leu Thr Arg Lys Asn Ile

420 425 430

Ser Ala Ile Thr Leu Ser Val Glu Asp Trp Leu Trp Val His Phe Met

435 440 445

Leu Ile Lys Asp Gly Ile Ser Ser Asp Asp Pro Val Tyr Glu Arg Tyr

450 455 460

Ser Leu Val Asp Phe Gln Asn Ile Ile Thr Ser Tyr Gly Ser Ser Ser

465 470 475 480

Phe Asn Asn His Tyr Leu Gln Val Leu Leu Leu Ser Gly Gln Tyr Glu

485 490 495

Leu Ala Val Gln Tyr Ala Tyr Thr Ile Asn Glu Ile Asp Ala Val His

500 505 510

Leu Ala Ile Ala Leu Ala Asp Tyr Lys Leu Leu Lys Val Ala Ala Asn

515 520 525

Val Thr Asp Asp Glu Phe Val Thr Ser Pro Thr Gly Glu Arg Lys Ile

530 535 540

Asn Phe Ala Lys Ile Leu Gly Asn Tyr Thr Lys Ser Phe Lys Phe Ser

545 550 555 560

Asp Pro Arg Val Ala Val Glu Tyr Leu Leu Leu Ile Ala Leu Ala His

565 570 575

Glu Asp Ser Gln Ile Glu Leu Ala His Glu Ala Leu Arg Glu Leu Val

580 585 590

Leu Asp Thr Lys Glu Phe Thr Ile Leu Leu Gly Lys Ile Asn Arg Asp

595 600 605

Gly Thr Arg Ile Pro Gly Ile Ile Glu Glu Arg Gln Pro Leu Leu Tyr

610 615 620

Leu Ala Asp Lys Glu Asp Phe Leu His Lys Ile Thr Glu Gln Ala Ala

625 630 635 640

Arg Arg Ala Asp Glu Asn Gly Arg Val Tyr Asp Ser Leu Leu Leu Tyr

645 650 655

Gln Leu Ala Glu Glu Tyr Asp Ile Val Ile Ser Ile Val Asn Lys Leu

660 665 670

Leu Ser Glu Met Leu Ser Asn Thr Asp Leu Ala Gln Pro Leu Met Arg

675 680 685

Gln Asp Asp Asn Ser Glu Thr Asn Pro Val Leu Ile Ala Lys Lys Leu

690 695 700

Ile Asp Val Tyr Ile Lys Asn Leu Glu Ile Ser Lys Lys Val His Arg

705 710 715 720

Lys Asn Lys Glu Thr Cys Ile Leu Leu Leu Lys Leu Val Asp Ile Arg

725 730 735

Arg Thr Tyr Ile Ala Arg Gln Trp Gln Asn Thr Leu Gln Gln Ile Glu

740 745 750

Glu Leu Asp Leu Leu Pro Ser Val Glu Asp Ser Ser Pro Arg Lys Lys

755 760 765

Ala Gln Glu Phe His Asn Leu Asp Asp Cys Ile Ile Lys Asn Val Pro

770 775 780

Asn Leu Leu Ile Ile Ala Met Thr Cys Val Ser Asn Leu Ile Lys Gln

785 790 795 800

Leu Ser Lys Gly Pro Phe Ser Asn Gly Ala Thr Gln Ala Gln Val Glu

805 810 815

Ala Leu Lys Lys Val Ala Asn Asn Tyr Met Ile Tyr Arg Gly Met Ile

820 825 830

Gln Tyr Lys Met Pro Arg Glu Val Tyr Asn Thr Leu Ile Asn Ile Glu

835 840 845

Val Asp Leu

850

<210> 38

<211> 997

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 174

<400> 38

gatcatgacc tgcagatgga gctgctccag aatctcgcgc ttcgctatct cgtattttat 60

ccgctccgtc tcctcgctct cacgccccag cggcccctcc tccgcacgca gcccgctgta 120

ttcgtcgtcg cccagggaaa gctcgtgcgg cgacttcggt gtcgccacct ggtaatatgc 180

cggccctgcg ccgatgtgtg ggcgccccgt gatatcctcc gagtccatga gtaatacttg 240

cggagatgat attagctgac cgctgtcgct atgtgcaatg ggctcctctg ccccgtggaa 300

ttcttccgag cttcgatgct gaatcctgct atgttgtctc ttgttccgaa gcgtccccgt 360

taatgtcatt gcacccggcg ttctgtagta taaccgcgtt cctgcgtcgt gctgcccagc 420

gcctcaaatt tgatgttccc gactgttcct gcaagtaatt accgaagact gcgcccgctt 480

agtctccagg atagaatacg ctcatataaa cccggtcctg actatagaga gtgtgcagag 540

agtgcgaaga gcttcgcttg ccctgctcct acgcttgaag agccccatag aaaaaagcgg 600

aaacgagaag cctcttcctt tgatgcattt cagccgttcc cgcagtcacg cgaccgcaac 660

tctcagatat ttacagcttt tcagtgcttc atttcattag tggcaacatg gccgagcggt 720

taaggcgaaa gattagaaat cttttgggct atgcccgcgc aggttcgagt cctgctgttg 780

tcgttatttt ttgccgaatc catgtataca tggtcacgtg catgagaggt cacaccacgt 840

atgtaagtat agtaaagatt ccgttagagt aaggattaca tattcttctg agcctcaagc 900

tccgcaagga ggtcgtcgag cagcgcaacc gtttcctgtg gcgacgatat ccacgctgtg 960

cagcctgcga gccgtgcctt gaagtcattc gcagatc 997

<210> 39

<211> 58

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 174

<400> 39

Tyr Gln Val Ala Thr Pro Lys Ser Pro His Glu Leu Ser Leu Gly Asp

1 5 10 15

Asp Glu Tyr Ser Gly Leu Arg Ala Glu Glu Gly Pro Leu Gly Arg Glu

20 25 30

Ser Glu Glu Thr Glu Arg Ile Lys Tyr Glu Ile Ala Lys Arg Glu Ile

35 40 45

Leu Glu Gln Leu His Leu Gln Val Met Ile

50 55

<210> 40

<211> 3197

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (964)..(2589)

<223>

<220>

<221> misc_feature

<223> Oligo 174

<400> 40

tgcgcctgcc tgcggagacc ccagatcatc cgcaaggtgc gcgcagtggc cattgtaccg 60

ggggcagcgc tttgaccggg agtgggccgc gaaattcggc gtgccgctgt tgcgcgagac 120

gctggaggaa atcgttctta cagtccaggg tcggctcgag aacttcccac ccgcgcagcg 180

tgtgcaattc agctgtttcg acggcggcag cgacgtgtgg tgcgacatcg gcggcaagga 240

cctcggtgta gccttcctgc agcggttcta ctcgccggag cgtccaatcc gccctgagca 300

gtccttgcat gtcggtgacc agttcgcccc cgtcggatct gcgaatgact tcaaggcacg 360

gctcgcaggc tgcacagcgt ggatatcgtc gccacaggaa acggttgcgc tgctcgacga 420

cctccttgcg gagcttgagg ctcagaagaa tatgtaatcc ttactctaac ggaatcttta 480

ctatacttac atacgtggtg tgacctctca tgcacgtgac catgtataca tggattcggc 540

aaaaaataac gacaacagca ggactcgaac ctgcgcgggc atagcccaaa agatttctaa 600

tctttcgcct taaccgctcg gccatgttgc cactaatgaa atgaagcact gaaaagctgt 660

aaatatctga gagttgcggt cgcgtgactg cgggaacggc tgaaatgcat caaaggaaga 720

ggcttctcgt ttccgctttt ttctatgggg ctcttcaagc gtaggagcag ggcaagcgaa 780

gctcttcgca ctctctgcac actctctata gtcaggaccg ggtttatatg agcgtattct 840

atcctggaga ctaagcgggc gcagtcttcg gtaattactt gcaggaacag tcgggaacat 900

caaatttgag gcgctgggca gcacgacgca ggaacgcggt tatactacag aacgccgggt 960

gca atg aca tta acg ggg acg ctt cgg aac aag aga caa cat agc agg 1008

Met Thr Leu Thr Gly Thr Leu Arg Asn Lys Arg Gln His Ser Arg

1 5 10 15

att cag cat cga agc tcg gaa gaa ttc cac ggg gca gag gag ccc att 1056

Ile Gln His Arg Ser Ser Glu Glu Phe His Gly Ala Glu Glu Pro Ile

20 25 30

gca cat agc gac agc ggt cag cta ata tca tct ccg caa gta tta ctc 1104

Ala His Ser Asp Ser Gly Gln Leu Ile Ser Ser Pro Gln Val Leu Leu

35 40 45

atg gac tcg gag gat atc acg ggg cgc cca cac atc ggc gca ggg ccg 1152

Met Asp Ser Glu Asp Ile Thr Gly Arg Pro His Ile Gly Ala Gly Pro

50 55 60

gca tat tac cag gtg gcg aca ccg aag tcg ccg cac gag ctt tcc ctg 1200

Ala Tyr Tyr Gln Val Ala Thr Pro Lys Ser Pro His Glu Leu Ser Leu

65 70 75

ggc gac gac gaa tac agc ggg ctg cgt gcg gag gag ggg ccg ctg ggg 1248

Gly Asp Asp Glu Tyr Ser Gly Leu Arg Ala Glu Glu Gly Pro Leu Gly

80 85 90 95

cgt gag agc gag gag acg gag cgg ata aaa tac gag ata gcg aag cgc 1296

Arg Glu Ser Glu Glu Thr Glu Arg Ile Lys Tyr Glu Ile Ala Lys Arg

100 105 110

gag att ctg gag cag ctc cat ctg cag gtc atg atc aag cat cga gaa 1344

Glu Ile Leu Glu Gln Leu His Leu Gln Val Met Ile Lys His Arg Glu

115 120 125

atg gac gac ctg gaa gcc gag tca cgt ggc tta ggg gcg aaa ttg gag 1392

Met Asp Asp Leu Glu Ala Glu Ser Arg Gly Leu Gly Ala Lys Leu Glu

130 135 140

gtg ctg gag atg ttg cat gat gat ccg gag ctc ttg gcc aag gtg gac 1440

Val Leu Glu Met Leu His Asp Asp Pro Glu Leu Leu Ala Lys Val Asp

145 150 155

gcg cac cag gag caa cag gca cag ctg cgt ttg gca cag tta gaa gca 1488

Ala His Gln Glu Gln Gln Ala Gln Leu Arg Leu Ala Gln Leu Glu Ala

160 165 170 175

cgt cgc agg agg gaa caa cag atg gca gcg cta ccc cct gct ccg ctt 1536

Arg Arg Arg Arg Glu Gln Gln Met Ala Ala Leu Pro Pro Ala Pro Leu

180 185 190

tcg tcg agc agt ggt ggt gag tac tat tac cac acc cgc agt aag agc 1584

Ser Ser Ser Ser Gly Gly Glu Tyr Tyr Tyr His Thr Arg Ser Lys Ser

195 200 205

atg aac tcc cat cag ggg agg tct tct acc ctc cgg ccg gct gac ggg 1632

Met Asn Ser His Gln Gly Arg Ser Ser Thr Leu Arg Pro Ala Asp Gly

210 215 220

aat gta atc ggc ctc cgt gtt ttg ggc tca aag acg gtt gca gac gct 1680

Asn Val Ile Gly Leu Arg Val Leu Gly Ser Lys Thr Val Ala Asp Ala

225 230 235

agt agt ccc gga acc acg tca cca cct ttc agc tcc ttt cag cag gca 1728

Ser Ser Pro Gly Thr Thr Ser Pro Pro Phe Ser Ser Phe Gln Gln Ala

240 245 250 255

gac cct ttc gcg ccg caa cat ttc aac cag cat cac agg agg aac tac 1776

Asp Pro Phe Ala Pro Gln His Phe Asn Gln His His Arg Arg Asn Tyr

260 265 270

agc agt aat tgt att tca agc aac agc ggc gta gtc ggg aaa acc gac 1824

Ser Ser Asn Cys Ile Ser Ser Asn Ser Gly Val Val Gly Lys Thr Asp

275 280 285

agc ggg gat gcg att ttt agg cgc ctt gat ggg ctc ctg atc gtt atc 1872

Ser Gly Asp Ala Ile Phe Arg Arg Leu Asp Gly Leu Leu Ile Val Ile

290 295 300

acg tgc tgc aag tgc ggt aag tcg gga ttt acg tct gca caa ggc atc 1920

Thr Cys Cys Lys Cys Gly Lys Ser Gly Phe Thr Ser Ala Gln Gly Ile

305 310 315

gtc aac cac tcc agg ctg aaa cat gcc aat tct tat tcc agt cag cca 1968

Val Asn His Ser Arg Leu Lys His Ala Asn Ser Tyr Ser Ser Gln Pro

320 325 330 335

cta gct gta ctt cat aac cag sga att ctt ccg gaa gag cag caa aat 2016

Leu Ala Val Leu His Asn Gln Xaa Ile Leu Pro Glu Glu Gln Gln Asn

340 345 350

aaa att gtc atg gat aaa ttt aag gaa ctg cat ctt gac cct cag acg 2064

Lys Ile Val Met Asp Lys Phe Lys Glu Leu His Leu Asp Pro Gln Thr

355 360 365

gag tac ctg ccg aat ccc aca gtg gtc tcc caa agc cca tcc tct agt 2112

Glu Tyr Leu Pro Asn Pro Thr Val Val Ser Gln Ser Pro Ser Ser Ser

370 375 380

gct aat tca aat gca tgt ata aca gct act gaa ggg cgc gat ata tca 2160

Ala Asn Ser Asn Ala Cys Ile Thr Ala Thr Glu Gly Arg Asp Ile Ser

385 390 395

cca aca cat atc tca tcg tcc tta tcg cct acc tgc gtg cag cca gtt 2208

Pro Thr His Ile Ser Ser Ser Leu Ser Pro Thr Cys Val Gln Pro Val

400 405 410 415

tcc caa ttt cac tca act aaa cat ctg gag aag ctc tat ggc aaa gag 2256

Ser Gln Phe His Ser Thr Lys His Leu Glu Lys Leu Tyr Gly Lys Glu

420 425 430

gga ttc cag cag ata gtg gac tat gtc aag gag tct aaa gat gat ttg 2304

Gly Phe Gln Gln Ile Val Asp Tyr Val Lys Glu Ser Lys Asp Asp Leu

435 440 445

ggc ccc ccc atg cgg gtc gat tcg gat atc gat aac gat acg aat tcg 2352

Gly Pro Pro Met Arg Val Asp Ser Asp Ile Asp Asn Asp Thr Asn Ser

450 455 460

ctg cac ccg ggg gat caa tcg gat ttg ctt tca caa cat aat tcg gag 2400

Leu His Pro Gly Asp Gln Ser Asp Leu Leu Ser Gln His Asn Ser Glu

465 470 475

cgt tca acc gac cta aag cgt cgc gca tcg cct aat atg gac aaa gct 2448

Arg Ser Thr Asp Leu Lys Arg Arg Ala Ser Pro Asn Met Asp Lys Ala

480 485 490 495

ctt cgt gaa cga atg cgg cct gct gag aag cgg gtg agg ccc gac gct 2496

Leu Arg Glu Arg Met Arg Pro Ala Glu Lys Arg Val Arg Pro Asp Ala

500 505 510

atg gca ctc gtc gag ctc cct gct gaa gag aag aga tca tcg cat tac 2544

Met Ala Leu Val Glu Leu Pro Ala Glu Glu Lys Arg Ser Ser His Tyr

515 520 525

aac tta agg gcc agg tca aag cta aaa tca ctc tcg aaa cat gaa 2589

Asn Leu Arg Ala Arg Ser Lys Leu Lys Ser Leu Ser Lys His Glu

530 535 540

taatatgtaa gtattgttga gacctcattt cccttcgcat ataagtacta tgtattaata 2649

tcatagattc tggacggatc atcacttgtt tgctcggact ctctcaggaa acgcttgggc 2709

gacataggga aatatgtaac gtaccctaac ttcgcgcata ctaaatcaca gccgaaaaca 2769

ctggggctga acagcatata acacatgttc ctttcgcaac acaagaggag tatagttcga 2829

aggatttgtt ggccctcggc taagcgctac cattcggaac tggtgcgctg gttctacgcc 2889

acggattcgc ccttggaaaa gccgtacgaa ccgaactata agcctgtaaa gcctccggta 2949

aactttatac cgttctctaa aagcgattcc gaccgtttag agcggttcta tagccttgaa 3009

aggccctctg agaatcccat ttcggtcaat gaggattatc tgttcgaagt atacttggaa 3069

gcaagacgtc tgaagcccgc gtattgggat ggccctgttt acgaggtccg gagaggcacc 3129

tggcttaata gcgatggcat gcctctaagt gaagacctga gcgccgagct actgagctat 3189

cggccaag 3197

<210> 41

<211> 542

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<222> (343)..(343)

<223> Xaa' at position 343 represents Gly or Arg.

<220>

<221> misc_feature

<223> Oligo 174

<400> 41

Met Thr Leu Thr Gly Thr Leu Arg Asn Lys Arg Gln His Ser Arg Ile

1 5 10 15

Gln His Arg Ser Ser Glu Glu Phe His Gly Ala Glu Glu Pro Ile Ala

20 25 30

His Ser Asp Ser Gly Gln Leu Ile Ser Ser Pro Gln Val Leu Leu Met

35 40 45

Asp Ser Glu Asp Ile Thr Gly Arg Pro His Ile Gly Ala Gly Pro Ala

50 55 60

Tyr Tyr Gln Val Ala Thr Pro Lys Ser Pro His Glu Leu Ser Leu Gly

65 70 75 80

Asp Asp Glu Tyr Ser Gly Leu Arg Ala Glu Glu Gly Pro Leu Gly Arg

85 90 95

Glu Ser Glu Glu Thr Glu Arg Ile Lys Tyr Glu Ile Ala Lys Arg Glu

100 105 110

Ile Leu Glu Gln Leu His Leu Gln Val Met Ile Lys His Arg Glu Met

115 120 125

Asp Asp Leu Glu Ala Glu Ser Arg Gly Leu Gly Ala Lys Leu Glu Val

130 135 140

Leu Glu Met Leu His Asp Asp Pro Glu Leu Leu Ala Lys Val Asp Ala

145 150 155 160

His Gln Glu Gln Gln Ala Gln Leu Arg Leu Ala Gln Leu Glu Ala Arg

165 170 175

Arg Arg Arg Glu Gln Gln Met Ala Ala Leu Pro Pro Ala Pro Leu Ser

180 185 190

Ser Ser Ser Gly Gly Glu Tyr Tyr Tyr His Thr Arg Ser Lys Ser Met

195 200 205

Asn Ser His Gln Gly Arg Ser Ser Thr Leu Arg Pro Ala Asp Gly Asn

210 215 220

Val Ile Gly Leu Arg Val Leu Gly Ser Lys Thr Val Ala Asp Ala Ser

225 230 235 240

Ser Pro Gly Thr Thr Ser Pro Pro Phe Ser Ser Phe Gln Gln Ala Asp

245 250 255

Pro Phe Ala Pro Gln His Phe Asn Gln His His Arg Arg Asn Tyr Ser

260 265 270

Ser Asn Cys Ile Ser Ser Asn Ser Gly Val Val Gly Lys Thr Asp Ser

275 280 285

Gly Asp Ala Ile Phe Arg Arg Leu Asp Gly Leu Leu Ile Val Ile Thr

290 295 300

Cys Cys Lys Cys Gly Lys Ser Gly Phe Thr Ser Ala Gln Gly Ile Val

305 310 315 320

Asn His Ser Arg Leu Lys His Ala Asn Ser Tyr Ser Ser Gln Pro Leu

325 330 335

Ala Val Leu His Asn Gln Xaa Ile Leu Pro Glu Glu Gln Gln Asn Lys

340 345 350

Ile Val Met Asp Lys Phe Lys Glu Leu His Leu Asp Pro Gln Thr Glu

355 360 365

Tyr Leu Pro Asn Pro Thr Val Val Ser Gln Ser Pro Ser Ser Ser Ala

370 375 380

Asn Ser Asn Ala Cys Ile Thr Ala Thr Glu Gly Arg Asp Ile Ser Pro

385 390 395 400

Thr His Ile Ser Ser Ser Leu Ser Pro Thr Cys Val Gln Pro Val Ser

405 410 415

Gln Phe His Ser Thr Lys His Leu Glu Lys Leu Tyr Gly Lys Glu Gly

420 425 430

Phe Gln Gln Ile Val Asp Tyr Val Lys Glu Ser Lys Asp Asp Leu Gly

435 440 445

Pro Pro Met Arg Val Asp Ser Asp Ile Asp Asn Asp Thr Asn Ser Leu

450 455 460

His Pro Gly Asp Gln Ser Asp Leu Leu Ser Gln His Asn Ser Glu Arg

465 470 475 480

Ser Thr Asp Leu Lys Arg Arg Ala Ser Pro Asn Met Asp Lys Ala Leu

485 490 495

Arg Glu Arg Met Arg Pro Ala Glu Lys Arg Val Arg Pro Asp Ala Met

500 505 510

Ala Leu Val Glu Leu Pro Ala Glu Glu Lys Arg Ser Ser His Tyr Asn

515 520 525

Leu Arg Ala Arg Ser Lys Leu Lys Ser Leu Ser Lys His Glu

530 535 540

<210> 42

<211> 1086

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 51

<400> 42

gatctagtac cagctgagac tctctagttg cgtagtaact gtcgagcata cagcaggtga 60

ttaaaaatgg catctcgggt acaagcattc attaactgtg ctagtcagaa ggcctccttt 120

tttgtttcga agaccgtcta ctgcggcaag gttgccggtg aattgaccaa gacggtgtac 180

ttcaaagagg gcttgcagcc tccaaacata tctgacttcg agatggtata ctggagacta 240

ctaaagcagt tcaagaatgc ggctgcacac ccacagcaaa cattggcatc tgtgaagagc 300

ctagggaaga atgacctggt gaagtacggt gccgtcggtg tgcagatgct cggactgtac 360

tcgctaggcg aggcgatcgg tagaagacac cttgtcggct acacaaacta ctcctcgcac 420

cactagatat tccgcgcggc ggacggagcg ggccgggatt gcgcccattg tgtggggaag 480

ccaacctaga aaacgaattg atgaagcatt gattcgcaga tctcarggat ctgatgatac 540

atctataaat aacaactcta aactagttat caatcttata tattcgtact atttgtgttc 600

atactatgat acagtacgcg cgcgacactt gggcgccggc gagttatgca tgctcttcct 660

cgctggcgtc gccggcttgc tgggccagtt tgcgcttctt agatgcctct atcatttctt 720

tttgctgtcg tttcttctgc ttcagtatct ttggaaccgt gctgatctgc ttcctgtcca 780

cgaatgcctt gcccttctta atcatctcct tttccctctt ggtcatcttc gcgatattct 840

ccatccaagc ggcgacctcg cacccactct gcttcatgac gttgacaatc ggtttcacag 900

caawtgcgtc ctgctttgta aagaaaggtc actgctgtac ctttccgtcc gcctctgcct 960

gtacggccga tcctgtggac gtaagcctgt gctgagcgag gaacgtcata ggttgataac 1020

aagattgatg cccttgaaat ctataccacg tgccagaaca tcggtacaga tgaggcacca 1080

tagatc 1086

<210> 43

<211> 25

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 51

<400> 43

Asp Leu Trp Cys Leu Ile Cys Thr Asp Val Leu Ala Arg Gly Ile Asp

1 5 10 15

Phe Lys Gly Ile Asn Leu Val Ile Asn

20 25

<210> 44

<211> 36

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 51

<400> 44

Leu Leu Ser Thr Tyr Asp Val Pro Arg Ser Ala Gln Ala Tyr Val His

1 5 10 15

Arg Ile Gly Arg Thr Gly Arg Gly Gly Arg Lys Gly Thr Ala Val Thr

20 25 30

Phe Leu Tyr Lys

35

<210> 45

<211> 100

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 51

<220>

<221> misc_feature

<222> (29)..(29)

<223> unknown amino acid

<400> 45

His Arg Leu Thr Ser Thr Gly Ser Ala Val Gln Ala Glu Ala Asp Gly

1 5 10 15

Lys Val Gln Gln Pro Phe Phe Thr Lys Gln Asp Ala Xaa Ala Val Lys

20 25 30

Pro Ile Val Asn Val Met Lys Gln Ser Gly Cys Glu Val Ala Ala Trp

35 40 45

Met Glu Asn Ile Ala Lys Met Thr Lys Arg Glu Lys Glu Met Ile Lys

50 55 60

Lys Gly Lys Ala Phe Val Asp Arg Lys Gln Ile Ser Thr Val Pro Lys

65 70 75 80

Ile Leu Lys Gln Lys Lys Arg Gln Gln Lys Glu Met Ile Glu Ala Ser

85 90 95

Lys Lys Arg Lys

100

<210> 46

<211> 3296

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (502)..(2208)

<223>

<220>

<221> misc_ feature

<223> Oligo 51

<400> 46

atcgtcggga atctgcaacc cgtacttatc tagattgaat gcttgcttaa atttcgcctg 60

gtgccttgct gggtccccct cgttgtagtg cagccccgcc gggttcttaa gatcttcatt 120

ggggttcttc aggcctcgga ctttgatacc attaacgatg taagatgcat ggcgcgtaga 180

ctgtctagtg cctatcgata tcttagaaca caacatggtg ctatagacgg caactacagc 240

agattaacca cggctggtcg ctcttgaagg tggcctaagc caagagatga gcatccgcta 300

agctatcaaa attttcaaac taccccgggt aacaagttat ccgggtaacc acgtacaaaa 360

cgatatttga aaattttgta ttcacattgc tttttggtca ttagcctgcc cggtgcgcga 420

tgaggtccct acttgtcagt cagatcgtat ccgagctagt gagactgggt caggccaata 480

aggacattga gactcgaagt t atg gat att ttc agg gtt ctg aca cgt ggc 531

Met Asp Ile Phe Arg Val Leu Thr Arg Gly

1 5 10

gca tcg gtg aag aag gac ggg aag cag gtg gac ttc tcc aga gtg cag 579

Ala Ser Val Lys Lys Asp Gly Lys Gln Val Asp Phe Ser Arg Val Gln

15 20 25

agc cag cga aca gcg cgg cct gga cag cgc ggg aag gac gag gac gaa 627

Ser Gln Arg Thr Ala Arg Pro Gly Gln Arg Gly Lys Asp Glu Asp Glu

30 35 40

gag caa ttc tcg cgc gag ctg gat ttc ttt cgc acg agg cgg tct gtg 675

Glu Gln Phe Ser Arg Glu Leu Asp Phe Phe Arg Thr Arg Arg Ser Val

45 50 55

cct gct gcc gca gag agg cca gcg gaa gag acc ggg gat gtt cat agg 723

Pro Ala Ala Ala Glu Arg Pro Ala Glu Glu Thr Gly Asp Val His Arg

60 65 70

gac gag acc gag cag gac gag ggc gca gag gac gtg gca cca ccg ccc 771

Asp Glu Thr Glu Gln Asp Glu Gly Ala Glu Asp Val Ala Pro Pro Pro

75 80 85 90

cgg atc acg aca gca gaa gaa gcg tcg cag cta cgg cgc tcg tac cgg 819

Arg Ile Thr Thr Ala Glu Glu Ala Ser Gln Leu Arg Arg Ser Tyr Arg

95 100 105

ggc aag gtc aca ggc gcg gat gcg cct ctc ccc att ggg tcg ttt gag 867

Gly Lys Val Thr Gly Ala Asp Ala Pro Leu Pro Ile Gly Ser Phe Glu

110 115 120

gac ctg gtg aca cgc ttc aag ctg gac aag agg ctg cta tcg aac ctg 915

Asp Leu Val Thr Arg Phe Lys Leu Asp Lys Arg Leu Leu Ser Asn Leu

125 130 135

att gag aac aac ttt acg gag ccg acg ccc atc cag tgc gag gcc atc 963

Ile Glu Asn Asn Phe Thr Glu Pro Thr Pro Ile Gln Cys Glu Ala Ile

140 145 150

ccc atc agt ctg cag aac cgg gac ata gtg gcg tgt gcg ccg acc ggt 1011

Pro Ile Ser Leu Gln Asn Arg Asp Ile Val Ala Cys Ala Pro Thr Gly

155 160 165 170

agt ggt aag act ctg gcc ttt ttg ata cct ctt tta cag cag gtg ata 1059

Ser Gly Lys Thr Leu Ala Phe Leu Ile Pro Leu Leu Gln Gln Val Ile

175 180 185

tcc gac aag gcc gtt ggc acc ggc gtg aag ggc ttg att atc tcg ccc 1107

Ser Asp Lys Ala Val Gly Thr Gly Val Lys Gly Leu Ile Ile Ser Pro

190 195 200

aca aaa gaa ctt gcc aac cag atc ttt gac gag tgc tcg aag ctt gct 1155

Thr Lys Glu Leu Ala Asn Gln Ile Phe Asp Glu Cys Ser Lys Leu Ala

205 210 215

cag cgg atc ttc ctc gag aaa aag cgc ccg ttg tca gtg gca ttg ctc 1203

Gln Arg Ile Phe Leu Glu Lys Lys Arg Pro Leu Ser Val Ala Leu Leu

220 225 230

tcc aag tct ctc gcg gcg aag ctg aaa aac cag atc gtg agc gac aag 1251

Ser Lys Ser Leu Ala Ala Lys Leu Lys Asn Gln Ile Val Ser Asp Lys

235 240 245 250

aaa tat gat atc atc ata tcg act cct ctg cga ctc ata gat ata gtg 1299

Lys Tyr Asp Ile Ile Ile Ser Thr Pro Leu Arg Leu Ile Asp Ile Val

255 260 265

aag agc gaa tcg ctt gac cta agc gct gtc aag tac ctg atc ttt gat 1347

Lys Ser Glu Ser Leu Asp Leu Ser Ala Val Lys Tyr Leu Ile Phe Asp

270 275 280

gaa gcc gac aag cta ttt gac aaa acc ttt gtg gaa cag acg gac gac 1395

Glu Ala Asp Lys Leu Phe Asp Lys Thr Phe Val Glu Gln Thr Asp Asp

285 290 295

atc cta agc gca tgt agc cac cca aat att agc aag gtg ctg ttc tcg 1443

Ile Leu Ser Ala Cys Ser His Pro Asn Ile Ser Lys Val Leu Phe Ser

300 305 310

gcc acc ctg ccc tcc agt gtc gaa gag ctt gca cag tcg atc atg acc 1491

Ala Thr Leu Pro Ser Ser Val Glu Glu Leu Ala Gln Ser Ile Met Thr

315 320 325 330

gac ccc gtc aga gta atc att ggc cac aag gag gcc gct aat acg aac 1539

Asp Pro Val Arg Val Ile Ile Gly His Lys Glu Ala Ala Asn Thr Asn

335 340 345

att gaa cag aaa tta gta ttc tgc gga aac gaa gaa ggt aag ttg gtt 1587

Ile Glu Gln Lys Leu Val Phe Cys Gly Asn Glu Glu Gly Lys Leu Val

350 355 360

gcc atc agg cag cta ata cag gaa ggg atg ttc cgc cct ccc gta ata 1635

Ala Ile Arg Gln Leu Ile Gln Glu Gly Met Phe Arg Pro Pro Val Ile

365 370 375

atc ttt ttg gaa tcc atc acc aga gcc aaa gca tta ttc cat gag cta 1683

Ile Phe Leu Glu Ser Ile Thr Arg Ala Lys Ala Leu Phe His Glu Leu

380 385 390

ttg tac gat aaa cta aat gtt gat gtt atc cac gct gag cgt acc caa 1731

Leu Tyr Asp Lys Leu Asn Val Asp Val Ile His Ala Glu Arg Thr Gln

395 400 405 410

gtt caa agg gag aag atc atc gaa cga ttc aag agc ggt gat cta tgg 1779

Val Gln Arg Glu Lys Ile Ile Glu Arg Phe Lys Ser Gly Asp Leu Trp

415 420 425

tgc ctc atc tgt acc gat gtt ctg gca cgt ggt ata gat ttc aag ggc 1827

Cys Leu Ile Cys Thr Asp Val Leu Ala Arg Gly Ile Asp Phe Lys Gly

430 435 440

atc aat ctt gtt atc aac tat gac gtt cct cgc tca gca cag gct tac 1875

Ile Asn Leu Val Ile Asn Tyr Asp Val Pro Arg Ser Ala Gln Ala Tyr

445 450 455

gtc cac agg atc ggc cgt aca ggc aga ggc gga cgg aaa ggt aca gca 1923

Val His Arg Ile Gly Arg Thr Gly Arg Gly Gly Arg Lys Gly Thr Ala

460 465 470

gtg act ttc ttt aca aag cag gac gca att gct gtg aaa ccg att gtc 1971

Val Thr Phe Phe Thr Lys Gln Asp Ala Ile Ala Val Lys Pro Ile Val

475 480 485 490

aac gtc atg aag cag agt ggg tgc gag gtc gcc gct tgg atg gag aat 2019

Asn Val Met Lys Gln Ser Gly Cys Glu Val Ala Ala Trp Met Glu Asn

495 500 505

atc gcg aag atg acc aag agg gaa aag gag atg att aag aag ggc aag 2067

Ile Ala Lys Met Thr Lys Arg Glu Lys Glu Met Ile Lys Lys Gly Lys

510 515 520

gca ttc gtg gac agg aag cag atc agc acg gtt cca aag ata ctg aag 2115

Ala Phe Val Asp Arg Lys Gln Ile Ser Thr Val Pro Lys Ile Leu Lys

525 530 535

cag aag aaa cga cag caa aaa gaa atg ata gag gca tct aag aag cgc 2163

Gln Lys Lys Arg Gln Gln Lys Glu Met Ile Glu Ala Ser Lys Lys Arg

540 545 550

aaa ctg gcc cag caa gcc ggc gac gcc agc gag gaa gag cat gca 2208

Lys Leu Ala Gln Gln Ala Gly Asp Ala Ser Glu Glu Glu His Ala

555 560 565

taactcgccg gcgcccaagt gtcgcgcgcg tactgtatca tagtatgaac acaaatagta 2268

cgaatatata agattgataa ctagtttaga gttgttattt atagatgtat catcagatcc 2328

ctgagatctg cgaatcaatg cttcatcaat tcgttttcta ggttggcttc cccacacaat 2388

gggcgcaatc ccggcccgct ccgtccgccg cgcggaatat ctagtggtgc gaggagtagt 2448

ttgtgtagcc gacaaggtgt cttctaccga tcgcctcgcc tagcgagtac agtccgagca 2508

tctgcacacc gacggcaccg tacttcacca ggtcattctt ccctaggctc ttcacagatg 2568

ccaatgtttg ctgtgggtgt gcagccgcat tcttgaactg ctttagtagt ctccagtata 2628

ccatctcgaa gtcagatatg tttggaggct gcaagccctc tttgaagtac accgtcttgg 2688

tcaattcacc ggcaaccttg ccgcagtaga cggtcttcga aacaaaaaag gaggccttct 2748

gactagcaca gttaatgaat gcttgtaccc gagatgccat ttttaatcac ctgctgtatg 2808

ctcgacagtt actacgcaac tagagagtct cagctggtac tagatcgctt atatcgtcaa 2868

aattttcaaa ggtggcagca ttcaggatat ccgggtaaca atataccaat ggccaatcag 2928

atgccggttt ttagcacgga gagtggtctt ataccggtga tggactttga tggccaggcg 2988

gaaggcgata tacggccgaa aagtagtgat tgagcgcgcg gtagtgtaat cgtttacttg 3048

cgtctgtcta ttcggcgaac gcggcgagca tagagctgtt tatcgcgcgt acaaagtccg 3108

aaacaattac gctaaaacag tgagaatagc tgagtgtggg cgcgcgagca aactagacac 3168

ggccgttcag gcgtatcgat ctccggacgc cgtcgccgag gaccactatc ttgtcttcct 3228

gctgcaggcg attcagagcg tctgaaatcg tcatgttgtc caggcgggcc tgagagttct 3288

cgttcagc 3296

<210> 47

<211> 569

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 51

<400> 47

Met Asp Ile Phe Arg Val Leu Thr Arg Gly Ala Ser Val Lys Lys Asp

1 5 10 15

Gly Lys Gln Val Asp Phe Ser Arg Val Gln Ser Gln Arg Thr Ala Arg

20 25 30

Pro Gly Gln Arg Gly Lys Asp Glu Asp Glu Glu Gln Phe Ser Arg Glu

35 40 45

Leu Asp Phe Phe Arg Thr Arg Arg Ser Val Pro Ala Ala Ala Glu Arg

50 55 60

Pro Ala Glu Glu Thr Gly Asp Val His Arg Asp Glu Thr Glu Gln Asp

65 70 75 80

Glu Gly Ala Glu Asp Val Ala Pro Pro Pro Arg Ile Thr Thr Ala Glu

85 90 95

Glu Ala Ser Gln Leu Arg Arg Ser Tyr Arg Gly Lys Val Thr Gly Ala

100 105 110

Asp Ala Pro Leu Pro Ile Gly Ser Phe Glu Asp Leu Val Thr Arg Phe

115 120 125

Lys Leu Asp Lys Arg Leu Leu Ser Asn Leu Ile Glu Asn Asn Phe Thr

130 135 140

Glu Pro Thr Pro Ile Gln cys Glu Ala Ile Pro Ile Ser Leu Gln Asn

145 150 155 160

Arg Asp Ile Val Ala Cys Ala Pro Thr Gly Ser Gly Lys Thr Leu Ala

165 170 175

Phe Leu Ile Pro Leu Leu Gln Gln Val Ile Ser Asp Lys Ala Val Gly

180 185 190

Thr Gly Val Lys Gly Leu Ile Ile Ser Pro Thr Lys Glu Leu Ala Asn

195 200 205

Gln Ile Phe Asp Glu Cys Ser Lys Leu Ala Gln Arg Ile Phe Leu Glu

210 215 220

Lys Lys Arg Pro Leu Ser Val Ala Leu Leu Ser Lys Ser Leu Ala Ala

225 230 235 240

Lys Leu Lys Asn Gln Ile Val Ser Asp Lys Lys Tyr Asp Ile Ile Ile

245 250 255

Ser Thr Pro Leu Arg Leu Ile Asp Ile Val Lys Ser Glu Ser Leu Asp

260 265 270

Leu Ser Ala Val Lys Tyr Leu Ile Phe Asp Glu Ala Asp Lys Leu Phe

275 280 285

Asp Lys Thr Phe Val Glu Gln Thr Asp Asp Ile Leu Ser Ala Cys Ser

290 295 300

His Pro Asn Ile Ser Lys Val Leu Phe Ser Ala Thr Leu Pro Ser Ser

305 310 315 320

Val Glu Glu Leu Ala Gln Ser Ile Met Thr Asp Pro Val Arg Val Ile

325 330 335

Ile Gly His Lys Glu Ala Ala Asn Thr Asn Ile Glu Gln Lys Leu Val

340 345 350

Phe Cys Gly Asn Glu Glu Gly Lys Leu Val Ala Ile Arg Gln Leu Ile

355 360 365

Gln Glu Gly Met Phe Arg Pro Pro Val Ile Ile Phe Leu Glu Ser Ile

370 375 380

Thr Arg Ala Lys Ala Leu Phe His Glu Leu Leu Tyr Asp Lys Leu Asn

385 390 395 400

Val Asp Val Ile His Ala Glu Arg Thr Gln Val Gln Arg Glu Lys Ile

405 410 415

Ile Glu Arg Phe Lys Ser Gly Asp Leu Trp Cys Leu Ile Cys Thr Asp

420 425 430

Val Leu Ala Arg Gly Ile Asp Phe Lys Gly Ile Asn Leu Val Ile Asn

435 440 445

Tyr Asp Val Pro Arg Ser Ala Gln Ala Tyr Val His Arg Ile Gly Arg

450 455 460

Thr Gly Arg Gly Gly Arg Lys Gly Thr Ala Val Thr Phe Phe Thr Lys

465 470 475 480

Gln Asp Ala Ile Ala Val Lys Pro Ile Val Asn Val Met Lys Gln Ser

485 490 495

Gly Cys Glu Val Ala Ala Trp Met Glu Asn Ile Ala Lys Met Thr Lys

500 505 510

Arg Glu Lys Glu Met Ile Lys Lys Gly Lys Ala Phe Val Asp Arg Lys

515 520 525

Gln Ile Ser Thr Val Pro Lys Ile Leu Lys Gln Lys Lys Arg Gln Gln

530 535 540

Lys Glu Met Ile Glu Ala Ser Lys Lys Arg Lys Leu Ala Gln Gln Ala

545 550 555 560

Gly Asp Ala Ser Glu Glu Glu His Ala

565

<210> 48

<211> 528

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 30

<220>

<221> misc_feature

<222> (113)..(113)

<223> unknown nucleotide

<400> 48

gacgctttca acgcatccat taagaaaaat aacattccgg atgattggac ctttattcat 60

aacgaagaga gtaccaacgg gagcagcgaa aacgattctt ccacaggaat gcngcaaggc 120

ccggtctcta ggccactggg ttgacggcaa cggtaagcag ctggacggaa aactcaaatt 180

tactgttaaa aatgtctaca ctgcgggcag aatggtgtcc ctcgaggggt cactgctaag 240

tgaaggctat cacaggtcgc aagcagaaaa cctgcctgtc gtttccaaca ccaagatcat 300

ctttgatgac gaggtctctc aagagaacaa ggaatctcat aaggacttgg aacttagcgt 360

tctaaaagaa gataatggcg acgagatcat gtatgaaaaa gattccagcg attcaaacag 420

cgacagcgac agcgactaag ttgcctctca tatttagttg ccttctacgt ccgcatatta 480

catatataaa acatgttttg aatactgaag cacaatgagt cgcatcgc 528

<210> 49

<211> 34

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 30

<400> 49

Asp Ala Phe Asn Ala Ser Ile Lys Lys Asn Asn Ile Pro Asp Asp Trp

1 5 10 15

Thr Phe Ile His Asn Glu Glu Ser Thr Asn Gly Ser Ser Glu Asn Asp

20 25 30

Ser Ser

<210> 50

<211> 105

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 30

<400> 50

Arg Ser Leu Gly His Trp Val Asp Gly Asn Gly Lys Gln Leu Asp Gly

1 5 10 15

Lys Leu Lys Phe Thr Val Lys Asn Val Tyr Thr Ala Gly Arg Met Val

20 25 30

Ser Leu Glu Gly Ser Leu Leu Ser Glu Gly Tyr His Arg Ser Gln Ala

35 40 45

Glu Asn Leu Pro Val Val Ser Asn Thr Lys Ile Ile Phe Asp Asp Glu

50 55 60

Val Ser Gln Glu Asn Lys Glu Ser His Lys Asp Leu Glu Leu Ser Val

65 70 75 80

Leu Lys Glu Asp Asn Gly Asp Glu Ile Met Tyr Glu Lys Asp Ser Ser

85 90 95

Asp Ser Asn Ser Asp Ser Asp Ser Asp

100 105

<210> 51

<211> 1489

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (198)..(1073)

<223>

<220>

<221> misc_feature

<223> Oligo 30

<400> 51

caatttttgt atcacgtgac acataccatg gaagttttgt atccattttt tacgttacaa 60

tacgtacaga aaattttcac attagacgcc agtcctcatg cgatgagatg agcttcaaga 120

gcagtagctg tcgctgaacg gacatcaacg tggttatact gtcctgcttg ctaaggtagt 180

tacgcaaatt ggttaga atg ggt gtc aaa aaa agc ccg ctc aaa cgg tcc 230

Met Gly Val Lys Lys Ser Pro Leu Lys Arg Ser

1 5 10

aag acc gcc gag ttc atc aac aag cat cgc aag cta tgc aag aac ccc 278

Lys Thr Ala Glu Phe Ile Asn Lys His Arg Lys Leu Cys Lys Asn Pro

15 20 25

atc agc agt gaa gat aac aca tca cag tgc atc acg cgg ctt cca gtg 326

Ile Ser Ser Glu Asp Asn Thr Ser Gln Cys Ile Thr Arg Leu Pro Val

30 35 40

tct atg tac gtc tcg cta gct ccc ctt tac cag caa cat cct ctg gag 374

Ser Met Tyr Val Ser Leu Ala Pro Leu Tyr Gln Gln His Pro Leu Glu

45 50 55

ggc ata aaa cgg caa cat ctg aat ccc atg gtc atg aaa tac aac gga 422

Gly Ile Lys Arg Gln His Leu Asn Pro Met Val Met Lys Tyr Asn Gly

60 65 70 75

gat gtt gga ggt gta ata ctg gga tat gag aat gtg gcg att atg aat 470

Asp Val Gly Gly Val Ile Leu Gly Tyr Glu Asn Val Ala Ile Met Asn

80 85 90

gaa ggc gct gcg aaa gat gaa gaa ctg cta gtc aag ttg acg cca gat 518

Glu Gly Ala Ala Lys Asp Glu Glu Leu Leu Val Lys Leu Thr Pro Asp

95 100 105

acc cct ttt gcg ttt acc tgg tgc agc gta gat ctt gtg gta tgg tcg 566

Thr Pro Phe Ala Phe Thr Trp Cys Ser Val Asp Leu Val Val Trp Ser

110 115 120

cca cat atc ggc gat gtg ctc gag gga tgg atc ttc ata cag tca gcc 614

Pro His Ile Gly Asp Val Leu Glu Gly Trp Ile Phe Ile Gln Ser Ala

125 130 135

tca cac att ggt ctg ctg atc cac gac gct ttc aac gca tcc att aag 662

Ser His Ile Gly Leu Leu Ile His Asp Ala Phe Asn Ala Ser Ile Lys

140 145 150 155

aaa aat aac att ccg gat gat tgg acc ttt att cat aac gaa gag agt 710

Lys Asn Asn Ile Pro Asp Asp Trp Thr Phe Ile His Asn Glu Glu Ser

160 165 170

acc aac ggg agc agc gaa aac gat tct tcc aca gga atg cgc aag gcc 758

Thr Asn Gly Ser Ser Glu Asn Asp Ser Ser Thr Gly Met Arg Lys Ala

175 180 185

cgg tct cta ggc cac tgg gtt gac ggc aac ggt aag cag ctg gac gga 806

Arg Ser Leu Gly His Trp Val Asp Gly Asn Gly Lys Gln Leu Asp Gly

190 195 200

aaa ctc aaa ttt act gtt aaa aat gtc tac act gcg ggc aga atg gtg 854

Lys Leu Lys Phe Thr Val Lys Asn Val Tyr Thr Ala Gly Arg Met Val

205 210 215

tcc ctc gag ggg tca ctg cta agt gaa ggc tat cac agg tcg caa gca 902

Ser Leu Glu Gly Ser Leu Leu Ser Glu Gly Tyr His Arg Ser Gln Ala

220 225 230 235

gaa aac ctg cct gtc gtt tcc aac acc aag atc atc ttt gat gac gag 950

Glu Asn Leu Pro Val Val Ser Asn Thr Lys Ile Ile Phe Asp Asp Glu

240 245 250

gtc tct caa gag aac aag gaa tct cat aag gac ttg gaa ctt agc gtt 998

Val Ser Gln Glu Asn Lys Glu Ser His Lys Asp Leu Glu Leu Ser Val

255 260 265

cta aaa gaa gat aat ggc gac gag atc atg tat gaa aaa gat tcc agc 1046

Leu Lys Glu Asp Asn Gly Asp Glu Ile Met Tyr Glu Lys Asp Ser Ser

270 275 280

gat tca aac agc gac agc gac agc gac taagttgcct ctcatattta 1093

Asp Ser Asn Ser Asp Ser Asp Ser Asp

285 290

gttgccttct acgtccgcat attacatata taaaacatgt tttgaatact gaagcacaat 1153

gagtcgcatc gcagttagtg tatagataga acatgaaagt ccaattgaga caatttacat 1213

ccatatttta gtgagttaaa agcgacatta aacttggaag caatatatat accttatttc 1273

cacgcaataa ctaaattcca aaatgtcgat tattttaagg ctaggctaca acgggtaccc 1333

aatagcttat gatcgtgaat acaacgacgt aaatacctaa taatatgctc tttttatgtc 1393

caaattcact actcattacc tggtataatc attatatacg aaaatacttt atatatgtca 1453

tctgaagact gtctctctac caatcattgc accaag 1489

<210> 52

<211> 292

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 30

<400> 52

Met Gly Val Lys Lys Ser Pro Leu Lys Arg Ser Lys Thr Ala Glu Phe

1 5 10 15

Ile Asn Lys His Arg Lys Leu Cys Lys Asn Pro Ile Ser Ser Glu Asp

20 25 30

Asn Thr Ser Gln Cys Ile Thr Arg Leu Pro Val Ser Met Tyr Val Ser

35 40 45

Leu Ala Pro Leu Tyr Gln Gln His Pro Leu Glu Gly Ile Lys Arg Gln

50 55 60

His Leu Asn Pro Met Val Met Lys Tyr Asn Gly Asp Val Gly Gly Val

65 70 75 80

Ile Leu Gly Tyr Glu Asn Val Ala Ile Met Asn Glu Gly Ala Ala Lys

85 90 95

Asp Glu Glu Leu Leu Val Lys Leu Thr Pro Asp Thr Pro Phe Ala Phe

100 105 110

Thr Trp Cys Ser Val Asp Leu Val Val Trp Ser Pro His Ile Gly Asp

115 120 125

Val Leu Glu Gly Trp Ile Phe Ile Gln Ser Ala Ser His Ile Gly Leu

130 135 140

Leu Ile His Asp Ala Phe Asn Ala Ser Ile Lys Lys Asn Asn Ile Pro

145 150 155 160

Asp Asp Trp Thr Phe Ile His Asn Glu Glu Ser Thr Asn Gly Ser Ser

165 170 175

Glu Asn Asp Ser Ser Thr Gly Met Arg Lys Ala Arg Ser Leu Gly His

180 185 190

Trp Val Asp Gly Asn Gly Lys Gln Leu Asp Gly Lys Leu Lys Phe Thr

195 200 205

Val Lys Asn Val Tyr Thr Ala Gly Arg Met Val Ser Leu Glu Gly Ser

210 215 220

Leu Leu Ser Glu Gly Tyr His Arg Ser Gln Ala Glu Asn Leu Pro Val

225 230 235 240

Val Ser Asn Thr Lys Ile Ile Phe Asp Asp Glu Val Ser Gln Glu Asn

245 250 255

Lys Glu Ser His Lys Asp Leu Glu Leu Ser Val Leu Lys Glu Asp Asn

260 265 270

Gly Asp Glu Ile Met Tyr Glu Lys Asp Ser Ser Asp Ser Asn Ser Asp

275 280 285

Ser Asp Ser Asp

290

<210> 53

<211> 1289

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 124

<400> 53

gatcaactac gatatgccta gtgaggcaga ccaatacctc cacagagtcg gtagagcggg 60

cagattcggt accaaaggtc tggctatttc ccttgtgtcc tcaaaagatg atgaggaggt 120

tttagctaag atccaagaac gttttgaccg tgaagatcac cgaatttcca gaagaaggtg 180

tcgacccatc tacctatttg aacacttgaa caacaccgct tctccccatt tccgatacta 240

tatacgaaaa gggctaacgt aaataagaga agaactcaac aaagggaaat tttaaccttt 300

ttcttggttt tttgtatcta atataatcat caaggactgg gacgtatggt ttagcctagt 360

ctagctgaat actattagcg tcgcttctcc gtcggaaagt actgatctaa taaaccttcc 420

aaaatgtttt actgacattg atggcctagc tctagaattt cttgtactcg tgacttcttt 480

acacccagtc gcttttgctt ttacgtaaaa ccaaaactga atgttttact tctcatataa 540

aactgcatct ttaaatctta aatcgggcct catattacat atgtgatttt ttttcccgga 600

aagtgcatcg aacttcaatg caggagtggt aatacgccaa gagcaatgac gggatcttca 660

gcgttccttg cggatgcgca agtaaaacat aaacttagaa tatcactaca aataaacagc 720

aactaggtta caagcactat tataaacggc cacatgaaga catctctcgg ctgtaaccca 780

gcgctcatgt ttgctctggg gtgtcgcgtg cgctgacgtc aggaggctgc ggtaagatga 840

tctgcatatc ttgctgtgca ctgggctttt cgttagactt ggatgataga accttggaaa 900

ctttctctag acctttggtg gaattaggca tgctgtttaa atgatgtgca tgaatattac 960

ttaacgggag ttccttgggg agttcttcaa tgtggcttcg ggccttcaaa gcacgagggg 1020

cagcagctaa caatgacgga gaactatcat gccaattgga gtgatcagac tcagcaatat 1080

tagttacagg cgttgaatct gattcctcgt ctaggacgcg cgatgccagg atcccaaggg 1140

tggttagcat atgggaaccg tttttgcttc cgctgtcgct gcaagttatt ccactattaa 1200

cagagccact gttacttttt gccttcacac taggcgttgt gggcatgtac tgggcagtct 1260

tattcagaat gcgcatgagc ctgttgatc 1289

<210> 54

<211> 49

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 124

<400> 54

Ile Asn Tyr Asp Met Pro Ser Glu Ala Asp Gln Tyr Leu His Arg Val

1 5 10 15

Gly Arg Ala Gly Arg Phe Gly Thr Lys Gly Leu Ala Ile Ser Leu Val

20 25 30

Ser Ser Lys Asp Asp Glu Glu Val Leu Ala Lys Ile Gln Glu Arg Phe

35 40 45

Asp

<210> 55

<211> 18

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 124

<400> 55

Val Lys Ile Thr Glu Phe Pro Glu Glu Gly Val Asp Pro Ser Thr Tyr

1 5 10 15

Leu Asn

<210> 56

<211> 3228

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (465)..(1775)

<223>

<220>

<221> misc_feature

<223> Oligo 124

<400> 56

ccagacgagg atgatgcccc ggactcccgc cgctgcagat gttgctgtcg ctattctggg 60

gcattcgcct ccgctggaac gaacgccatc cgcagaacgc gcatcaacag atccgcagac 120

tatgcatgcc caattgaagg cgttggtggc taccattgat gaccatgagc cacagcttta 180

taacaatatg gcagatcccg catcaaaagt tctactgaat aacatcttca accgtcttcg 240

agccatggtg agtgatgatg cgtcctagtc tcgatcacgt gattcaaaag cgtatcctgt 300

tacccggccc tctaaaggtg gcaaactgtc gaaaattgaa agcgtaatga aaaatcagcg 360

aaaagaacag gatttgtttg gaacgccata ttatctttca ttctttgaaa gcataaagcg 420

ctattttagc tgtcaggtca ttctttagtt aatcgattat cagg atg tca cac gaa 476

Met Ser His Glu

1

ggc gag gaa gat tta ttg gaa tat tcc gat aac gaa cag gaa atc cag 524

Gly Glu Glu Asp Leu Leu Glu Tyr Ser Asp Asn Glu Gln Glu Ile Gln

5 10 15 20

gtt gac aac acg aag gcc act gag gta gcc ggt aac gga gaa gag gca 572

Val Asp Asn Thr Lys Ala Thr Glu Val Ala Gly Asn Gly Glu Glu Ala

25 30 35

gct gac gga aag gat gga gac aag aag gga tcg tac gta ggt att cac 620

Ala Asp Gly Lys Asp Gly Asp Lys Lys Gly Ser Tyr Val Gly Ile His

40 45 50

tcg aca ggt ttt aag gat ttt ctg ttg aag cca gag ctt tcc cgg gcc 668

Ser Thr Gly Phe Lys Asp Phe Leu Leu Lys Pro Glu Leu Ser Arg Ala

55 60 65

att att gac tgt ggt ttg gaa cat cca tgt gag gtc caa caa cac acc 716

Ile Ile Asp Cys Gly Leu Glu His Pro Cys Glu Val Gln Gln His Thr

70 75 80

atc cca caa tca att cac ggt acg gac gtt cta tgt cag gca aag tcc 764

Ile Pro Gln Ser Ile His Gly Thr Asp Val Leu Cys Gln Ala Lys Ser

85 90 95 100

ggt ctc ggg aaa act gct gtg ttt gtg ttg tca acg ctg cag caa ttg 812

Gly Leu Gly Lys Thr Ala Val Phe Val Leu Ser Thr Leu Gln Gln Leu

105 110 115

gac cct gtt cca ggc gag gta tca gtg gtc gtt ctg tgc aat gcc aga 860

Asp Pro Val Pro Gly Glu Val Ser Val Val Val Leu Cys Asn Ala Arg

120 125 130

gaa ttg gct tat cag atc aga aac gag tac ttg aga ttc tcg aaa tat 908

Glu Leu Ala Tyr Gln Ile Arg Asn Glu Tyr Leu Arg Phe Ser Lys Tyr

135 140 145

atg ccg gac gtg aaa aca gct gtt ttc tat ggt ggc aca gac act agg 956

Met Pro Asp Val Lys Thr Ala Val Phe Tyr Gly Gly Thr Asp Thr Arg

150 155 160

aag gac att gag ttg tta aaa aac aaa gat act gcg cca cat att gta 1004

Lys Asp Ile Glu Leu Leu Lys Asn Lys Asp Thr Ala Pro His Ile Val

165 170 175 180

gtt gca acg ccg gga cgg ttg aag gcg ctg gta cgg gac aac aat att 1052

Val Ala Thr Pro Gly Arg Leu Lys Ala Leu Val Arg Asp Asn Asn Ile

185 190 195

gac ttg tct cac gtt aag aac ttt gtt atc gat gag tgt gac aag gtg 1100

Asp Leu Ser His Val Lys Asn Phe Val Ile Asp Glu Cys Asp Lys Val

200 205 210

ttg gag gag ctc gat atg aga aga gat gtg caa gac att ttc agg gca 1148

Leu Glu Glu Leu Asp Met Arg Arg Asp Val Gln Asp Ile Phe Arg Ala

215 220 225

act cct aga gat aag cag gtg atg atg ttc tct gct acg cta tct caa 1196

Thr Pro Arg Asp Lys Gln Val Met Met Phe Ser Ala Thr Leu Ser Gln

230 235 240

gag atc aga ccg atc tgt aga cgt ttc ttg caa aac cct ctg gag att 1244

Glu Ile Arg Pro Ile Cys Arg Arg Phe Leu Gln Asn Pro Leu Glu Ile

245 250 255 260

ttt gtt gat gac gaa gct aag ttg acc ttg cac ggt ttg cag cag tac 1292

Phe Val Asp Asp Glu Ala Lys Leu Thr Leu His Gly Leu Gln Gln Tyr

265 270 275

tat atc agg ctt gag gaa cgt gag aag aac cgt aag ctg gct caa ttg 1340

Tyr Ile Arg Leu Glu Glu Arg Glu Lys Asn Arg Lys Leu Ala Gln Leu

280 285 290

ttg gat gat ttg gaa ttt aac cag gtt att atc ttc gta aaa tcg aca 1388

Leu Asp Asp Leu Glu Phe Asn Gln Val Ile Ile Phe Val Lys Ser Thr

295 300 305

ctt aga gca aac gaa ttg act aag ctg ttg aat gct tcc aac ttc cct 1436

Leu Arg Ala Asn Glu Leu Thr Lys Leu Leu Asn Ala Ser Asn Phe Pro

310 315 320

gca att act gtt cac ggt cac atg aga cag gaa gag cgt att gcc cgc 1484

Ala Ile Thr Val His Gly His Met Arg Gln Glu Glu Arg Ile Ala Arg

325 330 335 340

tac aag gcc ttc aag gaa ttt gaa aag cgt atc tgt gtg tca aca gac 1532

Tyr Lys Ala Phe Lys Glu Phe Glu Lys Arg Ile Cys Val Ser Thr Asp

345 350 355

gtt ttc ggt agg ggt att gat atc gag cgt atc aac cta gcg atc aac 1580

Val Phe Gly Arg Gly Ile Asp Ile Glu Arg Ile Asn Leu Ala Ile Asn

360 365 370

tac gat atg cct agt gag gca gac caa tac ctc cac aga gtc ggt aga 1628

Tyr Asp Met Pro Ser Glu Ala Asp Gln Tyr Leu His Arg Val Gly Arg

375 380 385

gcg ggc aga ttc ggt acc aaa ggt ctg gct att tcc ctt gtg tcc tca 1676

Ala Gly Arg Phe Gly Thr Lys Gly Leu Ala Ile Ser Leu Val Ser Ser

390 395 400

aaa gat gat gag gag gtt tta gct aag atc caa gaa cgt ttt gac gtg 1724

Lys Asp Asp Glu Glu Val Leu Ala Lys Ile Gln Glu Arg Phe Asp Val

405 410 415 420

aag atc acc gaa ttt cca gaa gaa ggt gtc gac cca tct acc tat ttg 1772

Lys Ile Thr Glu Phe Pro Glu Glu Gly Val Asp Pro Ser Thr Tyr Leu

425 430 435

aac acttgaacaa caccgcttct ccccatttcc gatactatat acgaaaaggg 1825

Asn

ctaacgtaaa taagagaaga actcaacaaa gggaaatttt aacctttttc ttggtttttt 1885

gtatctaata taatcatcaa ggactgggac gtatggttta gcctagtcta gctgaatact 1945

attagcgtcg cttctccgtc ggaaagtact gatctaataa accttccaaa atgttttact 2005

gacattgatg gcctagctct agaatttctt gtactcgtga cttctttaca cccagtcgct 2065

tttgctttta cgtaaaacca aaactgaatg ttttacttct catataaaac tgcatcttta 2125

aatcttaaat cgggcctcat attacatatg tgattttttt tcccggaaag tgcatcgaac 2185

ttcaatgcag gagtggtaat acgccaagag caatgacggg atcttcagcg ttccttgcgg 2245

atgcgcaagt aaaacataaa cttagaatat cactacaaat aaacagcaac taggttacaa 2305

gcactattat aaacggccac atgaagacat ctctcggctg taacccagcg ctcatgtttg 2365

ctctggggtg tcgcgtgcgc tgacgtcagg aggctgcggt aagatgatct gcatatcttg 2425

ctgtgcactg ggcttttcgt tagacttgga tgatagaacc ttggaaactt tctctagacc 2485

tttggtggaa ttaggcatgc tgtttaaatg atgtgcatga atattactta acgggagttc 2545

cttggggagt tcttcaatgt ggcttcgggc cttcaaagca cgaggggcag cagctaacaa 2605

tgacggagaa ctatcatgcc aattggagtg atcagactca gcaatattag ttacaggcgt 2665

tgaatctgat tcctcgtcta ggacgcgcga tgccaggatc ccaagggtgg ttagcatatg 2725

ggaaccgttt ttgcttccgc tgtcgctgca agttattcca ctattaacag agccactgtt 2785

actttttgcc ttcacactag gcgttgtggg catgtactgg gcagtcttat tcagaatgcg 2845

catgagcctg ttgatctgtt tattcttctc cataatggta tcacgatatt ttgagatttt 2905

atgatctcta gtgcgaatct gttccttcaa ctcttgctta tctatattga gcttgcattt 2965

cacgtgttcg tacatatgct tggacaaatt aagctcaact tcagaacggg aatccacatt 3025

ctttaacgtt gaattcagca ccaggttttt gaacttgagt tgccggatct gctcttgtga 3085

ctttgaaagc aggcttacca acaattctaa ggtcttcgag agttcatcgt tggatgggac 3145

aaacagttcg agtggtgtct gttgcctcga ctctgatggt tccgtcctat caaattcgtt 3205

acatcgccgt gtcgacaagt tgt 3228

<210> 57

<211> 437

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 124

<400> 57

Met Ser His Glu Gly Glu Glu Asp Leu Leu Glu Tyr Ser Asp Asn Glu

1 5 10 15

Gln Glu Ile Gln Val Asp Asn Thr Lys Ala Thr Glu Val Ala Gly Asn

20 25 30

Gly Glu Glu Ala Ala Asp Gly Lys Asp Gly Asp Lys Lys Gly Ser Tyr

35 40 45

Val Gly Ile His Ser Thr Gly Phe Lys Asp Phe Leu Leu Lys Pro Glu

50 55 60

Leu Ser Arg Ala Ile Ile Asp Cys Gly Leu Glu His Pro Cys Glu Val

65 70 75 80

Gln Gln His Thr Ile Pro Gln Ser Ile His Gly Thr Asp Val Leu Cys

85 90 95

Gln Ala Lys Ser Gly Leu Gly Lys Thr Ala Val Phe Val Leu Ser Thr

100 105 110

Leu Gln Gln Leu Asp Pro Val Pro Gly Glu Val Ser Val Val Val Leu

115 120 125

Cys Asn Ala Arg Glu Leu Ala Tyr Gln Ile Arg Asn Glu Tyr Leu Arg

130 135 140

Phe Ser Lys Tyr Met Pro Asp Val Lys Thr Ala Val Phe Tyr Gly Gly

145 150 155 160

Thr Asp Thr Arg Lys Asp Ile Glu Leu Leu Lys Asn Lys Asp Thr Ala

165 170 175

Pro His Ile Val Val Ala Thr Pro Gly Arg Leu Lys Ala Leu Val Arg

180 185 190

Asp Asn Asn Ile Asp Leu Ser His Val Lys Asn Phe Val Ile Asp Glu

195 200 205

Cys Asp Lys Val Leu Glu Glu Leu Asp Met Arg Arg Asp Val Gln Asp

210 215 220

Ile Phe Arg Ala Thr Pro Arg Asp Lys Gln Val Met Met Phe Ser Ala

225 230 235 240

Thr Leu Ser Gln Glu Ile Arg Pro Ile Cys Arg Arg Phe Leu Gln Asn

245 250 255

Pro Leu Glu Ile Phe Val Asp Asp Glu Ala Lys Leu Thr Leu His Gly

260 265 270

Leu Gln Gln Tyr Tyr Ile Arg Leu Glu Glu Arg Glu Lys Asn Arg Lys

275 280 285

Leu Ala Gln Leu Leu Asp Asp Leu Glu Phe Asn Gln Val Ile Ile Phe

290 295 300

Val Lys Ser Thr Leu Arg Ala Asn Glu Leu Thr Lys Leu Leu Asn Ala

305 310 315 320

Ser Asn Phe Pro Ala Ile Thr Val His Gly His Met Arg Gln Glu Glu

325 330 335

Arg Ile Ala Arg Tyr Lys Ala Phe Lys Glu Phe Glu Lys Arg Ile Cys

340 345 350

Val Ser Thr Asp Val Phe Gly Arg Gly Ile Asp Ile Glu Arg Ile Asn

355 360 365

Leu Ala Ile Asn Tyr Asp Met Pro Ser Glu Ala Asp Gln Tyr Leu His

370 375 380

Arg Val Gly Arg Ala Gly Arg Phe Gly Thr Lys Gly Leu Ala Ile Ser

385 390 395 400

Leu Val Ser Ser Lys Asp Asp Glu Glu Val Leu Ala Lys Ile Gln Glu

405 410 415

Arg Phe Asp Val Lys Ile Thr Glu Phe Pro Glu Glu Gly Val Asp Pro

420 425 430

Ser Thr Tyr Leu Asn

435

<210> 58

<211> 545

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 139

<220>

<221> misc_feature

<222> (358)..(358)

<223> unknown nucleotide

<220>

<221> misc_feature

<222> (418)..(418)

<223> unknown nucleotide

<220>

<221> misc_feature

<222> (461)..(461)

<223> unknown nucleotide

<220>

<221> misc_feature

<222> (506)..(506)

<223> unknown nucleotide

<220>

<221> misc_feature

<222> (539)..(539)

<223> unknown nucleotide

<400> 58

gatccacacg ccaaccgaca ggcagaacat ctacgattta ctgaaatatc tcctggagaa 60

cgagccgaar gattcgttwg cctgagtcat gtactatatt attacacttt gagtwgttta 120

tgtataggtt gggacaacat gcagacaggc acctacacac ctggattgga cagcgtagcc 180

gctgccgcgg cctgttgctt cttagtaacc ctctttttgg ggactgccgc cccatctgcc 240

tttttggtgg gcttccgctg ccttttcggt ttggatttgt cgtcgctgtc cgcggtgccg 300

gcgcgcttgc cgtttgcctc gctcggcgcg gtgtgcgcac tgcccatccc cataccgncc 360

ggcccaacat tctgcagcat ggccccggcc tgctgcttca caggctgtgt agtaagangg 420

gtcgacatcg agatgcggtg attatacccc ggcggggcca ngttcgcgtt gagtgcctgc 480

tgcttcttct tctctttggt caagcngaac tgctcgacga attttagtct gctgaaccng 540

gtatt 545

<210> 59

<211> 27

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 139

<220>

<221> misc_feature

<222> (26)..(26)

<223> unknown amino acid

<400> 59

Ile His Thr Pro Thr Asp Arg Gln Asn Ile Tyr Asp Leu Leu Lys Tyr

1 5 10 15

Leu Leu Glu Asn Glu Pro Lys Asp Ser Xaa Ala

20 25

<210> 60

<211> 1396

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (402)..(638)

<223>

<220>

<221> misc_feature

<223> Oligo 139

<220>

<221> CDS

<222> (663)..(974)

<223>

<400> 60

cttccccgga atgaaagccg ggctcacgct gagttgcagg cgtggcagcc gcgcggcaat 60

atttgcacgg ttctctcagc actgcgccag tcaacccctt aactctcgag ggtgccgccc 120

ctcccgtagg cgcagtcggc caaggaggaa gcggcggcac atggtgccgg ccgccaccgc 180

ggagcaggat caccggaatg acggatgtca cgtgcaggcc gagggaccga ctaagcgcgg 240

tgttcgacag cgcgctcgtg gcggcaggca cagccgtcgg ttaatgtgca cacacgtgat 300

ggtcacgtgc gcaacggccg ggcctttttg tgtggtcaaa gccagagcac actcgggcag 360

atccacagca tacacaccat agagggacag tgccgatgac g acc gcg cgt gac aat 416

Thr Ala Arg Asp Asn

1 5

agc gcg acg acg ctc gag ctg tat cgc aag acg ctg aat ttc aat gtg 464

Ser Ala Thr Thr Leu Glu Leu Tyr Arg Lys Thr Leu Asn Phe Asn Val

10 15 20

atc ggc aga tat gac ccc aaa ata aag cag ctg ctg ttc cac acg ccg 512

Ile Gly Arg Tyr Asp Pro Lys Ile Lys Gln Leu Leu Phe His Thr Pro

25 30 35

cat gcg acg gtg tac aag tgg gag gcc ggc gag aac aag tgg aac aag 560

His Ala Thr Val Tyr Lys Trp Glu Ala Gly Glu Asn Lys Trp Asn Lys

40 45 50

ctg gag tac cag ggc gtg ctg gct ata tat cta cgc gat gtg cgc gag 608

Leu Glu Tyr Gln Gly Val Leu Ala Ile Tyr Leu Arg Asp Val Arg Glu

55 60 65

cag gcg gag ctg ccg gtg ccg cac cag gag gcgagtgcag gcgcggaggg 658

Gln Ala Glu Leu Pro Val Pro His Gln Glu

70 75

gcgg tgc ggc gag gtg ctc agc ggg cgc gac atc tac aac tac gcg ctg 707

Cys Gly Glu Val Leu Ser Gly Arg Asp Ile Tyr Asn Tyr Ala Leu

80 85 90

atc gtg ctc aac cgg atc aac ccc gag aac ttc tcg att gca att gcg 755

Ile Val Leu Asn Arg Ile Asn Pro Glu Asn Phe Ser Ile Ala Ile Ala

95 100 105 110

ccg aac agc gtt gtg aac aag cgg cgt ctc ttt tcg ccg gag gag aac 803

Pro Asn Ser Val Val Asn Lys Arg Arg Leu Phe Ser Pro Glu Glu Asn

115 120 125

gtg cag cag ccg ctg gag cca atg gac gtt gag gtc aaa gat gaa ctg 851

Val Gln Gln Pro Leu Glu Pro Met Asp Val Glu Val Lys Asp Glu Leu

130 135 140

gtg atc atc aaa aac ctg cgg aag gag gtg tac ggc atc tgg atc cac 899

Val Ile Ile Lys Asn Leu Arg Lys Glu Val Tyr Gly Ile Trp Ile His

145 150 155

acg cca acc gac agg cag aac atc tac gat tta ctg aaa tat ctc ctg 947

Thr Pro Thr Asp Arg Gln Asn Ile Tyr Asp Leu Leu Lys Tyr Leu Leu

160 165 170

gag aac gag ccg aag gat tcg ttt gcc tgagtcatgt actatattat 994

Glu Asn Glu Pro Lys Asp Ser Phe Ala

175 180

tacactttga gttgtttatg tataggttgg gacaacatgc agacaggcac ctacacacct 1054

ggattggaca gcgtagccgc tgccgcggcc tgttgcttct tagtaaccct ctttttgggg 1114

actgccgccc catctgcctt tttggtgggc ttccgctgcc ttttcggttt ggatttgtcg 1174

tcgctgtccg cggtgccggc gcgcttgccg tttgcctcgc tcggcgcggt gtgcgcactg 1234

cccatcccca taccgcccgg cccaacattc tgcagcatgg ccccggcctg ctgcttcaca 1294

ggctgtgtag taagaggggt cgacatcgag atgcggtggt tataccccgg cggggccagg 1354

ttcgcgttga gtgcctgctg ctcttcttct ctttggtcaa gc 1396

<210> 61

<211> 79

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 139

<400> 61

Thr Ala Arg Asp Asn Ser Ala Thr Thr Leu Glu Leu Tyr Arg Lys Thr

1 5 10 15

Leu Asn Phe Asn Val Ile Gly Arg Tyr Asp Pro Lys Ile Lys Gln Leu

20 25 30

Leu Phe His Thr Pro His Ala Thr Val Tyr Lys Trp Glu Ala Gly Glu

35 40 45

Asn Lys Trp Asn Lys Leu Glu Tyr Gln Gly Val Leu Ala Ile Tyr Leu

50 55 60

Arg Asp Val Arg Glu Gln Ala Glu Leu Pro Val Pro His Gln Glu

65 70 75

<210> 62

<211> 104

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 139

<400> 62

Cys Gly Glu Val Leu Ser Gly Arg Asp Ile Tyr Asn Tyr Ala Leu Ile

1 5 10 15

Val Leu Asn Arg Ile Asn Pro Glu Asn Phe Ser Ile Ala Ile Ala Pro

20 25 30

Asn Ser Val Val Asn Lys Arg Arg Leu Phe Ser Pro Glu Glu Asn Val

35 40 45

Gln Gln Pro Leu Glu Pro Met Asp Val Glu Val Lys Asp Glu Leu Val

50 55 60

Ile Ile Lys Asn Leu Arg Lys Glu Val Tyr Gly Ile Trp Ile His Thr

65 70 75 80

Pro Thr Asp Arg Gln Asn Ile Tyr Asp Leu Leu Lys Tyr Leu Leu Glu

85 90 95

Asn Glu Pro Lys Asp Ser Phe Ala

100

<210> 63

<211> 944

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 144

<400> 63

gatctgagaa tgttaacgat aaccctgcta ttgcgaagaa cgttgtgagc ttctacgcgc 60

ctgagaagaa ggtcgacaag aagggtgtca tcattgacaa agagctcagc atcttcaaga 120

agtggaaact tgtgcgtgcc atcgacggca agttctccaa cgaaattacc tggtcacctg 180

ctggacgttt tgtatgcgtc gctgctattg gtaagattgg ttcccgtaac gagaacattg 240

atttctacga tatggactat ccaaacactg aaaagatcat taacactgct actgacgtta 300

acgctaccct gagagacgtt gcccacatca actacgccag tgccactgat tacgaatggg 360

acccaagtgg acggtacctt gccttctggt cttccgcgtg gaagcacaag gccgaaaatg 420

gatacaaggt attcaacttg gccggtgcca tcgtgcgtga ggagctcatc accgacttta 480

acaacttctt ctggagacca agaccagact ctctactatc caactctgag aagaagaagg 540

tcagaaagaa cctcaaggaa tggtccgcgc actttgaaga gcaggatgct atggaggcgg 600

acagtgctac aagagagtta atcctaaaga gacgcaactg gttggatgaa tggtccaagt 660

acagagaggc ttgcaagcaa accctatccg aaagtggatt gtccatttgc gattgtgtcg 720

aactgtcaac taaggatgag gactgtgagc ttgtcgagga gattagagag actgtggttg 780

aggagtccac cgaggaagtg ccatttttcg aggagtaatc gggtttcggc tgttgttgta 840

taatagtggc aacactggta attgattatg tatatataca agtactacat ccagcgtata 900

atgttttctt ttccgcagca gcgtctcgtc gcactcatgc gatc 944

<210> 64

<211> 260

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 144

<400> 64

Asn Pro Ala Ile Ala Lys Asn Val Val Ser Phe Tyr Ala Pro Glu Lys

1 5 10 15

Lys Val Asp Lys Lys Gly Val Ile Ile Asp Lys Glu Leu Ser Ile Phe

20 25 30

Lys Lys Trp Lys Leu Val Arg Ala Ile Asp Gly Lys Phe Ser Asn Glu

35 40 45

Ile Thr Trp Ser Pro Ala Gly Arg Phe Val Cys Val Ala Ala Ile Gly

50 55 60

Lys Ile Gly Ser Arg Asn Glu Asn Ile Asp Phe Tyr Asp Met Asp Tyr

65 70 75 80

Pro Asn Thr Glu Lys Ile Ile Asn Thr Ala Thr Asp Val Asn Ala Thr

85 90 95

Leu Arg Asp Val Ala His Ile Asn Tyr Ala Ser Ala Thr Asp Tyr Glu

100 105 110

Trp Asp Pro Ser Gly Arg Tyr Leu Ala Phe Trp Ser Ser Ala Trp Lys

115 120 125

His Lys Ala Glu Asn Gly Tyr Lys Val Phe Asn Leu Ala Gly Ala Ile

130 135 140

Val Arg Glu Glu Leu Ile Thr Asp Phe Asn Asn Phe Phe Trp Arg Pro

145 150 155 160

Arg Pro Asp Ser Leu Leu Ser Asn Ser Glu Lys Lys Lys Val Arg Lys

165 170 175

Asn Leu Lys Glu Trp Ser Ala His Phe Glu Glu Gln Asp Ala Met Glu

180 185 190

Ala Asp Ser Ala Thr Arg Glu Leu Ile Leu Lys Arg Arg Asn Trp Leu

195 200 205

Asp Glu Trp Ser Lys Tyr Arg Glu Ala Cys Lys Gln Thr Leu Ser Glu

210 215 220

Ser Gly Leu Ser Ile Cys Asp Cys Val Glu Leu Ser Thr Lys Asp Glu

225 230 235 240

Asp Cys Glu Leu Val Glu Glu Ile Arg Glu Thr Val Val Glu Glu Ser

245 250 255

Thr Glu Glu Val

260

<210> 65

<211> 3397

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (468)..(2675)

<223>

<220>

<221> misc_feature

<223> Oligo 144

<400> 65

actccattgc cctcttgcgc tgccatcgtc tcactttcgc ctgcaaatat ccactttggt 60

cccgtcgcct gctccattca gcacagattg gattatcgcc tctaacacct ccatgagtct 120

tgctgaagct tgcttggtgc tttgcgcgta ctgtttttgg acatctatgt aaacaagcga 180

aaatacccac ttctgtgtcg cgtggataag gtgcgcgccc acattgcgaa tcccacggca 240

cggccacttg ctagtaaacc ccaaagcgta acgctagttc tctagccggg cactgagacc 300

gttaatgcag cagccaatgg cctgcatttg ggatgaggtt ggtcagatgg aatcacgtga 360

tatagatgct tggcgaggtc aaggtgaaaa attgttaagt taaaattttg gacgcatcac 420

cagttccagc gagagaagat cagtcaagca gcaggtatag agcgaag atg gca gcg 476

Met Ala Ala

1

gta ttc gac gat ata agg ctt gag gac atc cct gtg gat gat gtg gat 524

Val Phe Asp Asp Ile Arg Leu Glu Asp Ile Pro Val Asp Asp Val Asp

5 10 15

atg cag gac ctt gag gag acg tat gct gta gag cgg agc atc gaa ttt 572

Met Gln Asp Leu Glu Glu Thr Tyr Ala Val Glu Arg Ser Ile Glu Phe

20 25 30 35

gac aga tac gtg gtt gtc gac ggg gcg ccg gtg gcg ccg gag gcg aag 620

Asp Arg Tyr Val Val Val Asp Gly Ala Pro Val Ala Pro Glu Ala Lys

40 45 50

gtg ggc gcg ctg cag aag gtg ctg acg aag cta ttt tcg cag gcg ggg 668

Val Gly Ala Leu Gln Lys Val Leu Thr Lys Leu Phe Ser Gln Ala Gly

55 60 65

tca gtg gtg gac atg gat gtg cct gtc gaa gag ggg cgg acg aag gga 716

Ser Val Val Asp Met Asp Val Pro Val Glu Glu Gly Arg Thr Lys Gly

70 75 80

cac ctt ttc att gag ttt gag gac gcc ggc gct gcg cgg cgg gcg atc 764

His Leu Phe Ile Glu Phe Glu Asp Ala Gly Ala Ala Arg Arg Ala Ile

85 90 95

aag atg ttc aac ggg aag aag ttg gac gtc aag cac cgc ctg tgg gtg 812

Lys Met Phe Asn Gly Lys Lys Leu Asp Val Lys His Arg Leu Trp Val

100 105 110 115

aac gga cta gac gac atg gag cgc tac ggg cgg ccg gac ttt tca acg 860

Asn Gly Leu Asp Asp Met Glu Arg Tyr Gly Arg Pro Asp Phe Ser Thr

120 125 130

gaa tac cgg gag cct gtg gtg ccg gag ttc gag gcg acg gaa tac cca 908

Glu Tyr Arg Glu Pro Val Val Pro Glu Phe Glu Ala Thr Glu Tyr Pro

135 140 145

cgg tcg tgg ttg cag gac gag acg ggt cgg gac cag ttt gtg ctg cag 956

Arg Ser Trp Leu Gln Asp Glu Thr Gly Arg Asp Gln Phe Val Leu Gln

150 155 160

aag ggc gag atg acg gct gta ttc tgg aac cgg aac aac ctg cag ccg 1004

Lys Gly Glu Met Thr Ala Val Phe Trp Asn Arg Asn Asn Leu Gln Pro

165 170 175

gag aac gtt gtc gaa ccg cgc cgc aac tgg tcg aac tct atc ttg aac 1052

Glu Asn Val Val Glu Pro Arg Arg Asn Trp Ser Asn Ser Ile Leu Asn

180 185 190 195

ttc tcg cct cac ggt acg tac cta ttc tcg ttc cac gac cag ggc att 1100

Phe Ser Pro His Gly Thr Tyr Leu Phe Ser Phe His Asp Gln Gly Ile

200 205 210

gcg tcc tgg ggt ggg ccg cag ttc aag cgt ctc cgt cgg ttt gcg cac 1148

Ala Ser Trp Gly Gly Pro Gln Phe Lys Arg Leu Arg Arg Phe Ala His

215 220 225

cct gat gtt aag gcg atc tcc atg tcc tcg acc gag aag tac ctg gtt 1196

Pro Asp Val Lys Ala Ile Ser Met Ser Ser Thr Glu Lys Tyr Leu Val

230 235 240

acc ttt tcg tcg gaa cct cta gaa gtc tcg gat gaa cct aac gag gct 1244

Thr Phe Ser Ser Glu Pro Leu Glu Val Ser Asp Glu Pro Asn Glu Ala

245 250 255

tgt cca ttc ggg ccc gag tcg cgg ggc cac cag cta tgt ata tgg gat 1292

Cys Pro Phe Gly Pro Glu Ser Arg Gly His Gln Leu Cys Ile Trp Asp

260 265 270 275

gtg gca aca ggt gtc tgc gtg aag acc ttt gcg ctg ccg cct cag cag 1340

Val Ala Thr Gly Val Cys Val Lys Thr Phe Ala Leu Pro Pro Gln Gln

280 285 290

cag ctg caa tgg cct atg gtc aag tgg tcc ttt gac gac aag ttc tgc 1388

Gln Leu Gln Trp Pro Met Val Lys Trp Ser Phe Asp Asp Lys Phe Cys

295 300 305

gct cgt ctt ggc cct ggc gca att gct gtg tac gag acc gag aag aac 1436

Ala Arg Leu Gly Pro Gly Ala Ile Ala Val Tyr Glu Thr Glu Lys Asn

310 315 320

ttc cag ctg ttg ggc ggt aag gtg atg aag atc gag gat gtt cag gac 1484

Phe Gln Leu Leu Gly Gly Lys Val Met Lys Ile Glu Asp Val Gln Asp

325 330 335

ttc tcc ttt gct cct aag ggc atc aag ttg gcg tca aac aga ccc aac 1532

Phe Ser Phe Ala Pro Lys Gly Ile Lys Leu Ala Ser Asn Arg Pro Asn

340 345 350 355

gac cca cca tct act gtc atg gta tac tgg act cca gag tcg aac aac 1580

Asp Pro Pro Ser Thr Val Met Val Tyr Trp Thr Pro Glu Ser Asn Asn

360 365 370

cag tcg tgt aaa gct gtc ctg att gag cta ccg aac cgc cgt gtt ctg 1628

Gln Ser Cys Lys Ala Val Leu Ile Glu Leu Pro Asn Arg Arg Val Leu

375 380 385

cgt acc atc aac ttg gtg cag gtt act gat gtc tcc ttc cat tgg cag 1676

Arg Thr Ile Asn Leu Val Gln Val Thr Asp Val Ser Phe His Trp Gln

390 395 400

aac cag gca gag ttc ctc tgt gta cag gtg gac cgt cac acg aag tct 1724

Asn Gln Ala Glu Phe Leu Cys Val Gln Val Asp Arg His Thr Lys Ser

405 410 415

agg aag acc atc ttc acc aac atg gag att tgc tct ttg act gcc aga 1772

Arg Lys Thr Ile Phe Thr Asn Met Glu Ile Cys Ser Leu Thr Ala Arg

420 425 430 435

gag ttt cca ttt gag aag gtg gag att aag gac cgc tgc atg cgc ttt 1820

Glu Phe Pro Phe Glu Lys Val Glu Ile Lys Asp Arg Cys Met Arg Phe

440 445 450

gca tgg gaa cct aat agc gac cgt ttc gtg atc att tcg aga tct gag 1868

Ala Trp Glu Pro Asn Ser Asp Arg Phe Val Ile Ile Ser Arg Ser Glu

455 460 465

aat gtt aac gat aac cct gct att gcg aag aac gtt gtg agc ttc tac 1916

Asn Val Asn Asp Asn Pro Ala Ile Ala Lys Asn Val Val Ser Phe Tyr

470 475 480

gcg cct gag aag aag gtc gac aag aag ggt gtc atc att gac aaa gag 1964

Ala Pro Glu Lys Lys Val Asp Lys Lys Gly Val Ile Ile Asp Lys Glu

485 490 495

ctc agc atc ttc aag aag tgg aaa ctt gtg cgt gcc atc gac ggc aag 2012

Leu Ser Ile Phe Lys Lys Trp Lys Leu Val Arg Ala Ile Asp Gly Lys

500 505 510 515

ttc tcc aac gaa att acc tgg tca cct gct gga cgt ttt gta tgc gtc 2060

Phe Ser Asn Glu Ile Thr Trp Ser Pro Ala Gly Arg Phe Val Cys Val

520 525 530

gct gct att ggt aag att ggt tcc cgt aac gag aac att gat ttc tac 2108

Ala Ala Ile Gly Lys Ile Gly Ser Arg Asn Glu Asn Ile Asp Phe Tyr

535 540 545

gat atg gac tat cca aac act gaa aag atc att aac act gct act gac 2156

Asp Met Asp Tyr Pro Asn Thr Glu Lys Ile Ile Asn Thr Ala Thr Asp

550 555 560

gtt aac gct acc ctg aga gac gtt gcc cac atc aac tac gcc agt gcc 2204

Val Asn Ala Thr Leu Arg Asp Val Ala His Ile Asn Tyr Ala Ser Ala

565 570 575

act gat tac gaa tgg gac cca agt gga cgg tac ctt gcc ttc tgg tct 2252

Thr Asp Tyr Glu Trp Asp Pro Ser Gly Arg Tyr Leu Ala Phe Trp Ser

580 585 590 595

tcc gcg tgg aag cac aag gcc gaa aat gga tac aag gta ttc aac ttg 2300

Ser Ala Trp Lys His Lys Ala Glu Asn Gly Tyr Lys Val Phe Asn Leu

600 605 610

gcc ggt gcc atc gtg cgt gag gag ctc atc acc gac ttt aac aac ttc 2348

Ala Gly Ala Ile Val Arg Glu Glu Leu Ile Thr Asp Phe Asn Asn Phe

615 620 625

ttc tgg aga cca aga cca gac tct cta cta tcc aac tct gag aag aag 2396

Phe Trp Arg Pro Arg Pro Asp Ser Leu Leu Ser Asn Ser Glu Lys Lys

630 635 640

aag gtc aga aag aac ctc aag gaa tgg tcc gcg cac ttt gaa gag cag 2444

Lys Val Arg Lys Asn Leu Lys Glu Trp Ser Ala His Phe Glu Glu Gln

645 650 655

gat gct atg gag gcg gac agt gct aca aga gag tta atc cta aag aga 2492

Asp Ala Met Glu Ala Asp Ser Ala Thr Arg Glu Leu Ile Leu Lys Arg

660 665 670 675

cgc aac tgg ttg gat gaa tgg tcc aag tac aga gag gct tgc aag caa 2540

Arg Asn Trp Leu Asp Glu Trp Ser Lys Tyr Arg Glu Ala Cys Lys Gln

680 685 690

acc cta tcc gaa agt gga ttg tcc att tgc gat tgt gtc gaa ctg tca 2588

Thr Leu Ser Glu Ser Gly Leu Ser Ile Cys Asp Cys Val Glu Leu Ser

695 700 705

act aag gat gag gac tgt gag ctt gtc gag gag att aga gag act gtg 2636

Thr Lys Asp Glu Asp Cys Glu Leu Val Glu Glu Ile Arg Glu Thr Val

710 715 720

gtt gag gag tcc acc gag gaa gtg cca ttt ttc gag gag taatcgggtt 2685

Val Glu Glu Ser Thr Glu Glu Val Pro Phe Phe Glu Glu

725 730 735

tcggctgttg ttgtataata gtggcaacac tggtaattga ttatgtatat atacaagtac 2745

tacatccagc gtataatgtt ttcttttccg cagcagcgtc tcgtcgcact catgcgatct 2805

ggctgcggcc gccaccacgc agaccgcgga ccgccttcgg tgagatcatt accatacaat 2865

ctagagaagc aataaatagg tttttcgttt attcagcttt ggtacgcagc tgcagcccat 2925

gccagcgttt gatgtcgtcc gcaagcacgc ggttgacgag cttgtgctgc tgcaccatgc 2985

tcttgccctt gaagtgtgtg ctccggatat cgatcgtgaa catcgaacca caacccccgc 3045

tcacatcagt gactttcaca taccccggcg caagcgcgtc ggttagcttt tgcgtaatga 3105

gcgcctcttc cggcgtgcta gtataatagc gcgcgaagag acgggagtaa gagcccacaa 3165

gccgacctct agctaacatg gggtcgtttg agcggcaaat aatgaaacgg gagcagcaga 3225

tgaagtttaa atctgcgtga tctgcagata tacgtgtgtg cgcgtgagca gtcagcctga 3285

gaacttttcg gaacatcgct tttgccggca acgtggggga agccctactt gccttacaag 3345

tcaactcatg gcacaccggc tacccttctg atgtttgcat tcccagccaa ga 3397

<210> 66

<211> 736

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 144

<400> 66

Met Ala Ala Val Phe Asp Asp Ile Arg Leu Glu Asp Ile Pro Val Asp

1 5 10 15

Asp Val Asp Met Gln Asp Leu Glu Glu Thr Tyr Ala Val Glu Arg Ser

20 25 30

Ile Glu Phe Asp Arg Tyr Val Val Val Asp Gly Ala Pro Val Ala Pro

35 40 45

Glu Ala Lys Val Gly Ala Leu Gln Lys Val Leu Thr Lys Leu Phe Ser

50 55 60

Gln Ala Gly Ser Val Val Asp Met Asp Val Pro Val Glu Glu Gly Arg

65 70 75 80

Thr Lys Gly His Leu Phe Ile Glu Phe Glu Asp Ala Gly Ala Ala Arg

85 90 95

Arg Ala Ile Lys Met Phe Asn Gly Lys Lys Leu Asp Val Lys His Arg

100 105 110

Leu Trp Val Asn Gly Leu Asp Asp Met Glu Arg Tyr Gly Arg Pro Asp

115 120 125

Phe Ser Thr Glu Tyr Arg Glu Pro Val Val Pro Glu Phe Glu Ala Thr

130 135 140

Glu Tyr Pro Arg Ser Trp Leu Gln Asp Glu Thr Gly Arg Asp Gln Phe

145 150 155 160

Val Leu Gln Lys Gly Glu Met Thr Ala Val Phe Trp Asn Arg Asn Asn

165 170 175

Leu Gln Pro Glu Asn Val Val Glu Pro Arg Arg Asn Trp Ser Asn Ser

180 185 190

Ile Leu Asn Phe Ser Pro His Gly Thr Tyr Leu Phe Ser Phe His Asp

195 200 205

Gln Gly Ile Ala Ser Trp Gly Gly Pro Gln Phe Lys Arg Leu Arg Arg

210 215 220

Phe Ala His Pro Asp Val Lys Ala Ile Ser Met Ser Ser Thr Glu Lys

225 230 235 240

Tyr Leu Val Thr Phe Ser Ser Glu Pro Leu Glu Val Ser Asp Glu Pro

245 250 255

Asn Glu Ala Cys Pro Phe Gly Pro Glu Ser Arg Gly His Gln Leu Cys

260 265 270

Ile Trp Asp Val Ala Thr Gly Val Cys Val Lys Thr Phe Ala Leu Pro

275 280 285

Pro Gln Gln Gln Leu Gln Trp Pro Met Val Lys Trp Ser Phe Asp Asp

290 295 300

Lys Phe Cys Ala Arg Leu Gly Pro Gly Ala Ile Ala Val Tyr Glu Thr

305 310 315 320

Glu Lys Asn Phe Gln Leu Leu Gly Gly Lys Val Met Lys Ile Glu Asp

325 330 335

Val Gln Asp Phe Ser Phe Ala Pro Lys Gly Ile Lys Leu Ala Ser Asn

340 345 350

Arg Pro Asn Asp Pro Pro Ser Thr Val Met Val Tyr Trp Thr Pro Glu

355 360 365

Ser Asn Asn Gln Ser Cys Lys Ala Val Leu Ile Glu Leu Pro Asn Arg

370 375 380

Arg Val Leu Arg Thr Ile Asn Leu Val Gln Val Thr Asp Val Ser Phe

385 390 395 400

His Trp Gln Asn Gln Ala Glu Phe Leu Cys Val Gln Val Asp Arg His

405 410 415

Thr Lys Ser Arg Lys Thr Ile Phe Thr Asn Met Glu Ile Cys Ser Leu

420 425 430

Thr Ala Arg Glu Phe Pro Phe Glu Lys Val Glu Ile Lys Asp Arg Cys

435 440 445

Met Arg Phe Ala Trp Glu Pro Asn Ser Asp Arg Phe Val Ile Ile Ser

450 455 460

Arg Ser Glu Asn Val Asn Asp Asn Pro Ala Ile Ala Lys Asn Val Val

465 470 475 480

Ser Phe Tyr Ala Pro Glu Lys Lys Val Asp Lys Lys Gly Val Ile Ile

485 490 495

Asp Lys Glu Leu Ser Ile Phe Lys Lys Trp Lys Leu Val Arg Ala Ile

500 505 510

Asp Gly Lys Phe Ser Asn Glu Ile Thr Trp Ser Pro Ala Gly Arg Phe

515 520 525

Val Cys Val Ala Ala Ile Gly Lys Ile Gly Ser Arg Asn Glu Asn Ile

530 535 540

Asp Phe Tyr Asp Met Asp Tyr Pro Asn Thr Glu Lys Ile Ile Asn Thr

545 550 555 560

Ala Thr Asp Val Asn Ala Thr Leu Arg Asp Val Ala His Ile Asn Tyr

565 570 575

Ala Ser Ala Thr Asp Tyr Glu Trp Asp Pro Ser Gly Arg Tyr Leu Ala

580 585 590

Phe Trp Ser Ser Ala Trp Lys His Lys Ala Glu Asn Gly Tyr Lys Val

595 600 605

Phe Asn Leu Ala Gly Ala Ile Val Arg Glu Glu Leu Ile Thr Asp Phe

610 615 620

Asn Asn Phe Phe Trp Arg Pro Arg Pro Asp Ser Leu Leu Ser Asn Ser

625 630 635 640

Glu Lys Lys Lys Val Arg Lys Asn Leu Lys Glu Trp Ser Ala His Phe

645 650 655

Glu Glu Gln Asp Ala Met Glu Ala Asp Ser Ala Thr Arg Glu Leu Ile

660 665 670

Leu Lys Arg Arg Asn Trp Leu Asp Glu Trp Ser Lys Tyr Arg Glu Ala

675 680 685

Cys Lys Gln Thr Leu Ser Glu Ser Gly Leu Ser Ile Cys Asp Cys Val

690 695 700

Glu Leu Ser Thr Lys Asp Glu Asp Cys Glu Leu Val Glu Glu Ile Arg

705 710 715 720

Glu Thr Val Val Glu Glu Ser Thr Glu Glu Val Pro Phe Phe Glu Glu

725 730 735

<210> 67

<211> 1136

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 168

<400> 67

gatcggaggg gtgaagtgct cgggttggct ttcaggagaa acacggacca actgtatgcg 60

gcctgtgccg attataaaat acgtacgttc gcaattaacc aattttcgca gcctggaggt 120

tctatatggt caccaagata tcwgtcgcag atatttcggc cctgaatatg gagcgctgcg 180

ttacggtcgg gtccagggat aggacctgta tgctgtggaa gattgcagac gaaacacgct 240

tgaccttcag aggcggtgac gatcctgaaa agctgctcag aagatggcag aaggcgaaca 300

gtgaacagga aaacaaggat gcagacgaca atactccagc ggagccgccc gtcttttacg 360

gcgagggaag catagactgc atcaccatgc tcgacgattc acacttcatc tcgggctcgg 420

acaatggaaa catatcgctt tggtccctat ccaagaaaaa gccgctcttc gttcagcgag 480

ttgcccatgg agtgcagcca cagccagata ataccaagat cagcggcgag cgggacccag 540

ctgtgcgtac gcagcaggcc caaggcaacc gtctcgcgca gccgtactgg ataactgccc 600

tgcacgccgt cccctacagc aatgtattct tcagtgggtc ctggaacgga accatgaagg 660

tctggaagct gcacgaaaac atgcgctctt tcgagccact cggcgaactg gatggctgca 720

agggcctggt gacgaagatc cagacggtgg aggccggtaa gagcggcagg gaaacacttc 780

gcgtcctcgc cagtgtcagc aaggagcacc ggcttggtcg gtggatgggc aagcttcccg 840

gcgccagaaa cggactgttt tccgcagtca tcgaccaggc tggcttctga gcaccagtcg 900

cctgaagaac actcgacaca ctgcagcttg cacacacacc gttccaacag tctctctgca 960

agcgagctgc tatcggcatt atgtttgtat ggatacatag acagacatag aagcaataaa 1020

aaaaggcgca ccatggcgct acgaactaat cgcaaaaggg aagcctgctg ggctccacat 1080

gtctagagaa cttcggattt tgcattcgct cgcacctttc gcgctcaaca tagatc 1136

<210> 68

<211> 34

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 168

<400> 68

Arg Gly Glu Val Leu Gly Leu Ala Phe Arg Arg Asn Thr Asp Gln Leu

1 5 10 15

Tyr Ala Ala Cys Ala Asp Tyr Lys Ile Arg Thr Phe Ala Ile Asn Gln

20 25 30

Phe Ser

<210> 69

<211> 239

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 168

<400> 69

Ser Val Ala Asp Ile Ser Ala Leu Asn Met Glu Arg Cys Val Thr Val

1 5 10 15

Gly Ser Arg Asp Arg Thr Cys Met Leu Trp Lys Ile Ala Asp Glu Thr

20 25 30

Arg Leu Thr Phe Arg Gly Gly Asp Asp Pro Glu Lys Leu Leu Arg Arg

35 40 45

Trp Gln Lys Ala Asn Ser Glu Gln Glu Asn Lys Asp Ala Asp Asp Asn

50 55 60

Thr Pro Ala Glu Pro Pro Val Phe Tyr Gly Glu Gly Ser Ile Asp Cys

65 70 75 80

Ile Thr Met Leu Asp Asp Ser His Phe Ile Ser Gly Ser Asp Asn Gly

85 90 95

Asn Ile Ser Leu Trp Ser Leu Ser Lys Lys Lys Pro Leu Phe Val Gln

100 105 110

Arg Val Ala His Gly Val Gln Pro Gln Pro Asp Asn Thr Lys Ile Ser

115 120 125

Gly Glu Arg Asp Pro Ala Val Arg Thr Gln Gln Ala Gln Gly Asn Arg

130 135 140

Leu Ala Gln Pro Tyr Trp Ile Thr Ala Leu His Ala Val Pro Tyr Ser

145 150 155 160

Asn Val Phe Phe Ser Gly Ser Trp Asn Gly Thr Met Lys Val Trp Lys

165 170 175

Leu His Glu Asn Met Arg Ser Phe Glu Pro Leu Gly Glu Leu Asp Gly

180 185 190

Cys Lys Gly Leu Val Thr Lys Ile Gln Thr Val Glu Ala Gly Lys Ser

195 200 205

Gly Arg Glu Thr Leu Arg Val Leu Ala Ser Val Ser Lys Glu His Arg

210 215 220

Leu Gly Arg Trp Met Gly Lys Leu Pro Gly Ala Arg Asn Gly Leu

225 230 235

<210> 70

<211> 2681

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (660)..(2432)

<223>

<220>

<221> misc_feature

<223> Oligo 168

<400> 70

acagccagaa gacattcatg tctggccact cggcctgaac tatgacatgt taaattacat 60

tctaatcaaa ggcaagaatc tgtggcccgg cttccccttg ccgctgcctt cagaaatgga 120

aatcaggctt ccacttctcg ataaaacgca ggtgctgaaa aacgacacat ctcccgacga 180

agaagtggtg atccctcccg cactagcagc agaagaggaa ttcctccgct ctcaggtcct 240

tgcaggactc cttgcagata cactaaagta tgatggagag ttttttggga atgagaatga 300

gatactggcg aacttgaatg gggttcgtga caaatcgttg ctacgcctct ttgcctccgc 360

ttgttcagac cacaacaccg agaaggcgct atcactggtg aaggaattga agcaagataa 420

agcattgaat gccgctcaaa aaatagcaga acgcgctgaa ttattaagac tggtaagcag 480

catcaacgac atcagaaact ctcgtttcga gtcagaattg aacaacttat agcactgaac 540

caacactgta actagtcctg tcgcttagtt aatatagttt tttttgaagg ttcgcggaat 600

cgtttctttt tatatgaaaa ttttgaaggt cctttggaag gcgatgagaa gaaacgccg 659

atg agg aag gct tta tct cca ctg gtt agt agg aac tct cat ata gca 707

Met Arg Lys Ala Leu Ser Pro Leu Val Ser Arg Asn Ser His Ile Ala

1 5 10 15

cgc acc atg gca cgg ggt tcg gat agc gcg tct cgc aag aga act agg 755

Arg Thr Met Ala Arg Gly Ser Asp Ser Ala Ser Arg Lys Arg Thr Arg

20 25 30

cag aag act acg gaa aag aat aat gtt gtg gac gag gaa att tcg ggt 803

Gln Lys Thr Thr Glu Lys Asn Asn Val Val Asp Glu Glu Ile Ser Gly

35 40 45

gtg tca tct aac gag gaa gga tca tcg tca gat gaa caa gaa tta gag 851

Val Ser Ser Asn Glu Glu Gly Ser Ser Ser Asp Glu Gln Glu Leu Glu

50 55 60

gat gta gct gac gct gag ttg gat tct gat gag gaa ttt gcg aat gag 899

Asp Val Ala Asp Ala Glu Leu Asp Ser Asp Glu Glu Phe Ala Asn Glu

65 70 75 80

aac ccc gct gac aag cgg agg cga ctc gcc aaa caa tac cta gaa aat 947

Asn Pro Ala Asp Lys Arg Arg Arg Leu Ala Lys Gln Tyr Leu Glu Asn

85 90 95

att aag gaa gag gct aat gaa att atg cat ggc gat tct gag gca cat 995

Ile Lys Glu Glu Ala Asn Glu Ile Met His Gly Asp Ser Glu Ala His

100 105 110

gca gac gat gcc caa ggt caa ggt ttt gct gat gca tac aat aac ttt 1043

Ala Asp Asp Ala Gln Gly Gln Gly Phe Ala Asp Ala Tyr Asn Asn Phe

115 120 125

gac gcg cgc gac ctg gat cgt gac att atc tca gca aga ttg aag caa 1091

Asp Ala Arg Asp Leu Asp Arg Asp Ile Ile Ser Ala Arg Leu Lys Gln

130 135 140

gac gtt gct gag caa agg gga tct gtg tac cgc tgg atc gcc gat aag 1139

Asp Val Ala Glu Gln Arg Gly Ser Val Tyr Arg Trp Ile Ala Asp Lys

145 150 155 160

ctg cta cta tct gag gcc aag aag tcc ttc acc agg gtc ggg gag aag 1187

Leu Leu Leu Ser Glu Ala Lys Lys Ser Phe Thr Arg Val Gly Glu Lys

165 170 175

aac ctg act gcg ctc agc tgc tat cag caa gca atg aat aaa ttt tca 1235

Asn Leu Thr Ala Leu Ser Cys Tyr Gln Gln Ala Met Asn Lys Phe Ser

180 185 190

cat agg gaa atc caa agc aaa agc aag gga cta atg ttt gcc tat act 1283

His Arg Glu Ile Gln Ser Lys Ser Lys Gly Leu Met Phe Ala Tyr Thr

195 200 205

gtc agt aag gac atg caa ctg acg aaa tac gat atc acc gac ttc aat 1331

Val Ser Lys Asp Met Gln Leu Thr Lys Tyr Asp Ile Thr Asp Phe Asn

210 215 220

gct aga ccg acg aag gta aag tac act aag gga ggg cgc aaa tac atc 1379

Ala Arg Pro Thr Lys Val Lys Tyr Thr Lys Gly Gly Arg Lys Tyr Ile

225 230 235 240

cca gag ggt aac cag ggc ttt cag aac acg acg gag gga cac tat gat 1427

Pro Glu Gly Asn Gln Gly Phe Gln Asn Thr Thr Glu Gly His Tyr Asp

245 250 255

gag att ctg acg gtt gca gct tct cca gat ggt aag tac gtt gtc acg 1475

Glu Ile Leu Thr Val Ala Ala Ser Pro Asp Gly Lys Tyr Val Val Thr

260 265 270

gga ggg aga gac aaa aag ctt atc gtg tgg agc act gag tcg ttg gca 1523

Gly Gly Arg Asp Lys Lys Leu Ile Val Trp Ser Thr Glu Ser Leu Ala

275 280 285

cca gtt aaa gtt ata cca acc aaa gat cgg agg ggt gaa gtg ctc ggg 1571

Pro Val Lys Val Ile Pro Thr Lys Asp Arg Arg Gly Glu Val Leu Gly

290 295 300

ttg gct ttc agg aga aac acg gac caa ctg tat gcg gcc tgt gcc gat 1619

Leu Ala Phe Arg Arg Asn Thr Asp Gln Leu Tyr Ala Ala Cys Ala Asp

305 310 315 320

tat aaa ata cgt acg ttc gca att aac caa ttt tcg cag ctg gag gtt 1667

Tyr Lys Ile Arg Thr Phe Ala Ile Asn Gln Phe Ser Gln Leu Glu Val

325 330 335

cta tat ggt cac caa gat atc gtc gca gat att tcg gcc ctg aat atg 1715

Leu Tyr Gly His Gln Asp Ile Val Ala Asp Ile Ser Ala Leu Asn Met

340 345 350

gag cgc tgc gtt acg gtc ggg tcc agg gat agg acc tgt atg ctg tgg 1763

Glu Arg Cys Val Thr Val Gly Ser Arg Asp Arg Thr Cys Met Leu Trp

355 360 365

aag att gca gac gaa aca cgc ttg acc ttc aga ggc ggt gac gat cct 1811

Lys Ile Ala Asp Glu Thr Arg Leu Thr Phe Arg Gly Gly Asp Asp Pro

370 375 380

gaa aag ctg ctc aga aga tgg cag aag gcg aac agt gaa cag gaa aac 1859

Glu Lys Leu Leu Arg Arg Trp Gln Lys Ala Asn Ser Glu Gln Glu Asn

385 390 395 400

aag gat gca gac gac aat act cca gcg gag ccg ccc gtc ttt tac ggc 1907

Lys Asp Ala Asp Asp Asn Thr Pro Ala Glu Pro Pro Val Phe Tyr Gly

405 410 415

gag gga agc ata gac tgc atc acc atg ctc gac gat tca cac ttc atc 1955

Glu Gly Ser Ile Asp Cys Ile Thr Met Leu Asp Asp Ser His Phe Ile

420 425 430

tcg ggc tcg gac aat gga aac ata tcg ctt tgg tcc cta tcc aag aaa 2003

Ser Gly Ser Asp Asn Gly Asn Ile Ser Leu Trp Ser Leu Ser Lys Lys

435 440 445

aag ccg ctc ttc gtt cag cga gtt gcc cat gga gtg cag cca cag cca 2051

Lys Pro Leu Phe Val Gln Arg Val Ala His Gly Val Gln Pro Gln Pro

450 455 460

gat aat acc aag atc agc ggc gag cgg gac cca gct gtg cgt acg cag 2099

Asp Asn Thr Lys Ile Ser Gly Glu Arg Asp Pro Ala Val Arg Thr Gln

465 470 475 480

cag gcc caa ggc aac cgt ctc gcg cag ccg tac tgg ata act gcc ctg 2147

Gln Ala Gln Gly Asn Arg Leu Ala Gln Pro Tyr Trp Ile Thr Ala Leu

485 490 495

cac gcc gtc ccc tac agc aat gta ttc ttc agt ggg tcc tgg aac gga 2195

His Ala Val Pro Tyr Ser Asn Val Phe Phe Ser Gly Ser Trp Asn Gly

500 505 510

acc atg aag gtc tgg aag ctg cac gaa aac atg cgc tct ttc gag cca 2243

Thr Met Lys Val Trp Lys Leu His Glu Asn Met Arg Ser Phe Glu Pro

515 520 525

ctc ggc gaa ctg gat ggc tgc aag ggc ctg gtg acg aag atc cag acg 2291

Leu Gly Glu Leu Asp Gly Cys Lys Gly Leu Val Thr Lys Ile Gln Thr

530 535 540

gtg gag gcc ggt aag agc ggc agg gaa aca ctt cgc gtc ctc gcc agt 2339

Val Glu Ala Gly Lys Ser Gly Arg Glu Thr Leu Arg Val Leu Ala Ser

545 550 555 560

gtc agc aag gag cac cgg ctt ggt cgg tgg atg ggc aag ctt ccc ggc 2387

Val Ser Lys Glu His Arg Leu Gly Arg Trp Met Gly Lys Leu Pro Gly

565 570 575

gcc aga aac gga ctg ttt tcc gca gtc atc gac cag gct ggc ttc 2432

Ala Arg Asn Gly Leu Phe Ser Ala Val Ile Asp Gln Ala Gly Phe

580 585 590

tgagcaccag tcgcctgaag aacactcgac acactgcagc ttgcacacac accgttccaa 2492

cagtctctct gcaagcgagc tgctatcggc attatgtttg tatggataca tagacagaca 2552

tagaagcaat aaaaaaaggc gcaccatggc gctacgaact aatcgcaaaa gggaagcctg 2612

ctgggctcca catgtctaga gaacttcgga ttttgcattc gctcgcacct ttcgcgctca 2672

acatagatc 2681

<210> 71

<211> 591

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 168

<400> 71

Met Arg Lys Ala Leu Ser Pro Leu Val Ser Arg Asn Ser His Ile Ala

1 5 10 15

Arg Thr Met Ala Arg Gly Ser Asp Ser Ala Ser Arg Lys Arg Thr Arg

20 25 30

Gln Lys Thr Thr Glu Lys Asn Asn Val Val Asp Glu Glu Ile Ser Gly

35 40 45

Val Ser Ser Asn Glu Glu Gly Ser Ser Ser Asp Glu Gln Glu Leu Glu

50 55 60

Asp Val Ala Asp Ala Glu Leu Asp Ser Asp Glu Glu Phe Ala Asn Glu

65 70 75 80

Asn Pro Ala Asp Lys Arg Arg Arg Leu Ala Lys Gln Tyr Leu Glu Asn

85 90 95

Ile Lys Glu Glu Ala Asn Glu Ile Met His Gly Asp Ser Glu Ala His

100 105 110

Ala Asp Asp Ala Gln Gly Gln Gly Phe Ala Asp Ala Tyr Asn Asn Phe

115 120 125

Asp Ala Arg Asp Leu Asp Arg Asp Ile Ile Ser Ala Arg Leu Lys Gln

130 135 140

Asp Val Ala Glu Gln Arg Gly Ser Val Tyr Arg Trp Ile Ala Asp Lys

145 150 155 160

Leu Leu Leu Ser Glu Ala Lys Lys Ser Phe Thr Arg Val Gly Glu Lys

165 170 175

Asn Leu Thr Ala Leu Ser Cys Tyr Gln Gln Ala Met Asn Lys Phe Ser

180 185 190

His Arg Glu Ile Gln Ser Lys Ser Lys Gly Leu Met Phe Ala Tyr Thr

195 200 205

Val Ser Lys Asp Met Gln Leu Thr Lys Tyr Asp Ile Thr Asp Phe Asn

210 215 220

Ala Arg Pro Thr Lys Val Lys Tyr Thr Lys Gly Gly Arg Lys Tyr Ile

225 230 235 240

Pro Glu Gly Asn Gln Gly Phe Gln Asn Thr Thr Glu Gly His Tyr Asp

245 250 255

Glu Ile Leu Thr Val Ala Ala Ser Pro Asp Gly Lys Tyr Val Val Thr

260 265 270

Gly Gly Arg Asp Lys Lys Leu Ile Val Trp Ser Thr Glu Ser Leu Ala

275 280 285

Pro Val Lys Val Ile Pro Thr Lys Asp Arg Arg Gly Glu Val Leu Gly

290 295 300

Leu Ala Phe Arg Arg Asn Thr Asp Gln Leu Tyr Ala Ala Cys Ala Asp

305 310 315 320

Tyr Lys Ile Arg Thr Phe Ala Ile Asn Gln Phe Ser Gln Leu Glu Val

325 330 335

Leu Tyr Gly His Gln Asp Ile Val Ala Asp Ile Ser Ala Leu Asn Met

340 345 350

Glu Arg Cys Val Thr Val Gly Ser Arg Asp Arg Thr Cys Met Leu Trp

355 360 365

Lys Ile Ala Asp Glu Thr Arg Leu Thr Phe Arg Gly Gly Asp Asp Pro

370 375 380

Glu Lys Leu Leu Arg Arg Trp Gln Lys Ala Asn Ser Glu Gln Glu Asn

385 390 395 400

Lys Asp Ala Asp Asp Asn Thr Pro Ala Glu Pro Pro Val Phe Tyr Gly

405 410 415

Glu Gly Ser Ile Asp Cys Ile Thr Met Leu Asp Asp Ser His Phe Ile

420 425 430

Ser Gly Ser Asp Asn Gly Asn Ile Ser Leu Trp Ser Leu Ser Lys Lys

435 440 445

Lys Pro Leu Phe Val Gln Arg Val Ala His Gly Val Gln Pro Gln Pro

450 455 460

Asp Asn Thr Lys Ile Ser Gly Glu Arg Asp Pro Ala Val Arg Thr Gln

465 470 475 480

Gln Ala Gln Gly Asn Arg Leu Ala Gln Pro Tyr Trp Ile Thr Ala Leu

485 490 495

His Ala Val Pro Tyr Ser Asn Val Phe Phe Ser Gly Ser Trp Asn Gly

500 505 510

Thr Met Lys Val Trp Lys Leu His Glu Asn Met Arg Ser Phe Glu Pro

515 520 525

Leu Gly Glu Leu Asp Gly Cys Lys Gly Leu Val Thr Lys Ile Gln Thr

530 535 540

Val Glu Ala Gly Lys Ser Gly Arg Glu Thr Leu Arg Val Leu Ala Ser

545 550 555 560

Val Ser Lys Glu His Arg Leu Gly Arg Trp Met Gly Lys Leu Pro Gly

565 570 575

Ala Arg Asn Gly Leu Phe Ser Ala Val Ile Asp Gln Ala Gly Phe

580 585 590

<210> 72

<211> 510

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 160

<400> 72

gatctgcgtg ggcctcgctg ggcgggcggc gggcttcaac aacatgtggg ccggggtaga 60

gagcaaagtc ggttagagga tataaacgta ggtaggggca gcagcctcar gcaggcctcc 120

gaggccttgg atctcttggc tagccttggc ctgggccttg gggcccatga caccagccac 180

cccagtgctt tctggattcg tcgtacttct cgatgaagtt ggcgttgacg gcggagacga 240

gcttggcaag ggcagcctcg tcctcggcgc ggacctcggt gagggcggcg acggcggagg 300

tcttctggtt gaccagggtg cctaggcggg ccttgccctt gacgatggcg tatgggacgc 360

ccatcttctt gcacagcgca ggcaggaaga tgacaagctc gatggggtcg acgtcgttgg 420

cgatgagcac gagcttggcc ttcttgttct cgacgaggga gacgacgtgg ttcaagccga 480

acttgacggc gtatggctta agcgaagcct 510

<210> 73

<211> 111

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 160

<400> 73

Ala Ser Leu Lys Pro Tyr Ala Val Lys Phe Gly Leu Asn His Val Val

1 5 10 15

Ser Leu Val Glu Asn Lys Lys Ala Lys Leu Val Leu Ile Ala Asn Asp

20 25 30

Val Asp Pro Ile Glu Leu Val Ile Phe Leu Pro Ala Leu Cys Lys Lys

35 40 45

Met Gly Val Pro Tyr Ala Ile Val Lys Gly Lys Ala Arg Leu Gly Thr

50 55 60

Leu Val Asn Gln Lys Thr Ser Ala Val Ala Ala Leu Thr Glu Val Arg

65 70 75 80

Ala Glu Asp Glu Ala Ala Leu Ala Lys Leu Val Ser Ala Val Asn Ala

85 90 95

Asn Phe Ile Glu Lys Tyr Asp Glu Ser Arg Lys His Trp Gly Gly

100 105 110

<210> 74

<211> 1437

<212> DNA

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 18

<400> 74

gatcagtgaa ttttcatcat tatggccaaa actgcgtctg gggctttaat cgaagtcatc 60

ttcatcataa tcatcaccat aagcccgata ttgcaagtcc ctgtcaacgc caccatagct 120

gcgctcatac agcagatcct cagcgccgag atcaagcctt gctcatctga gtgagatagt 180

tgaattcaga ttcctcatca ttgttgcgcc tagtttcaat tttccgggcc tgctcgcgac 240

tctgctcata attaggcacg cgcctacgag tctgaccccc attgccacca aagtcgcgcc 300

tacgctgacc cgagccgtac tcgtcgcggc cactgcgtct gcgttcttgc cccgatccac 360

caaagtcgcg cctacgctga ccccagccgt actcgtcgcg gccactgcgt ctgcgttctt 420

gccccgatcc accaaagtcg tgctgacgtt cttgccccta actgggattg ccaccatcat 480

aaggctttgg ctctgcaccc tctatttcct cttgcaaagc cggctccaaa gcaaggatag 540

agcttgcacc tgttaacaaa caaataatac tggataattt tatcttgctg atggttatta 600

ggctattcaa tgtagaaacc tattccacaa ataacataga gtgcttttat atgccacatt 660

tcatatcctt ccgcaagctt gatggcaaaa tgctgagtca taataaggat agagtaaagt 720

ggcccatctg atacactctt aatgacttcg catttttagt aactttgccc aggcatatca 780

tatattctac cccttcataa aagttatgta acgaataatt tgaaggagcc tattatttcc 840

tggaagagta taaccgtctt tttattccaa gtatccaaaa catcactata ggaacatata 900

ctaaaaagtg ggaacgttcc gatattcaca taactactgt tgttttagat caaaaaattg 960

tctattttat tggcgttgga tggatccttt gtatcacgaa atacgcaaat gttggaattt 1020

ttaactttgg aatacctatt gaagcaacat atgtcatttt acaaggagca tcactttaca 1080

ggcaccgaga aagaaagaaa ttgacaaaaa agaagggata agtgaaggca gttgcacagg 1140

caccgaccaa gacagtgatt tacaatagtt acaggtcaat aataagaaag acaactatgt 1200

ggcattatgc agtacgctct aagggcgcag ttccgatgat tgtttcaccc agctacacca 1260

gtagctcgca ctgccattta tttacagtgc ttgtgtcaca gacccaaaat ggataagtgt 1320

gagagcgact gtcgcttctg tatagctcaa cgccaatgtg tgtctgcact gggacaacac 1380

cggatttagt acattgacac caacyttcgc cgcctgctga cagatgcagc wccgatc 1437

<210> 75

<211> 1437

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (958)..(1272)

<223>

<220>

<221> misc_feature

<223> Oligo 18

<400> 75

gatcggwgct gcatctgtca gcaggcggcg aargttggtg tcaatgtact aaatccggtg 60

ttgtcccagt gcagacacac attggcgttg agctatacag aagcgacagt cgctctcaca 120

cttatccatt ttgggtctgt gacacaagca ctgtaaataa atggcagtgc gagctactgg 180

tgtagctggg tgaaacaatc atcggaactg cgcccttaga gcgtactgca taatgccaca 240

tagttgtctt tcttattatt gacctgtaac tattgtaaat cactgtcttg gtcggtgcct 300

gtgcaactgc cttcacttat cccttctttt ttgtcaattt ctttctttct cggtgcctgt 360

aaagtgatgc tccttgtaaa atgacatatg ttgcttcaat aggtattcca aagttaaaaa 420

ttccaacatt tgcgtatttc gtgatacaaa ggatccatcc aacgccaata aaatagacaa 480

ttttttgatc taaaacaaca gtagttatgt gaatatcgga acgttcccac tttttagtat 540

atgttcctat agtgatgttt tggatacttg gaataaaaag acggttatac tcttccagga 600

aataataggc tccttcaaat tattcgttac ataactttta tgaaggggta gaatatatga 660

tatgcctggg caaagttact aaaaatgcga agtcattaag agtgtatcag atgggccact 720

ttactctatc cttattatga ctcagcattt tgccatcaag cttgcggaag gatatgaaat 780

gtggcatata aaagcactct atgttatttg tggaataggt ttctacattg aatagcctaa 840

taaccatcag caagataaaa ttatccagta ttatttgttt gttaacaggt gcaagctcta 900

tccttgcttt ggagccggct ttgcaagagg aaatagaggg tgcagagcca aagcctt 957

atg atg gtg gca atc cca gtt agg ggc aag aac gtc agc acg act ttg 1005

Met Met Val Ala Ile Pro Val Arg Gly Lys Asn Val Ser Thr Thr Leu

1 5 10 15

gtg gat cgg ggc aag aac gca gac gca gtg gcc gcg acg agt acg gct 1053

Val Asp Arg Gly Lys Asn Ala Asp Ala Val Ala Ala Thr Ser Thr Ala

20 25 30

ggg gtc agc gta ggc gcg act ttg gtg gat cgg ggc aag aac gca gac 1101

Gly Val Ser Val Gly Ala Thr Leu Val Asp Arg Gly Lys Asn Ala Asp

35 40 45

gca gtg gcc gcg acg agt acg gct cgg gtc agc gta ggc gcg act ttg 1149

Ala Val Ala Ala Thr Ser Thr Ala Arg Val Ser Val Gly Ala Thr Leu

50 55 60

gtg gca atg ggg gtc aga ctc gta ggc gcg tgc cta att atg agc aga 1197

Val Ala Met Gly Val Arg Leu Val Gly Ala Cys Leu Ile Met Ser Arg

65 70 75 80

gtc gcg agc agg ccc gga aaa ttg aaa cta ggc gca aca atg atg agg 1245

Val Ala Ser Arg Pro Gly Lys Leu Lys Leu Gly Ala Thr Met Met Arg

85 90 95

aat ctg aat tca act atc tca ctc aga tgagcaaggc ttgatctcgg 1292

Asn Leu Asn Ser Thr Ile Ser Leu Arg

100 105

cgctgaggat ctgctgtatg agcgcagcta tggtggcgtt gacagggact tgcaatatcg 1352

ggcttatggt gatgattatg atgaagatga cttcgattaa agccccagac gcagttttgg 1412

ccataatgat gaaaattcac tgatc 1437

<210> 76

<211> 105

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 18

<400> 76

Met Met Val Ala Ile Pro Val Arg Gly Lys Asn Val Ser Thr Thr Leu

1 5 10 15

Val Asp Arg Gly Lys Asn Ala Asp Ala Val Ala Ala Thr Ser Thr Ala

20 25 30

Gly Val Ser Val Gly Ala Thr Leu Val Asp Arg Gly Lys Asn Ala Asp

35 40 45

Ala Val Ala Ala Thr Ser Thr Ala Arg Val Ser Val Gly Ala Thr Leu

50 55 60

Val Ala Met Gly Val Arg Leu Val Gly Ala Cys Leu Ile Met Ser Arg

65 70 75 80

Val Ala Ser Arg Pro Gly Lys Leu Lys Leu Gly Ala Thr Met Met Arg

85 90 95

Asn Leu Asn Ser Thr Ile Ser Leu Arg

100 105

<210> 77

<211> 2602

<212> DNA

<213> Ashbya gossypii

<220>

<221> CDS

<222> (1531)..(1845)

<223>

<220>

<221> misc_feature

<223> Oligo 18

<400> 77

tcagcggtgt ggtgtatggg tctctcagcg gtgtggtgta tgggtctctc agcggtgtgg 60

tgtatgggtc tctcagcggt gtggtgtatg gtctctcagc ggtgtggtgt atgggtctct 120

cagcggtgtg gtgtatgggt ctctcagcgg tgtggtgtat gggtctctca gcggtgtggt 180

gtatgggtct ctcagcggtg tggtgtatgg gtctctcagc ggtgtggtgt atcagagggt 240

tttaaaccta aatctgacgc cttaaccact cggccaaact ctcttatatg cttttatatg 300

aaaggcgtgc aatacgtacg agcaggttgg ccacgtgaac tggctgccca gggtcagagc 360

tgtatccgcg agtatcccaa aacccgtaca gtacgaaata cccacaccag tttcgcggta 420

atgtcagtta gcactcgcca cggtgatgac tgaactcgcg gctggcacga tcaacaacgt 480

agggcccgag ccttcagcag aggaggatcg tattattcat ataaaatgct cacacaagca 540

aacatggagg ccaggaaggg caccgtggtc ggtgatcggt gctgcatctg tcagcaggcg 600

gcgaaggttg gtgtcaatgt actaaatccg gtgttgtccc agtgcagaca cacattggcg 660

ttgagctata cagaagcgac agtcgctctc acacttatcc attttgggtc tgtgacacaa 720

gcactgtaaa taaatggcag tgcgagctac tggtgtagct gggtgaaaca atcatcggaa 780

ctgcgccctt agagcgtact gcataatgcc acatagttgt ctttcttatt attgacctgt 840

aactattgta aatcactgtc ttggtcggtg cctgtgcaac tgccttcact tatcccttct 900

tttttgtcaa tttctttctt tctcggtgcc tgtaaagtga tgctccttgt aaaatgacat 960

atgttgcttc aataggtatt ccaaagttaa aaattccaac atttgcgtat ttcgtgatac 1020

aaaggatcca tccaacgcca ataaaataga caattttttg atctaaaaca acagtagtta 1080

tgtgaatatc ggaacgttcc cactttttag tatatgttcc tatagtgatg ttttggatac 1140

ttggaataaa aagacggtta tactcttcca ggaaataata ggctccttca aattattcgt 1200

tacataactt ttatgaaggg gtagaatata tgatatgcct gggcaaagtt actaaaaatg 1260

cgaagtcatt aagagtgtat cagatgggcc actttactct atccttatta tgactcagca 1320

ttttgccatc aagcttgcgg aaggatatga aatgtggcat ataaaagcac tctatgttat 1380

ttgtggaata ggtttctaca ttgaatagcc taataaccat cagcaagata aaattatcca 1440

gtattatttg tttgttaaca ggtgcaagct ctatccttgc tttggagccg gctttgcaag 1500

aggaaataga gggtgcagag ccaaagcctt atg atg gtg gca atc cca gtt agg 1554

Met Met Val Ala Ile Pro Val Arg

1 5

ggc aag aac gtc agc acg act ttg gtg gat cgg ggc aag aac gca gac 1602

Gly Lys Asn Val Ser Thr Thr Leu Val Asp Arg Gly Lys Asn Ala Asp

10 15 20

gca gtg gcc gcg acg agt acg gct ggg gtc agc gta ggc gcg act ttg 1650

Ala Val Ala Ala Thr Ser Thr Ala Gly Val Ser Val Gly Ala Thr Leu

25 30 35 40

gtg gat cgg ggc aag aac gca gac gca gtg gcc gcg acg agt acg gct 1698

Val Asp Arg Gly Lys Asn Ala Asp Ala Val Ala Ala Thr Ser Thr Ala

45 50 55

cgg gtc agc gta ggc gcg act ttg gtg gca atg ggg gtc aga ctc gta 1746

Arg Val Ser Val Gly Ala Thr Leu Val Ala Met Gly Val Arg Leu Val

60 65 70

ggc gcg tgc cta att atg agc aga gtc gcg agc agg ccc gga aaa ttg 1794

Gly Ala Cys Leu Ile Met Ser Arg Val Ala Ser Arg Pro Gly Lys Leu

75 80 85

aaa cta ggc gca aca atg atg agg aat ctg aat tca act atc tca ctc 1842

Lys Leu Gly Ala Thr Met Met Arg Asn Leu Asn Ser Thr Ile Ser Leu

90 95 100

aga tgagcaaggc ttgatctcgg cgctgaggat ctgctgtatg agcgcagcta 1895

Arg

105

tggtggcgtt gacagggact tgcaatatcg ggcttatggt gatgattatg atgaagatga 1955

cttcgattaa agccccagac gcagttttgg ccataatgat gaaaattcac tgatcagact 2015

ataaagttgc tttggaaaat aagtcatatt ggcgatggac tcgtggcaag acacttagat 2075

acttgaattt cttcagtatt aaatgacatg acaactatcc acagtgaatt ctgaaaaata 2135

aacataaaaa caataatctt atgacccatt ttcatgtaaa ctgaatatag acgatagcaa 2195

tacattttct ctatattttg tactctggaa gcttctgcga gtgactacca tttgatggcc 2255

acgtttacct ccttcgtcag caggtatcca ggtacttaaa ttaatcactt cacagcacta 2315

aatgtggctt ttattcggtt ttattccaag aatacggatc tctgagacac ttgagcccct 2375

tagtttagtt acaacagtag cttccgaaag ctacgaataa tagttataga ttttctgctc 2435

tcacgtgatt cgcttacata aataacccga taaaaccatg agaaatatgt ccatgtagct 2495

gtgtgacgta tatatgggat aaggctgggt aggcctctag ccatctattt gcccgctgtc 2555

ttttggtaca atagcccaac gaaaatccga gcgtacctct acgtgtg 2602

<210> 78

<211> 105

<212> PRT

<213> Ashbya gossypii

<220>

<221> misc_feature

<223> Oligo 18

<400> 78

Met Met Val Ala Ile Pro Val Arg Gly Lys Asn Val Ser Thr Thr Leu

1 5 10 15

Val Asp Arg Gly Lys Asn Ala Asp Ala Val Ala Ala Thr Ser Thr Ala

20 25 30

Gly Val Ser Val Gly Ala Thr Leu Val Asp Arg Gly Lys Asn Ala Asp

35 40 45

Ala Val Ala Ala Thr Ser Thr Ala Arg Val Ser Val Gly Ala Thr Leu

50 55 60

Val Ala Met Gly Val Arg Leu Val Gly Ala Cys Leu Ile Met Ser Arg

65 70 75 80

Val Ala Ser Arg Pro Gly Lys Leu Lys Leu Gly Ala Thr Met Met Arg

85 90 95

Asn Leu Asn Ser Thr Ile Ser Leu Arg

100 105

Claims (24)

1. A polynucleotide isolatable from Ashbya gossypii encoding a protein involved in transcription, RNA processing and/or translation in Ashbya gossypii.

2. The polynucleotide of claim 1, which is associated with transcription, RNA processing and/or translation of Ashbya gossypii and has the structural and/or functional properties shown in Table 1.

3. Comprises the amino acid sequence of SEQ ID NO: 1, 6, 12, 17, 21, 26, 31, 38, 42, 48, 53, 58, 63, 67, 72 or 74, and preferably a polynucleotide of claim 1 or 2 isolated from Ashbya gossypii; a complementary polynucleotide thereof; and sequences derived from these polynucleotides by the degeneracy of the genetic code.

4. The polynucleotide of claim 3, comprising the nucleotide sequence of SEQ ID NO: 4, 10, 14, 19, 24, 29, 36, 40, 46, 51, 56, 60, 65, 70, 75, or 77 or a fragment thereof.

5. An oligonucleotide which is hybridizable, and in particular hybridizes under stringent conditions, to a polynucleotide of any preceding claim.

6. A polynucleotide which can hybridize, in particular under stringent conditions, with an oligonucleotide according to claim 5 and which codes for a gene product of a microorganism of the genus Ashbya or a functional equivalent of said gene product.

7. A polypeptide encoded by a polynucleotide comprising a nucleic acid sequence according to any one of claims 1 to 4 or a fragment thereof, or by a polynucleotide according to claim 6, and functional equivalents thereof, in particular having the activity as claimed in claim 2; or has a sequence comprising SEQ ID NO: 2, 3, 5, 7, 8, 9, 11, 13, 15, 16, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 34, 35, 37, 39, 41, 43, 44, 45, 47, 49, 50, 52, 54, 55, 57, 59, 61, 62, 64, 66, 68, 69, 71, 73, 76, or SEQ ID NO: 78 of at least 10 contiguous amino acid residues.

8. An expression cassette comprising a nucleic acid sequence according to any one of claims 1 to 6, wherein the nucleic acid sequence is operably linked to at least one regulatory nucleic acid sequence.

9. A recombinant vector comprising at least one expression cassette of claim 8.

10. A prokaryotic or eukaryotic host transformed with at least one vector according to claim 9.

11. A prokaryotic or eukaryotic host in which the functional expression of at least one gene encoding the polypeptide of claim 7 is modulated; or the biological activity of the polypeptide of claim 7 is reduced or increased.

12. The host of claim 10 or 11, which is from the genus Ashbya.

13. Use of an expression cassette according to claim 8, a vector according to claim 9 or a host according to any one of claims 10 to 12 for the microbial production of vitamin B2 and/or a precursor and/or derivative thereof.

14. Use of an expression cassette according to claim 8, a vector according to claim 9 or a host according to any one of claims 10 to 12 in the recombinant production of a polypeptide according to claim 7.

15. Method for the detection of an effector target which can be used for the modulation of the microbial production of vitamin B2 and/or precursors thereof and/or derivatives thereof, wherein a microorganism capable of producing vitamin B2 and/or precursors thereof and/or derivatives thereof microbiologically is treated with an effector which can interact with, in particular bind to, a target selected from the group consisting of the polypeptide of claim 7 or a nucleic acid coding sequence thereof; verifying the effect of the effector on the yield of vitamin B2 and/or a precursor and/or derivative thereof produced by a microbiological method; and isolating the target as appropriate.

16. A method for modulating microbial production of vitamin B2 and/or precursors thereof and/or derivatives thereof, wherein a microorganism capable of microbiologically producing vitamin B2 and/or precursors thereof and/or derivatives thereof is treated with an effector that can interact with a target selected from the group consisting of the polypeptide of claim 7 or nucleic acid encoding sequence thereof.

17. An effector against a target selected from the polypeptide of claim 7 or a nucleic acid encoding sequence thereof, wherein said effector is selected from the group consisting of:

a) an antibody or antigen-binding fragment thereof;

b) a polypeptide ligand other than a) that can interact with the polypeptide of claim 7;

c) a low molecular weight effector that can modulate the biological activity of the polypeptide of claim 7;

d) an antisense nucleic acid sequence.

18. A process for the microbial production of vitamin B2 and/or a precursor and/or a derivative thereof, wherein a host according to any one of claims 10 to 12 is cultured under conditions conducive to the production of vitamin B2 and/or a precursor and/or a derivative thereof, and the desired product is isolated from the culture mixture.

19. The method of claim 18, wherein the host is treated with the effector of claim 17 before and/or during culturing.

20. The method of claim 18 or 19, wherein the host is selected from a microorganism of the genus Ashbya.

21. The method of any one of claims 18 to 20, wherein the microorganism is a host of any one of claims 10 to 12.

22. Use of a polynucleotide according to any one of claims 1 to 4 and 6 or a polypeptide according to claim 7 as a target for modulating the production of vitamin B2 and/or a precursor and/or a derivative thereof in a microorganism of the genus ashbya.

23. Use of a polynucleotide according to any one of claims 1 to 4 and 6 or a polypeptide according to claim 7 in the microbial production of vitamin B2 and/or a precursor and/or derivative thereof, for use as a target in modulating transcription, RNA processing and/or translation of a microorganism of the genus ashbya during culture.

24. The host of claim 13, which has an enhanced adaptation to environmental and metabolic conditions, in particular has altered transcription, RNA processing and/or translation for improved production of vitamin B2.

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