Amino polyol amine oxidase polynucleotides and related polypeptides and methods of use

This application is a continuation of U.S. application Ser. No. 09/352,159, filed Jul. 12, 1999, now U.S. Pat. No. 6,211,434, which claims benefit to of Ser. No. 60/135,391, filed May 21, 1999 and Ser. No. 60/092,936, filed Jul. 15, 1998. This application is also a continuation of Ser. No. 09/352,168, filed Jul. 12, 1999, now U.S. Pat. No. 6, 211,435, all of which are hereby incorporated by reference.

The present invention relates generally to the detection and isolation of fumonisin and AP1 degrading enzymes and to compositions and methods for the in vivo detoxification or degradation of fumonisin or its hydrolysis product AP1. This method has broad application in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.

Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants.

These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop losses, is interference with the mechanism by which the pathogen causes injury to the host crop plant. Still another method, in the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, is interference with toxin production, storage, or activity. This invention falls into the latter two categories.

Since their discovery and structural elucidation in 1988 (Bezuidenhout et al., Journal Chem Soc, Chem Commun 1988: 743-745 (1988)), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are linked to several animal toxicoses including leukoencephalomalacia (Marasas, et al, Onderstepoort Journal of Veterinary Research 55: 197-204 (1988); Wilson, et at, American Association of Veterinary Laboratory Diagnosticians: Abstracts 33 rd Annual Meeting , Denver, Colo., Oct. 7-9, 1990, Madison, Wis., USA) and porcine pulmonary edema (Colvin, et al., Mycopathologia 117: 79-82 (1992)). Fumonisins are also suspected carcinogens (Geary W (1971) Coord Chem Rev 7: 81; Gelderblom, et al., Carcinogenesis 12: 1247-1251 (1991); Gelderblom, et al, Carcinogenesis 13: 433-437 (1992)). Fusarium isolates in section Liseola produce fumonisins in culture at levels from 2 to >4000 ppm (Leslie, et al., Phytopathology 82: 341-345 (1992)). Isolates from maize (predominantly mating population A) are among the highest producers of fumonisin. (Leslie et al., supra). Fumonisin levels detected in field-grown maize have fluctuated widely depending on location and growing season, but both preharvest and postharvest surveys of field maize have indicated that the potential for high levels of fumonisins exists (Murphy, et al., J Agr Food Chem 41: 263-266 (1993)). Surveys of food and feed products have also detected fumonisin (Holcomb, et al., J Agr Food Chem 41: 764-767 (1993); Hopmans, et al., J Agr Food Chem 41: 1655-1658 (1993); Sydenham, et al., J Agr Food Chem 39: 2014-2018 (1991)). The etiology of Fusarium ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson, Mycopathologia 117: 29-36 (1992)). Fusarium can be isolated from most field grown maize, even when no visible mold is present. The relationship between seedling infection and stalk and ear diseases caused by Fusarium is not clear. Genetic resistance to visible kernel mold has been identified (Gendloff, et al., Phytopathology 76: 684-688 (1986); Holley, et al., Plant Dis 73: 578-580 (1989)), but the relationship to visible mold to fumonisin production has yet to be elucidated.

Fumonisins have been shown in in vitro mammalian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphingosine N-acetyl transferase, resulting in the accumulation of the precursor sphinganine (Norred, et al., Mycopathologia 117: 73-78 (1992); Wang, et al., Biol Chem 266: 14486 (1991); Yoo, et al., Toxicol Appl Pharmacol 114: 9-15 (1992); Nelson, et al., Annu Rev Phytpathol 31:233-252 (1993)). It is likely that inhibition of this pathway accounts for at least some of fumonisin's toxicity, and support for this comes from measures of sphinganine: sphingosine ratios in animals fed purified fumonisin (Wang, et al., J Nutr 122: 1706-1716 (1992)). Fumonisins also affect plant cell growth (Abbas, et al., Weed Technol 6: 548-552 (1992); Vanasch, et al., Phytopathology 82: 1330-1332 (1992); Vesonder, et al., Arch Environ Contam Toxicol 23: 464-467 (1992)). Kuti et al., (Abstract, Annual Meeting American Phytopathological Society, Memphis, Tenn.: APS Press 1993) reported on the ability of exogenously added fumonisins to accelerate disease development and increase sporulation of Fusarium moniliforme and Fusarium oxysporum on tomato.

Enzymes that degrade the fungal toxin fumonisin to its de-esterified form (e.g. AP1 from FB1) have been identified in U.S. Pat. No. 5,716,820, issued Feb. 10, 1998, U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997, and all hereby incorporated by reference. It is understood that AP1 as used here designates the hydrolyzed form of any fumonisin, FB1, FB2, FB3, FB4, or any other AP1-like compounds, including synthetically produced AP1 like compounds, that contain a C-2 or C-1 amine group and one or more adjacent hydroxyl groups. Plants expressing a fumonisin esterase enzyme, infected by fumonisin producing fungus, and tested for fumonisin and AP1 were found to have low levels of fumonisin but high levels of AP1. AP1 is less toxic than fumonisins to plants and probably also to animals but contamination with AP1 is still a concern (Lamprecht, et al., Phytopathology , 84:383-391 (1991)). The preferred result would be complete detoxification of fumonisin to a non-toxic form. Therefore enzymes capable of degrading AP1 are necessary for the further detoxification of fumonisin.

The present invention provides newly discovered polynucleotides and related polypeptides of amino polyol amine oxidase (abbreviated APAO, formerly known as AP1 catabolase, U.S. Pat. No. 5,716,820, supra, U.S. Pat. No. 5,792,931 supra; pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, supra; trAPAO is the abbreviation for a truncated, but still functional APAO), capable of oxidatively deaminating the AP1 to a compound identified as the 2-oxo derivative of AP1 or its cyclic ketal form (abbreviated as 2-OP, formerly called AP1-N1, U.S. Pat. No. 5,716,820, and U.S. Pat. No. 5,792,931 supra; pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, supra), isolated from Exophiala spinifera , ATCC 74269. The partially purified APAO enzyme from Exophiala spinifera has little or no activity on intact FB1, a form of fumonisin. However, recombinant APAO enzyme from Exophiala spinifera , expressed in E. coli , has significant but reduced activity on intact FB1 and other B-series fumonisins. APAO or trAPAO thus could potentially be used without fumonisin esterase since the amine group is the major target for detoxification. Alternatively, fumonisin esterase and APAO (or trAPAO) can be used together for degrading toxins.

APAO is a type of flavin amine oxidase (EC 1.4.3.4, enzyme class nomeclature, see Enzyme Nomenclature 1992, Recommendations of the Nomenclature Committee of the IUBMB on the Nomenclature and Classification of Enzymes, Academic Press, Inc. (1992)). Flavin amine oxidases are known in mammals as monoamine oxidases, where they participate in the conversion of amines involved in neuronal function. A prokaryotic flavin amine oxidase that deaminates putrescine has been described (Ishizuka et al., J. Gen Microbiol . 139:425-432 (1993)). A single fungal gene, from Aspergillus niger has been cloned (Schilling et al., Mol Gen Genet . 247:430-438 (1995)). It deaminates a variety of alkyl and aryl amines, but when tested for its ability to oxidize AP1, was found to not contain AP1 oxidizing activity.

The toxicity of fumonisins and their potential widespread occurrence in food and feed makes it imperative to find detoxification or elimination strategies to remove the compound from the food chain.

The present invention provides polynucleotides, related polypeptides, and all conservatively modified variants of newly discovered APAOs. The nucleotide sequences of the APAOs comprise the sequence shown in SEQ ID NOS: 35, 37, 39, 41, 43, and 45. For expression in a plant, the polynuclcotide of the present invention can be operably linked to a targeting sequence. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention.

Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide comprising at least 20 contiguous bases of the polynucleotides of the present invention; (c) a polynucleotide having at least 50% sequence identity to the polynucleotides of the present invention; (d) a polynucleotide comprising at least 25 nucleotide in length which hybridizes under low stringency conditions to the polynucleotides of the present invention; and (e) a polynucleotide complementary to a polynucleotide of (a) through (e). The isolated nucleic acid can be DNA. The isolated nucleic acid can also be RNA.

In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.

In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.

In yet another aspect, the present invention relates to a transgenic plant or plant cell comprising a recombinant expression cassette with a promoter operably linked to any of the isolated nucleic acids of the present invention. Preferred plants containing the recombinant expression cassette of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, tomato, and millet. The present invention also provides transgenic seed from the transgenic plant.

In another aspect, the present invention relates to an isolated protein selected from the group consisting of (a) a polypeptide comprising at least 25 contiguous amino acids of SEQ ID NOS: 36, 38, 40, 42, 44, and 46; (b) a polypeptide comprising at least 55% sequence identity to SEQ ID NO: 36, 38, 40, 42, 44, and 46; (c) a polypeptide encoded by a nucleic acid of the present invention; (d) a polypeptide characterized by SEQ ID NO: 36, 38, 40, 42, 44, and 46; and (e) a conservatively modified variant of SEQ ID NO: 36, 38, 40, 42, 44, and 46.

Preferred embodiments of the subject invention include a host cell stably transformed by a polynucleotide construct as described above, and a method of producing a polypeptide of a recombinant gene comprising expressing a polynucleotide of the present invention in a recombinantly engineered cell and purifying the resulting polypeptide.

A number of expression systems using the said host cells could be used, such as but not limited to, microbial, bacterial, mammalian, insect, plant cells, yeast, or virus. In one embodiment the fumonisin degrading enzymes can be isolated and purified from the seeds or plant parts of a plant expressing the said enzyme.

Another embodiment of the subject invention comprises a method of reducing pathogenicity of a fungus producing fumonisin by transferring to a plant the nucleic acids of the present invention either by themselves or in combination with a nucleic acid coding for a fumonisin esterase.

This invention further provides methods of degrading fumonisin, a fumonisin degradative product, or a structurally related mycotoxin, comprising the step of reacting the mycotoxin with the degradative enzymes of the present invention. Additionally, fumonisins can be degraded to a less toxic form by application of both fumonisin esterase enzymes and APAO enzyme. Mycotoxins can be degraded in harvested grain, during the processing of harvested grain, in animal feed, or in plant tissue as, for example, during the use of the plant for silage or as a spray on grain, fruit or vegetables.

The polynucleotides of the present invention can also be used as a selectable marker for plant transformation. By transforming plant cells with an expression cassette containing the polynucleotide of the present invention and then placing the plant cells on media containing FB1, AP1 or a phytotoxic analog, only the plant cells expressing the polynucleotide of the present invention would survive.

Another embodiment of the present invention is the use of the enzyme fumonisin esterase and APAO by themselves or in combination as reagents for detecting fumonisin and structurally related toxins.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., J. H. Langenheim and K. V. Thimann, Botany: Plant Biology and Its Relation to Human Affairs (1982) John Wiley; Cell Culture and Somatic Cell Genetics of Plants , Vol. 1 (I. K. Vasil, ed. 1984); R. V. Stanier, J. L. Ingraham, M. L. Wheelis, and P. R. Painter, The Microbial World , (1986) 5th Ed., Prentice-Hall; O. D. Dhringra and J. B. Sinclair, Basic Plant Pathology Methods , (1985) CRC Press; Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning , Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); and the series Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.).

Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic and prokaryotic microorganisms), such as fungi, yeast, bacteria, actinomycetes, algae and protozoa, as well as other unicellular structures.

A “fumonisin-producing microbe” is any microbe capable of producing the mycotoxin fumonisin or analogs thereof. Such microbes are generally members of the fungal genus Fusarium, as well as recombinantly derived organisms, which have been genetically altered to enable them to produce fumonisin or analogs thereof

By “degrading fumonisin” is meant any modification to fumonisin, AP1, or any derivative of fumonisin or AP1 which causes a decrease or loss in its toxic activity, such as degradation to less than 1%, 5%, 10%, or 50% of original toxicity, with less than 10% being preferred. Such a change can comprise cleavage of any of the various bonds, oxidation, reduction, the addition or deletion of a chemical moiety, or any other change that affects the activity of the molecule. In a preferred embodiment, the modification includes hydrolysis of the ester linkage in the molecule as a first step and then oxidative deamination. Furthermore, chemically altered fumonisin can be isolated from cultures of microbes that produce an enzyme of this invention, such as growing the organisms on media containing radioactively-labeled fumonisin, tracing the label, and isolating the degraded toxin for further study. The degraded fumonisin can be compared to the active compound for its phytotoxicity or mammalian toxicity in known sensitive species, such as porcines, rabbits, and equines or in cell or tissue culture assays. Such toxicity assays are known in the art. For example, in plants a whole leaf bioassay can be used in which solutions of the active and inactive compound are applied to the leaves of sensitive plants. The leaves may be treated in situ or, alternatively, excised leaves may be used. The relative toxicity of the compounds can be estimated by grading the ensuing damage to the plant tissues and by measuring the size of lesions formed within a given time period. Other known assays can be performed at the cellular level, employing standard tissue culture methodologies e.g., using cell suspension cultures.

By “fumonisin esterase” is meant any enzyme capable of hydrolysis of the ester linkage in fumonisin or a structurally similar molecule such as AAL toxin. Two examples of such enzymes are ESPI and BESTI found in U.S. Pat. No. 5,716,820, issued Feb. 10, 1998, U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997.

By “structurally related mycotoxin” is meant any mycotoxin having a chemical structure related to a fumonisin or AP1 such as AAL toxin, fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin C1, fumonisin A1 and A2, and their analogs or hydrolyzed forms, as well as other mycotoxins having similar chemical structures, including synthetically made analogs that contain a C-2 or C-1 amine group and one or more adjacent hydroxyl groups, that would be expected to be degraded by the activity of an enzyme of the present invention. The present invention is the first flavin amine oxidase known to attack a primary amine not located at C-1 (i.e. C-2 of AP1) and resulting in a keto rather than an aldehydic product.

It is understood that “AP1” or “amino polyol” as used here is to designate the hydrolyzed form of any fumonisin, FB1, FB2, FB3, FB4, AAL, or any other AP1-like compound, including a compound made synthetically, that contains a C-2 or C-1 amine group and one or more adjacent hydroxyl groups.

By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications , D. H. Persing et at., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, one exception is Micrococcus rubens , for which GTG is the methionine codon (Oshizuka, et al, J Gen'l Microbiol , 139:425-432 (1993)) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated herein by reference.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” when the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%, preferably 60-90% of the native protein for it's native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine A), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins W. H. Freeman and Company.

As used herein, “consisting essentially of” means the inclusion of additional sequences to an object polynucleotide where the additional sequences do not selectively hybridize, under stringent hybridization conditions, to the same CDNA as the polynucleotide and where the hybridization conditions include a wash step in 0.1×SSC and 0.1% sodium dodecyl sulfate at 65° C.

By “encoding” or “encoded”, with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum ( Proc. Natl. Acad. Sci . ( USA ), 82: 2306-2309 (1985)), or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray et al. Nucl. Acids Res . 17: 477-498 (1989) and herein incorporated by reference). Thus, the maize preferred codon for a particular amino acid might be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra.

As used herein, “heterologous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

By “host cell” or “recombinantly engineered cell” is meant a cell, which contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli , or eukaryotic cells such as yeast, Pichia, insect, plant, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola, barley, millet, and tomato. A particularly preferred monocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.

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

The term “isolated” refers to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. Nucleic acids, which are “isolated”, as defined herein, are also referred to as “heterologous” nucleic acids.

Unless otherwise stated, the term “APAO nucleic acid” means a nucleic acid comprising a polynucleotide (“APAO polynucleotide”) encoding an APAO polypeptide. The term APAO, unless otherwise stated can encompass both APAO and the functional, truncated version of APAO designated trAPAO.

As used herein, “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNA molecules, which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology , Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual , 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology , F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).

As used herein “operably linked” includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants, which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum. A particularly preferred plant is Zea mays.

As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue preferred”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “regulatable” promoter is a promoter, which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development. Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter, which is active under most environmental conditions.

The term “APAO polypeptide or trAPAO polypeptide” refers to one or more amino acid sequences. The term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof. An “APAO or trAPAO protein” comprises an APAO or trAPAO polypeptide.

As used herein “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

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

The term “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.

The terms “stringent conditions” or “stringent hybridization conditions” include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20X SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem ., 138:267-284 (1984): T m =81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14,1 5, or 20° C. lower than the thermal melting point (T m ). Using the equation, hybridization and wash compositions, and desired T m , those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T m of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes , Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology , Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise stated, in the present application high stringency is defined as hybridization in 4×SCC, 5× Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and a wash in 0.1×SSC, 0.1% SDS at 65° C.

As used herein, “transgenic plant” includes reference to a plant, which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (Best Fit) of Smith and Waterman, Adv. Appl. Math may conduct optimal alignment of sequences for comparison 2: 482 (1981); by the homology alignment algorithm (GAP) of Needleman and Wunsch, J Mol. Biol . 48: 443 (1970); by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, Proc. Natl. Acad. Sci . 85: 2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et at, Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). The preferred program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, Journal of Molecular Evolution , 25:351-360 (1987) which is similar to the method described by Higgins and Sharp, CABIOS , 5:151-153 (1989) and hereby incorporated by reference). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology , Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al., Nucleic Acids Res . 25:3389-3402 (1997).

As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem ., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.

(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences, which differ by such conservative substitutions, arc said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci ., 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

(e) (i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 40-100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. The degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The terms “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with between 55-100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J Mol. Biol . 48: 443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. In addition, a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical. Peptides, which are “substantially similar” share sequences as, noted above except that residue positions, which are not identical, may differ by conservative amino acid changes.

Fumonisin Degrading Organisms

The present invention is based on the discovery of organisms with the ability to degrade the mycotoxin fumonisin. In a search for a biological means of detoxifying fumonisins, several dematiaceous hyphomycetes were isolated from field-grown maize kernels. The fungi were found to be capable of growing on fumonisins B1 or B2 (FB1 or FB2) as a sole carbon source, degrading it partially or completely in the process. One species, identified as Exophiala spinifera , a “black yeast”, was recovered from maize seed from diverse locations in the southeastern and south central US. The enzyme-active strain of Exophiala spinifera (ATCC 74269) was deposited (see U.S. patent application Ser. No. 5,716,820, issued Feb. 10, 1998, U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997). Other ernzyme-active strains of Exophiala spinifera were used to isolate APAO polynucleotides. Isolate ESP002 was isolated from palm trees (ATCC 26089) and isolate ESP003 was isolated from maize seed. Another fungus from which APAO polynucleotides were isolated was Rhinocladiella afrovirens (RAT 011).

Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising an APAO or trAPAO polynucleotide.

The present invention also includes polynucleotides optimized for expression in different organisms. For example, for expression of the polynucleotide in a maize plant, the sequence can be altered to account for specific codon preferences and to alter GC content as according to Murray et al, supra. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.

The APAO or trAPAO nucleic acids of the present invention comprise isolated APAO or trAPAO polynucleotides which, are inclusive of:

(a) a polynucleotide encoding an APAO or irAPAO polypeptide of the sequences shown in SEQ ID NOS: 36, 38, 40, 42, 44, and 46, and conservatively modified and polymorphic variants thereof;

(b) a polynucleotide which selectively hybridizes to a polynucleotide of (a) or (b);

(c) a polynucleotide having at least 50% sequence identity with polynucleotides of (a) or (b);

(d) complementary sequences of polynucleotides of (a), (b), or (c); and

(e) a polynucleotide comprising at least 25 contiguous nucleotides from a polynucleotide of (a), (b), (c), or (d).

In addition, polynucleotides are presented that are a fusion of an APAO or trAPAO polynucleotide and the polynucleotide of a fumonisin esterase. The invention encompasses the sequences from Exophiala or Rhinocladiella as well as sequences having sequence similarity with such sequences. It is recognized that the sequences of the invention can be used to isolate corresponding sequences in other organisms. Methods such as PCR, hybridization, and the like can be used to identify sequences having substantial sequence similarity to the sequences of the invention. See, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Planview, N.Y.) and Innis et al., (1990) PCR Protocols: Guide to Methods and Applications (Academic Press, New York). Coding sequences isolated based on their sequence identity to the entire fumonisin degrading coding sequences set forth herein or to fragments thereof are encompassed by the present invention.

It is recognized that the sequences of the invention can be used to isolate similar sequences from other fumonisin degrading organisms. Likewise sequences from other fumonisin degrading organisms may be used in combination with the sequences of the present invention. See, for example, copending application entitled “Compositions and Methods for Fumonisin Detoxification”, U.S. application Ser. No. 601092,953, filed concurrently herewith and herein incorporated by reference.

Plasmids containing the polynucleotide sequences of the invention were deposited with American Type Culture Collection (ATCC), Manassas, Va., and assigned Accession Nos. 98812, 98813, 98814, 98815, 98816, and PTA-32. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. § 112.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a fungus or bacteria.

The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. The nucleic acid of the present invention—excluding the polynucleotide sequence—is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. Exemplary nucleic acids include such vectors as: M13, lambda ZAP Express, lambda ZAPII, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/−, pSG5, pBK, pCR-Script, pET, pSPUTK, p3′SS, pGEM, pSK+/−, pGEX, pSPORTI and II, pOPRSVI CAT, pOP13 CAT, pXTI, pSG5, pPbac, pMbac, pMC1neo, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambda MOSElox. Optional vectors for the present invention, include but are not limited to, lambda ZAP II, and pGEX. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol . 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol . 68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett . 22: 1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts . 22(20): 1859-1862 (1981), e.g., using an automated synthesizer, e.g, as described in Needham-VanDevanter et al., Nucleic Acids Res ., 12: 6159-6168 (1984); and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated by specific sequence elements in the 5′ non-coding or untranslated region (5′ UTR) of the RNA. Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res . 15:8125 (1987)) and the 5<G>7 methyl GpppG RNA cap structure (Drummond et al., Nucleic Acids Res . 13:7375 (1985)). Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol . 8:284 (1988)). Accordingly, the present invention provides 5′ and/or 3′ UTR regions for modulation of translation of heterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host or to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res . 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides (3 nucleotides per amino acid) that can be used to determine a codon usage frequency can be any integer from 3 to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, J.-H., et al. Proc. Nail. Acad. Sci. USA 94:4504-4509 (1997) and Zhao, et al., Nature Biotech 16:258-261 (1998). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic, which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides, which comprise sequence regions, which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be an altered K m and/or K cat over the wild-type protein as provided herein. In other embodiments, a protein or polynucleotide generated from sequence shuffling will have a substrate binding affinity greater than the non-shuffled wild-type polynucleotide. In yet other embodiments, a protein or polynucleotide generated from sequence shuffling will have an altered pH optimum as compared to the non-shuffled wild-type polynuclcotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or greater than 150% of the wild-type value.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a polypeptide long enough to code for an active protein of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5′ and 3′ regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

A plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens , the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the GRP1-8 promoter, the 35S promoter from cauliflower mosaic virus (CaMV), as described in Odell et al., (1985), Nature , 313:810-812, rice actin (McElroy et al., (1990), Plant Cell , 163-171); ubiquitin (Christensen et al., (1992), Plant Mol. Biol . 12:619-632; and Christensen, et al., (1992), Plant Mol. Biol . 18:675-689); pEMU (Last, et al., (1991), Theor. Appl. Genet . 81:581-588); MAS (Velten et al., (1984), EMBO J . 3:2723-2730); and maize H3 histone (Lepetit et al., (1992), Mol. Gen. Genet . 231:276-285; and Atanassvoa et al., (1992), Plant Journal 2(3):291-300), the Rsyn7 as described in published PCT Application WO 97/44756, ALS promoter, as described in published PCT Application WO 96/30530, and other transcription initiation regions from various plant genes known to those of skill. For the present invention ubiquitin is the preferred promoter for expression in monocot plants.

Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as “inducible” promoters. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Adh1 promoter, which is inducible by hypoxia or cold stress, the Hsp70 promoter, which is inducible by heat stress, and the PPDK promoter, which is inducible by light.

Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.

If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′ end of a polynucleotide coding region. The polyadenylation region can be derived from a variety of plant genes, or from T-DNA. The 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene. Examples of such regulatory elements include, but are not limited to, 3′ termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan et al., (1983), Nucl. Acids Res . 12:369-385); the potato proteinase inhibitor II (PINII) gene (Keil, et al., (1986), Nucl. Acids Res . 14:5641-5650; and An et al., (1989), Plant Cell 1:115-122); and the CaMV 19S gene (Mogen et al., (1990), Plant Cell 2:1261-1272).

An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol . 8: 4395-4405 (1988); Callis et al., Genes Dev . 1: 1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook , Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., (1989), J. Biol. Chem . 264:4896-4900), the Nicotiana plumbaginifolia extension gene (DeLoose, et al., (1991), Gene 99:95-100), signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuka, et al., (1991), PNAS 88:834) and the barley lectin gene (Wilkins, et al., (1 990), Plant Cell , 2:301-313), signal peptides which cause proteins to be secreted such as that of PRIb (Lind, et al., (1992), Plant Mol. Biol . 18:47-53), or the barley alpha amylase (BAA) (Rahmatullah, et al., Plant Mot. Biol . 12:119 (1989)) and hereby incorporated by reference), or from the present invention the signal peptide from the ESP1 or BEST1 gene, or signal peptides which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase (Verwaert, et al., (1994), Plant Mol. Biol . 26:189-202) are useful in the invention. The barley alpha amylase signal sequence fused to the trAPAO or APAO polynucleotide is the preferred construct for expression in maize for the present invention.

The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene, which confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

Alternatively, the invention, itself, could be used as a method for selection of transformants, in other words as a selectable marker. An APAO or trAPAO polynucleotide operably linked to a promoter and then transformed into a plant cell by any of the methods described in the present application would express the degradative enzyme. When the plant cells are placed in the presence of fumonisin, AP1, or a phytotoxic analog in culture only the transformed cells would be able to grow. In another embodiment, the plant cell could be transformed with both a polynucleotide for APAO and a polynucleotide for fumonisin esterase. The selective agent in this case could be either AP1 or fumonisin or any structural analog. Thus, growth of plant cells in the presence of a mycotoxin favors the survival of plant cells that have been transformed to express the coding sequence that codes for one of the enzymes of this invention and degrades the toxin. When the APAO or trAPAO cassette with or without the fumonisin esterase polynucleotide, is co-transformed with another gene of interest and then placed in the presence of fumonisin, AP1 or a phytotoxic analog, this invention would allow for selection of only those plant cells that contain the gene of interest. In the past antibiotic resistance genes have been used as selectable markers. Given the current concerns by consumers and environmentalist over use of antibiotic genes and the possibility of resistant microorganisms arising due to this use, a non-antibiotic resistant selectable marker system such as the present invention, fulfills this very important need.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol., 153:253-277 (1987). These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene , 61:1-11 (1987) and Berger el al., Proc. Natl. Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein is plasmid pB1101.2 that is available from CLONTECH Laboratories, Inc. (Palo Alto, Calif.).

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression-of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or CDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter, such as ubiquitin, to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. Constitutive promoters are classified as providing for a range of constitutive expression. Thus, some are weak constitutive promoters, and others are strong constitutive promoters. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a “strong promoter” drives expression of a coding sequence at a “high level”, or about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts.

One of skill would recognize that modifications could be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli ; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction of the gene of interest into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., Gene 22: 229-235 (1983); Mosbach, et al., Nature 302: 543-545 (1983)). The pGEX-4T-1 plasmid vector from Pharmacia is the preferred E. coli expression vector for the present invention.

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.

Synthesis of heterologous proteins in yeast is well known. Sherman, F., et al., Methods in Yeast Genetics , Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast. Two widely utilized yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris . Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates or the pellets. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol. Rev . 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).

Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth, and Drosophila cell lines such as a Schneider cell line (See Schneider, J Embryol. Exp. Morphol . 27: 353-365 (1987).

As with yeast, when higher animal or plant host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol . 45: 773-781 (1983)). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M., Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol. II a Practical Approach , D. M. Glover, Ed., IRL Press, Arlington, Va. pp. 213-238 (1985).

In addition, one of the genes for fumonisin esterase or the APAO or trAPAO placed in the appropriate plant expression vector can be used to transform plant cells. The enzyme can then be isolated from plant callus or the transformed cells can be used to regenerate transgenic plants. Such transgenic plants can be harvested, and the appropriate tissues (seed or leaves, for example) can be subjected to large scale protein extraction and purification techniques, and the fumonisin degradation enzymes or APAO can be isolated for use in fumonisin and fumonisin hydrolysis product detoxification processes.

Plant Transformation Methods

Numerous methods for introducing foreign genes into plants are known and can be used to insert an APAO or trAPAO polynucleotide into a plant host including biological and physical plant transformation protocols. See, for example, Miki et al., (1993), “Procedure for Introducing Foreign DNA into Plants”, In: Methods in Plant Molecular Biology and Biotechnology , Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88. The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., (1985), Science 227:1229-31), electroporation, micro-injection, and biolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are known and available. See, for example, Gruber, et al., (1993), “Vectors for Plant Transformation” In: Methods in Plant Molecular Biology and Biotechnology , Glick and Thompson, eds. CRC Press, Inc., Boca Raton, pages 89-119.

Agrobacterium-mediated Transformation

The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria, which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes , respectively, carry genes responsible for genetic transformation of plants. See, for example, Kado, (1991), Crit. Rev. Plant Sci . 10:1. Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided in Gruber et al., supra; Miki, et al., supra; and Moloney et al., (1989), Plant Cell Reports 8:238.

Similarly, the gene can be inserted into the T-DNA region of a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes , respectively. Thus, expression cassettes can be constructed as above, using these plasmids. Many control sequences are known which when coupled to a heterologous coding sequence and transformed into a host organism show fidelity in gene expression with respect to tissue/organ specificity of the original coding sequence. See, e.g., Benfey, P. N., and Chua, N. H. (1989) Science 244: 174-181. Particularly suitable control sequences for use in these plasmids are promoters for constitutive leaf-specific expression of the gene in the various target plants. Other useful control sequences include a promoter and terminator from the nopaline synthase gene (NOS). The NOS promoter and terminator are present in the plasmid pARC2, available from the American Type Culture Collection and designated ATCC 67238. If such a system is used, the virulence (vir) gene from either the Ti or Ri plasmid must also be present, either along with the T-DNA portion, or via a binary system where the vir gene is present on a separate vector. Such systems, vectors for use therein, and methods of transforming plant cells are described in U.S. Pat. No. 4,658,082; U.S. application Ser. No. 913,914, filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993 to Robeson, et al.; and Simpson, R. B., et al. (1986) Plant Mol. Biol . 6: 403-415 (also referenced in the '306 patent); all incorporated by reference in their entirety.

Once constructed, these plasmids can be placed into A. rhizogenes or A. tumefaciens and these vectors used to transform cells of plant species, which are ordinarily susceptible to Fusarium or Alternaria infection. Several other transgenic plants are also contemplated by the present invention including but not limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton, melon and pepper. The selection of either A. tumefaciens or A. rhizogenes will depend on the plant being transformed thereby. In general A. tumefaciens is the preferred organism for transformation. Most dicotyledonous plants, some gymnosperms, and a few monocotyledonous plants (e.g. certain members of the Liliales and Arales) are susceptible to infection with A. tumefaciens. A. rhizogenes also has a wide host range, embracing most dicots and some gymnosperms, which includes members of the Leguminosae, Compositae, and Chenopodiaceae. Monocot plants can now be transformed with some success. European Patent Application Publication Number 604 662 A1 to Hiei et al. discloses a method for transforming monocots using Agrobacterium. Saito et al. discloses a method for transforming monocots with Agrobacterium using the scutellum of immature embryos (European Application 672 752 A1). Ishida et al discusses a method for transforming maize by exposing immature embryos to A. tumefaciens (Ishida et al., Nature Biotechnology , 1996, 14:745-750).

Once transformed, these cells can be used to regenerate transgenic plants, capable of degrading fumonisin. For example, whole plants can be infected with these vectors by wounding the plant and then introducing the vector into the wound site. Any part of the plant can be wounded, including leaves, stems and roots. Alternatively, plant tissue, in the form of an explant, such as cotyledonary tissue or leaf disks, can be inoculated with these vectors, and cultured under conditions, which promote plant regeneration. Roots or shoots transformed by inoculation of plant tissue with A. rhizogenes or A. tumefaciens , containing the gene coding for the fumonisin degradation enzyme, can be used as a source of plant tissue to regenerate fumonisin-resistant transgenic plants, either via somatic embryogenesis or organogenesis. Examples of such methods for regenerating plant tissue are disclosed in Shahin, E. A. (1985) Theor. Appl. Genet . 69:235-240; U.S. Pat. No. 4,658,082; Simpson, R. B., et al. (1986) Plant Mol. Biol . 6: 403-415; and U.S. Pat. applications Ser. Nos. 913,913 and 913,914, both filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993 to Robeson, et al.; the entire disclosures therein incorporated herein by reference.

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediated transformation is broad, some major cereal crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice (Hiei et al., (1994), The Plant Journal 6:271-282). Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate the plant cell walls and membranes. (Sanford et al., (1987), Part. Sci. Technol . 5:27; Sanford, 1988 , Trends Biotech 6:299; Sanford, (1990), Physiol. Plant 79:206; Klein et al., (1992), Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication of target cells as described in Zang et al., (1991), BioTechnology 9:996. Alternatively, liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, for example, Deshayes et al., (1985), EMBO J . 4:2731; and Christou et al., (1987), PNAS USA 84:3962. Direct uptake of DNA into protoplasts using CaCl 2 precipitation, polyvinyl alcohol, or poly-L-omithine has also been reported. See, for example, Hain et al., (1985), Mol. Gen. Genet . 199:161; and Draper et al., (1982), Plant Cell Physiol . 23:451.

Electroporation of protoplasts and whole cells and tissues has also been described. See, for example, Donn et al., (1990), In: Abstracts of the VIIth Int'l. Congress on Plant Cell and Tissue Culture IAPTC , A2-38, page 53; D'Halluin et al., (1992), Plant Cell 4:1495-1505; and Spencer et al., (1994), Plant Mol. Biol . 24:51-61.

Thus, polynucleotide encoding a polypeptide able to inactivate fumonisin or AP1 can be isolated and cloned in an appropriate vector and inserted into an organism normally sensitive to the Fusarium or its toxins. Furthermore, the polynucleotide imparting fumonisin or AP1 degradative activity can be transferred into a suitable plasmid, and transformed into a plant. Thus, a fumonisin or AP1 degrading transgenic plant can be produced. Organisms expressing the polynucleotide can be easily identified by their ability to degrade fumonisin or AP1. The protein capable of degrading fumonisin or AP1 can be isolated and characterized using techniques well known in the art.

APAO or trAPAO in a Transgenic Plant

Fumonisin esterase reduces but does not eliminate the toxicity of fumonisins. Therefore a second enzymatic modification to further reduce or abolish toxicity is desirable. The partially purified APAO enzyme from Exophiala spinifera has little or no activity on intact FB1, a form of fumonisin. However, recombinant APAO enzyme from Exophiala spinifera , expressed in E. coli , has significant but reduced activity on intact FB1 and other B-series fumonisins. APAO or trAPAO thus could potentially be used without fumonisin esterase since the amine group is the major target for detoxification. Alternatively, the two genes, fumonisin esterase and APAO (or trAPAO) can be used together for degrading toxins.

APAO is predicted to be an enzyme that, when by itself or co-expressed in a heterologous expression system along with fumonisin esterase (either ESP1 or BEST1), will result in the production of 2-oxo pentol (2-OP) from fumonisin B1. The substrate range of recombinant, E coli -expressed APAO is limited to fumonisins and their hydrolysis products and does not include amino acids, sphingolipid precursors such as phytosphingosine, or polyamines such as spermidine. Thus, APAO is highly specific for fumonisin-like amines, and thus would have little deleterious effect on other cellular metabolites. In addition, if it is extracellularly localized, it will limit any contact with biologically important amines that might also be substrates. The end result will be a more effective detoxification of fumonisins than can be achieved with esterase alone.

The oxidase activity of APAO is predicted to result in generation of hydrogen peroxide in stoichiometric amounts relative to AP1 or fumonisin oxidized. This may prove to be an additional benefit of this enzyme, since hydrogen peroxide is both antimicrobial and is thought to contribute to the onset of a defense response in plants (Przemylaw, Biochem J , 322:681-692 (1997), Lamb, el al., Ann Rev Plant Physiol Plant Mol Bio 48:251-275 (1997), and Alverez, et al., Oxidative Stress and the Molecular Biology of Antioxidant Defenses , Cold Spring Harbor Press, 815-839 (1997)).

Since one of the embodiments of the present invention is to have both a fumonisin esterase polynucleotide and an APAO or trAPAO polynucleotide present in a plant, there are several ways to introduce more than one polynucleotide in a plant. One way is to transform plant tissue with polynucleotides to both fumonisin esterase and APAO or trAPAO at the same time. In some tissue culture systems it is possible to transform callus with one polynucleotide and then after establishing a stable culture line containing the first polynucleotide, transform the callus a second time with the second polynucleotide. One could also transform plant tissue with one polynucleotide, regenerate whole plants, then transform the second polynucleotide into plant tissue and regenerate whole plants. The final step would then be to cross a plant containing the first polynucleotide with a plant containing the second polynucleotide and select for progeny containing both polynucleotides.

Another method is to create a fusion protein between esterase and APAO or trAPAO, preferably with a spacer region between the two polypeptides. Both enzymes would be active although tethered to each other. In addition, an enzyme cleavage site engineered in the spacer region, would allow cleavage by an endogenous or introduced protease.

Transgenic plants containing both a fumonisin esterase enzyme and/or the APAO enzyme and thus able to degrade fumonisin or a structurally related mycotoxin would be able to reduce or eliminate the pathogenicity of any microorganism that uses fumonisin or a structurally related mycotoxin as a mode of entry to infect a plant. Fungal pathogens frequently use toxins to damage plants and weaken cell integrity in order to gain entry and expand infection in a plant. By preventing the damage induced by a toxin, a plant would be able to prevent the establishment of the pathogen and thereby become tolerant or resistant to the pathogen.

Another benefit of fumonisin degradation is the production of hydrogen peroxide. When fumonisin or AP1 is oxididatively deaminated at C-2, as occurs by exposure to APAO or trAPAO enzyme, hydrogen peroxide is produced as a by-product. Hydrogen peroxide production can trigger enhanced resistance responses in a number of ways. 1) Hydrogen peroxide has direct antimicrobial activity. 2) Hydrogen peroxide acts as a substrate for peroxidases associated with lignin polymerization and hence cell wall strengthening. 3) Via still to be determined mechanisms, hydrogen peroxide acts as a signal for activation of expression of defense related genes, including those that result in stimulation of salicylic acid accumulation. Salicylic acid is thought to act an endogenous signal molecule that triggers expression of genes coding for several classes of pathogenesis-related proteins. Moreover, salicylic acid may set up the oxidative burst and thus act in a feedback loop enhancing its own synthesis. Salicylic acid may also be involved in hypersensitive cell death by acting as an inhibitor of catalase, an enzyme that removes hydrogen peroxide. 4) Hydrogen peroxide may trigger production of additional defense compounds of additional defense compounds such as phytoalexins, antimicrobial low molecular weight compounds. For a review on the role of the oxidative burst and SA please see Lamb, C. and Dixon, R. A., Ann. Rev. Plant Physiol. Plant Mol. Biol ., 48: 251-275 (1 997).

Detoxification of Harvested Grain, Silage, or Contaminated Food Crop

The present invention also relates to a method of detoxifying a fumonisin or a structurally related mycotoxin with an APAO enzyme during the processing of grain for animal or human food consumption, during the processing of plant material for silage, or food crops contaminated with a toxin producing microbe, such as but not limited to, tomato. Since the atmospheric ammoniation of corn has proven to be an ineffective method of detoxification (see B. Fitch Haumann, INFORM 6:248-257 (1995)), such a methodology during processing is particularly critical where transgenic detoxification is not applicable.

In one embodiment of the present invention, fumonisin degradative enzymes are presented to grain, plant material for silage, or a contaminated food crop, or during the processing procedure, at the appropriate stages of the procedure and in amounts effective for detoxification of fumonisins and structurally related mycotoxins. Detoxification by the enzymes, microbial strains, or an engineered microorganism can occur not only during the processing, but also any time prior or during the feeding of the grain or plant material to an animal or incorporation of the grain or food crop into a human food product, or before or during ingestion of the food crop.

Another embodiment of the present invention is the engineering of a bacterium or fungus to express the detoxification enzymes and then using the bacterium or fungus rather than the enzyme itself There are a number of microbes that could be engineered to express the polynucleotides of the present invention. One could also activate, either inducibly or constitutively, the endogenous genes for fumonisin esterase or APAO. By overexpressing the degradative enzymes and then treating plants, seed, or silage with the microorganism, it would be possible to degrade fumonisin in situ.

The polynucleotides of the invention can be introduced into microorganisms that multiply on plants (epiphytes) to deliver enzymes to potential target crops. Epiphytes can be gram-positive or gram-negative bacteria, for example.

The microorganisms that have been genetically altered to contain at least one degradative polynucleotide and resulting polypeptide may be used for protecting agricultural crops and products. In one aspect of the invention, whole, i.e. unlysed, cells of the transformed organism are treated with reagents that prolong the activity of the enzyme produced in the cell when the cell is applied to the environment of a target plant. A secretion leader may be used in combination with the gene of interest such that the resulting enzyme is secreted outside the host cell for presentation to the target plant.

The degradative enzymes can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray. Any suitable microorganism can be used for this purpose. See, for example, Gaertner, et al. (1993) in Advanced Engineered Pesticides , (ed. Kim, Marcel Dekker, New York).

The enzymes or microorganisms can be introduced during processing in appropriate manners, for example as a wash or spray, or in dried or lyophilized form or powered form, depending upon the nature of the milling process and/or the stage of processing at which the enzymatic treatment is carried out. See generally, Hoseney, R. C., Principles of Cereal Science and Technology , American Assn. of Cereal Chemists, Inc., 1990 (especially Chapters 5, 6 and 7); Jones, J. M., Food Safety , Eagan Press, St. Paul, Minn., 1992 (especially Chapters 7 and 9); and Jelen, P., Introduction to Food Processing , Restan Publ. Co., Reston, Va., 1985. Processed grain or silage to be used for animal feed can be treated with an effective amount of the enzymes in the form of an inoculant or probiotic additive, for example, or in any form recognized by those skilled in the art for use in animal feed. The enzymes of the present invention are expected to be particularly useful in detoxification during processing and/or in animal feed prior to its use, since the enzymes display relatively broad ranges of pH activity. The esterase from Exophiala spinifera , ATCC 74269, showed a range of activity from about pH 3 to about pH 6, and the esterase from the bacterium of ATCC 55552 showed a range of activity from about pH 6 to about pH 9 (U.S. Pat. No. 5,716,820, supra). The APAO enzyme from Exophiala spinifera (ATCC 74269) has a pH range of activity from pH 6 to pH 9.

Genetic Engineering of Ruminant Microorganisms

Ruminant microorganisms can be genetically engineered to contain and express either the fumonisin esterase enzymes or APAO, or a combination of the enzymes. The genetic engineering of microorganisms is now an art recognized technique, and ruminant microorganisms so engineered can be added to feed in any art recognized manner, for example as a probiotic or inoculant. In addition, microorganisms capable of functioning as bioreactors can be engineered so as to be capable of mass producing either the fumonisin esterases or the APAO enzyme.

Use of the Fumonisin Esterase and APAO Enzymes for Detection of Reagents for Fumonisins and Related Compounds

Another embodiment of the present invention is the use of the enzymes of the present invention as detection reagents for fumonisins and related compounds. The enzymes of the present invention can be used as detection reagents because of the high specificity of the esterase and deaminase enzymes, and the fact that hydrolysis followed by amine oxidation can be monitored by detection of hydrogen peroxide or ammonia using standard reagents (analogous to a glucose detection assay using glucose oxidase). Hydrogen peroxide is often measured by linking a hydrogen peroxide-dependent peroxidase reaction to a colored or otherwise detectable peroxidase product (e.g. Demmano, et al., European Journal of Biochemistry 238(3): 785-789 (1996)). Ammonia can be measured using ion-specific electrodes : Fritsche, et al., Analytica Chimica Acia 244(2): 179-182 (1991); West, et al., Analytical Chemistry 64(5): 533-540 (1992), a herein incorporated by reference) or by GC or other chromatographic method.

For example, recombinant or non-recombinant, active fumonisin esterase (ESP1 or BEST) and APAO proteins are added in catalytic amounts to a sample tube containing an unknown amount of fumonisins (FB1, FB2, FB3, FB4, or partial or complete hydrolysis products of these). The tube is incubated under pH and temperature conditions sufficient to convert any fumonisin in the sample to AP1, and correspondingly the AP1 to 2-OP, ammonia, and hydrogen peroxide. Alternatively, APAO or trAPAO is added in catalytic amounts to a sample tube containing an unknown amount of fumonisins (FB1, FB2, FB3, FB4, or partial or complete hydrolysis products of these). The tube is incubated under pH and temperature conditions sufficient to convert any fumonisin in the sample to 2-oxo FB1, ammonia, and hydrogen peroxide. Then suitable reagents are added for quantification of the hydrogen peroxide or ammonia that were generated stoichiometrically from fumonisins. By comparison with control tubes that received no esterase or APAO enzyme, the amount of fumonisin present can be calculated in direct molar proportion to the hydrogen peroxide or ammonia detected, relative to a standard curve.

This invention can be better understood by reference to the following non-limiting examples. It will be appreciated by those skilled in the art that other embodiments of the invention may be practiced without departing from the spirit and the scope of the invention as herein disclosed and claimed.

Exophiala spinifera

2

gaattttccg ccaatgcttg cttctcggcg ggaagaggtg gtgaaaatgt caaggtggga 60

tacaaggttg tcggtaacga aaccaccacc tttttgcttc ggaacacggc gcccgaggcc 120

gatcgtactg tacagccgga tgccgactgc tcaatttcag cgacgggggt gttgaggtgc 180

ac 182

3

29

Artificial Sequence

Designed oligonucleotide for 3′ RACE N21965

(Exophiala spinifera)

3

tggtttcgtt accgacaacc ttgtatccc 29

4

28

Artificial Sequence

Designed oligonucleotide for 5′ RACE 21968

4

gagttggtcc cagacagact tttgtcgt 28

5

gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt ttg 48

Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu

1 5 10 15

gag acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt ctt 96

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

20 25 30

gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg ggt 144

Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser Gly

35 40 45

ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat gac 192

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

50 55 60

agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg gag 240

Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu

65 70 75 80

ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa gac 288

Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp

85 90 95

ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag gag 336

Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu

100 105 110

gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg atc 384

Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile

115 120 125

gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag cgg 432

Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg

130 135 140

ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac ttg 480

Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu

145 150 155 160

cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc ggt 528

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

165 170 175

gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc aag 576

Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys

180 185 190

agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc ggg 624

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

195 200 205

cag tat atg cga tgc aaa aca ggt atg cag tcg att tgc cat gcc atg 672

Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met

210 215 220

tca aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc gct 720

Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val Ala

225 230 235 240

gaa att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg ggc 768

Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly

245 250 255

gcc gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc ttg 816

Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu

260 265 270

tat ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa gca 864

Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala

275 280 285

ttg gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc gta 912

Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val

290 295 300

tgg gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc caa 960

Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln

305 310 315 320

tcg agc tgt gac ccc atc tca ttt gcc aga gat acc agc atc gac gtc 1008

Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val

325 330 335

gat cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga cgg 1056

Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly Arg

340 345 350

aag tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg gac 1104

Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp

355 360 365

caa ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag ccg 1152

Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro

370 375 380

gcc aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa gga 1200

Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly

385 390 395 400

gct ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt tcg 1248

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

405 410 415

gcg ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag acg 1296

Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr

420 425 430

tct tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt caa 1344

Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln

435 440 445

cga ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 1389

Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

6

462

Exophiala spinifera

6

Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu

1 5 10 15

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

20 25 30

Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser Gly

35 40 45

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

50 55 60

Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu

65 70 75 80

Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp

85 90 95

Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu

100 105 110

Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile

115 120 125

Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg

130 135 140

Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu

145 150 155 160

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

165 170 175

Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys

180 185 190

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

195 200 205

Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met

210 215 220

Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val Ala

225 230 235 240

Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly

245 250 255

Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu

260 265 270

Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala

275 280 285

Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val

290 295 300

Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln

305 310 315 320

Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val

325 330 335

Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly Arg

340 345 350

Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp

355 360 365

Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro

370 375 380

Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly

385 390 395 400

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

405 410 415

Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr

420 425 430

Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln

435 440 445

Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

7

gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt ttg 48

Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu

1 5 10 15

gag acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt ctt 96

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

20 25 30

gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg ggt 144

Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser Gly

35 40 45

ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat gac 192

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

50 55 60

agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg gag 240

Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu

65 70 75 80

ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa gac 288

Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp

85 90 95

ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag gag 336

Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu

100 105 110

gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg atc 384

Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile

115 120 125

gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag cgg 432

Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg

130 135 140

ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac ttg 480

Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu

145 150 155 160

cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc ggt 528

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

165 170 175

gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc aag 576

Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys

180 185 190

agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc ggg 624

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

195 200 205

cag tat gtg cga tgc aaa aca ggtgcgtgtg gtgtcgtctc aggtggggga 675

Gln Tyr Val Arg Cys Lys Thr

210 215

ctcgtttctc agtggtcatt cca ggt atg cag tcg att tgc cat gcc atg tca 728

Gly Met Gln Ser Ile Cys His Ala Met Ser

220 225

aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc gct gaa 776

Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val Ala Glu

230 235 240

att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg ggc gcc 824

Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala

245 250 255

gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc ttg tat 872

Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr

260 265 270

ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa gca ttg 920

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

275 280 285

gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc gta tgg 968

Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp

290 295 300 305

gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc caa tcg 1016

Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser

310 315 320

agc tgt gac ccc atc tca ttt gcc aga gat acc agc atc gac gtc gat 1064

Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val Asp

325 330 335

cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga cgg aag 1112

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

340 345 350

tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg gac caa 1160

Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln

355 360 365

ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag ccg gcc 1208

Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Ala

370 375 380 385

aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa gga gct 1256

Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala

390 395 400

ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt tcg gcg 1304

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

405 410 415

ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag acg tct 1352

Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr Ser

420 425 430

tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt caa cga 1400

Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg

435 440 445

ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 1442

Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

8

462

Exophiala spinifera

8

Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu

1 5 10 15

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

20 25 30

Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser Gly

35 40 45

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

50 55 60

Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu

65 70 75 80

Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp

85 90 95

Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu

100 105 110

Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile

115 120 125

Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg

130 135 140

Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu

145 150 155 160

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

165 170 175

Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys

180 185 190

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

195 200 205

Gln Tyr Val Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met

210 215 220

Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val Ala

225 230 235 240

Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly

245 250 255

Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu

260 265 270

Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala

275 280 285

Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val

290 295 300

Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln

305 310 315 320

Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val

325 330 335

Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly Arg

340 345 350

Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp

355 360 365

Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro

370 375 380

Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly

385 390 395 400

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

405 410 415

Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr

420 425 430

Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln

435 440 445

Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

9

458

Exophiala spinifera

9

Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu

1 5 10 15

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

20 25 30

Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser Gly

35 40 45

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

50 55 60

Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu

65 70 75 80

Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp

85 90 95

Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu

100 105 110

Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile

115 120 125

Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg

130 135 140

Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu

145 150 155 160

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

165 170 175

Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys

180 185 190

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

195 200 205

Gln Tyr Val Arg Cys Lys Thr Gly Ala Cys Gly Val Val Ser Gly Gly

210 215 220

Gly Leu Val Ser Gln Trp Ser Phe Gln Val Cys Ser Arg Phe Ala Met

225 230 235 240

Pro Cys Gln Arg Asn Leu Phe Gln Ala Gln Cys Thr Ser Thr Pro Pro

245 250 255

Ser Leu Lys Leu Ser Ser Arg His Pro Ala Val Gln Tyr Asp Arg Pro

260 265 270

Arg Ala Pro Cys Ser Glu Ala Lys Arg Trp Trp Phe Arg Tyr Arg Gln

275 280 285

Pro Cys Ile Pro Pro His Phe His His Leu Phe Pro Pro Arg Ser Lys

290 295 300

His Trp Arg Lys Ile Leu Ser Trp Ala Thr Ile Ala Arg Ser Ser Tyr

305 310 315 320

Gly Thr Ser Arg Gly Gly Ala Asn Lys Ala Ser Arg Ala Ser Ser Asn

325 330 335

Arg Ala Val Thr Pro Ser His Leu Pro Glu Ile Pro Ala Ser Thr Ser

340 345 350

Ile Asp Asn Gly Pro Leu Pro Val Ser Trp Ser Glu Thr Arg Asp Gly

355 360 365

Ser Gly Pro Asn Ser Pro Ser Arg Tyr Asp Lys Ser Leu Ser Gly Thr

370 375 380

Asn Ser Ala Gln Pro Thr Arg Thr Pro Gly Pro Lys Ser Gln Ser Arg

385 390 395 400

Pro Thr Cys Ser Lys Ser Ser Gly Arg Ser Ser Ser Ile Ser Lys Glu

405 410 415

Leu Arg Ala Pro Ser Met Gly Thr Ile Ser Ser His Trp Val Arg Arg

420 425 430

Ser Glu Arg Arg Ser Arg Val Phe Ile Ser Leu Glu Arg Arg Arg Leu

435 440 445

Phe Gly Lys Gly Ile Trp Lys Gly Pro Tyr

450 455

10

aaa gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt 48

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

ttg gag acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt 96

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

20 25 30

ctt gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg 144

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

ggt ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat 192

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

50 55 60

gac agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg 240

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

gag ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa 288

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

gac ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag 336

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

gag gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg 384

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

atc gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag 432

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

cgg ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac 480

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

ttg cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc 528

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

165 170 175

ggt gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc 576

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

aag agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc 624

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

195 200 205

ggg cag tat atg cga tgc aaa aca ggt atg cag tcg att tgc cat gcc 672

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala

210 215 220

atg tca aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc 720

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

gct gaa att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg 768

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc 816

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

ttg tat ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa 864

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

gca ttg gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc 912

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

gta tgg gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc 960

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

caa tcg agc tgt gac ccc atc tca ttt gcc aga gat acc agc atc gac 1008

Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

gtc gat cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga 1056

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

cgg aag tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg 1104

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

gac caa ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag 1152

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

370 375 380

ccg gcc aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa 1200

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

gga gct ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt 1248

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

405 410 415

tcg gcg ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag 1296

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

acg tct tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt 1344

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

caa cga ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 1392

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

11

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

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

20 25 30

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

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

50 55 60

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

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

165 170 175

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

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

195 200 205

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala

210 215 220

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

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

370 375 380

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

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

405 410 415

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

12

34

Artificial Sequence

Primer sequence designed for cloning DNA into

expression vectors, N23256

12

ggggaattca aagacaacgt tgcggacgtg gtag 34

13

34

Artificial Sequence

Primer sequence designed for cloning DNA into

expression vectors N23256

13

ggggcggccg cctatgctgc tggcaccagg ctag 34

14

29

Artificial Sequence

Designed oligonucleotide for 3′ RACE, N21965

14

tggtttcgtt accgacaacc ttgtatccc 29

15

28

Artificial Sequence

Designed oligonucleotide for 5′ RACE, N21968

15

gagttggtcc cagacagact tttgtcgt 28

16

atg aga ttt cct tca att ttt act gct gtt tta ttc gca gca tcc tcc 48

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

1 5 10 15

gca tta gct gct cca gtc aac act aca aca gaa gat gaa acg gca caa 96

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

20 25 30

att ccg gct gaa gct gtc atc ggt tac tca gat tta gaa ggg gat ttc 144

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

gat gtt gct gtt ttg cca ttt tcc aac agc aca aat aac ggg tta ttg 192

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

ttt ata aat act act att gcc agc att gct gct aaa gaa gaa ggg gta 240

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

tct ctc gag aaa aga gag gct gaa gct gaa ttc aaa gac aac gtt gcg 288

Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Lys Asp Asn Val Ala

85 90 95

gac gtg gta gtg gtg ggc gct ggc ttg agc ggt ttg gag acg gca cgc 336

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

100 105 110

aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt ctt gag gcg atg gat 384

Lys Val Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp

115 120 125

cgt gta ggg gga aag act ctg agc gta caa tcg ggt ccc ggc agg acg 432

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

130 135 140

act atc aac gac ctc ggc gct gcg tgg atc aat gac agc aac caa agc 480

Thr Ile Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser

145 150 155 160

gaa gta tcc aga ttg ttt gaa aga ttt cat ttg gag ggc gag ctc cag 528

Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln

165 170 175

agg acg act gga aat tca atc cat caa gca caa gac ggt aca acc act 576

Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr

180 185 190

aca gct cct tat ggt gac tcc ttg ctg agc gag gag gtt gca agt gca 624

Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala

195 200 205

ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg atc gaa gag cat agc 672

Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser

210 215 220

ctt caa gac ctc aag gcg agc cct cag gcg aag cgg ctc gac agt gtg 720

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

225 230 235 240

agc ttc gcg cac tac tgt gag aag gaa cta aac ttg cct gct gtt ctc 768

Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu

245 250 255

ggc gta gca aac cag atc aca cgc gct ctg ctc ggt gtg gaa gcc cac 816

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

260 265 270

gag atc agc atg ctt ttt ctc acc gac tac atc aag agt gcc acc ggt 864

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

275 280 285

ctc agt aat att ttc tcg gac aag aaa gac ggc ggg cag tat atg cga 912

Leu Ser Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg

290 295 300

tgc aaa aca ggt atg cag tcg att tgc cat gcc atg tca aag gaa ctt 960

Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met Ser Lys Glu Leu

305 310 315 320

gtt cca ggc tca gtg cac ctc aac acc ccc gtc gct gaa att gag cag 1008

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

325 330 335

tcg gca tcc ggc tgt aca gta cga tcg gcc tcg ggc gcc gtg ttc cga 1056

Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg

340 345 350

agc aaa aag gtg gtg gtt tcg tta ccg aca acc ttg tat ccc acc ttg 1104

Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu

355 360 365

aca ttt tca cca cct ctt ccc gcc gag aag caa gca ttg gcg gaa aat 1152

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

370 375 380

tct atc ctg ggc tac tat agc aag ata gtc ttc gta tgg gac aag ccg 1200

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

385 390 395 400

tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc caa tcg agc tgt gac 1248

Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp

405 410 415

ccc atc tca ttt gcc aga gat acc agc atc gac gtc gat cga caa tgg 1296

Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val Asp Arg Gln Trp

420 425 430

tcc att acc tgt ttc atg gtc gga gac ccg gga cgg aag tgg tcc caa 1344

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

435 440 445

cag tcc aag cag gta cga caa aag tct gtc tgg gac caa ctc cgc gca 1392

Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala

450 455 460

gcc tac gag aac gcc ggg gcc caa gtc cca gag ccg gcc aac gtg ctc 1440

Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Ala Asn Val Leu

465 470 475 480

gaa atc gag tgg tcg aag cag cag tat ttc caa gga gct ccg agc gcc 1488

Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala

485 490 495

gtc tat ggg ctg aac gat ctc atc aca ctg ggt tcg gcg ctc aga acg 1536

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

500 505 510

ccg ttc aag agt gtt cat ttc gtt gga acg gag acg tct tta gtt tgg 1584

Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp

515 520 525

aaa ggg tat atg gaa ggg gcc ata cga tcg ggt caa cga ggt gct gca 1632

Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala

530 535 540

gaa gtt gtg gct agc ctg gtg cca gca gca taggcggccg c 1673

Glu Val Val Ala Ser Leu Val Pro Ala Ala

545 550

17

554

Exophiala spinifera

17

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

1 5 10 15

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

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Lys Asp Asn Val Ala

85 90 95

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

100 105 110

Lys Val Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp

115 120 125

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

130 135 140

Thr Ile Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser

145 150 155 160

Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln

165 170 175

Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr

180 185 190

Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala

195 200 205

Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser

210 215 220

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

225 230 235 240

Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu

245 250 255

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

260 265 270

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

275 280 285

Leu Ser Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg

290 295 300

Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met Ser Lys Glu Leu

305 310 315 320

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

325 330 335

Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg

340 345 350

Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu

355 360 365

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

370 375 380

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

385 390 395 400

Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp

405 410 415

Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val Asp Arg Gln Trp

420 425 430

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

435 440 445

Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala

450 455 460

Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Ala Asn Val Leu

465 470 475 480

Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala

485 490 495

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

500 505 510

Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp

515 520 525

Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala

530 535 540

Glu Val Val Ala Ser Leu Val Pro Ala Ala

545 550

18

2079

Unknown

GSTKtrAPAO 2079 for bacterial expression

18

atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc 48

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat ttg 96

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag ttt gaa ttg 144

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

ggt ttg gag ttt ccc aat ctt cct tat tat att gat ggt gat gtt aaa 192

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

tta aca cag tct atg gcc atc ata cgt tat ata gct gac aag cac aac 240

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

atg ttg ggt ggt tgt cca aaa gag cgt gca gag att tca atg ctt gaa 288

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

gga gcg gtt ttg gat att aga tac ggt gtt tcg aga att gca tat agt 336

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

aaa gac ttt gaa act ctc aaa gtt gat ttt ctt agc aag cta cct gaa 384

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

atg ctg aaa atg ttc gaa gat cgt tta tgt cat aaa aca tat tta aat 432

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

ggt gat cat gta acc cat cct gac ttc atg ttg tat gac gct ctt gat 480

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

gtt gtt tta tac atg gac cca atg tgc ctg gat gcg ttc cca aaa tta 528

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

gtt tgt ttt aaa aaa cgt att gaa gct atc cca caa att gat aag tac 576

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

ttg aaa tcc agc aag tat ata gca tgg cct ttg cag ggc tgg caa gcc 624

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

acg ttt ggt ggt ggc gac cat cct cca aaa tcg gat ctg gtt ccg cgt 672

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

210 215 220

gga tcc ccg gaa ttc aaa gac aac gtt gcg gac gtg gta gtg gtg ggc 720

Gly Ser Pro Glu Phe Lys Asp Asn Val Ala Asp Val Val Val Val Gly

225 230 235 240

gct ggc ttg agc ggt ttg gag acg gca cgc aaa gtc cag gcc gcc ggt 768

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

245 250 255

ctg tcc tgc ctc gtt ctt gag gcg atg gat cgt gta ggg gga aag act 816

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

260 265 270

ctg agc gta caa tcg ggt ccc ggc agg acg act atc aac gac ctc ggc 864

Leu Ser Val Gln Ser Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly

275 280 285

gct gcg tgg atc aat gac agc aac caa agc gaa gta tcc aga ttg ttt 912

Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe

290 295 300

gaa aga ttt cat ttg gag ggc gag ctc cag agg acg act gga aat tca 960

Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser

305 310 315 320

atc cat caa gca caa gac ggt aca acc act aca gct cct tat ggt gac 1008

Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp

325 330 335

tcc ttg ctg agc gag gag gtt gca agt gca ctt gcg gaa ctc ctc ccc 1056

Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro

340 345 350

gta tgg tct cag ctg atc gaa gag cat agc ctt caa gac ctc aag gcg 1104

Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala

355 360 365

agc cct cag gcg aag cgg ctc gac agt gtg agc ttc gcg cac tac tgt 1152

Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys

370 375 380

gag aag gaa cta aac ttg cct gct gtt ctc ggc gta gca aac cag atc 1200

Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Ala Asn Gln Ile

385 390 395 400

aca cgc gct ctg ctc ggt gtg gaa gcc cac gag atc agc atg ctt ttt 1248

Thr Arg Ala Leu Leu Gly Val Glu Ala His Glu Ile Ser Met Leu Phe

405 410 415

ctc acc gac tac atc aag agt gcc acc ggt ctc agt aat att ttc tcg 1296

Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser

420 425 430

gac aag aaa gac ggc ggg cag tat atg cga tgc aaa aca ggt atg cag 1344

Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln

435 440 445

tcg att tgc cat gcc atg tca aag gaa ctt gtt cca ggc tca gtg cac 1392

Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser Val His

450 455 460

ctc aac acc ccc gtc gct gaa att gag cag tcg gca tcc ggc tgt aca 1440

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

465 470 475 480

gta cga tcg gcc tcg ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt 1488

Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys Val Val Val

485 490 495

tcg tta ccg aca acc ttg tat ccc acc ttg aca ttt tca cca cct ctt 1536

Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu

500 505 510

ccc gcc gag aag caa gca ttg gcg gaa aat tct atc ctg ggc tac tat 1584

Pro Ala Glu Lys Gln Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr

515 520 525

agc aag ata gtc ttc gta tgg gac aag ccg tgg tgg cgc gaa caa ggc 1632

Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly

530 535 540

ttc tcg ggc gtc ctc caa tcg agc tgt gac ccc atc tca ttt gcc aga 1680

Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg

545 550 555 560

gat acc agc atc gac gtc gat cga caa tgg tcc att acc tgt ttc atg 1728

Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met

565 570 575

gtc gga gac ccg gga cgg aag tgg tcc caa cag tcc aag cag gta cga 1776

Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg

580 585 590

caa aag tct gtc tgg gac caa ctc cgc gca gcc tac gag aac gcc ggg 1824

Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly

595 600 605

gcc caa gtc cca gag ccg gcc aac gtg ctc gaa atc gag tgg tcg aag 1872

Ala Gln Val Pro Glu Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys

610 615 620

cag cag tat ttc caa gga gct ccg agc gcc gtc tat ggg ctg aac gat 1920

Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp

625 630 635 640

ctc atc aca ctg ggt tcg gcg ctc aga acg ccg ttc aag agt gtt cat 1968

Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His

645 650 655

ttc gtt gga acg gag acg tct tta gtt tgg aaa ggg tat atg gaa ggg 2016

Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly

660 665 670

gcc ata cga tcg ggt caa cga ggt gct gca gaa gtt gtg gct agc ctg 2064

Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu

675 680 685

gtg cca gca gca tag 2079

Val Pro Ala Ala

690

19

692

Unknown

GSTKtrAPAO 2079 for bacterial expression

19

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

210 215 220

Gly Ser Pro Glu Phe Lys Asp Asn Val Ala Asp Val Val Val Val Gly

225 230 235 240

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

245 250 255

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

260 265 270

Leu Ser Val Gln Ser Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly

275 280 285

Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe

290 295 300

Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser

305 310 315 320

Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp

325 330 335

Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro

340 345 350

Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala

355 360 365

Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys

370 375 380

Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Ala Asn Gln Ile

385 390 395 400

Thr Arg Ala Leu Leu Gly Val Glu Ala His Glu Ile Ser Met Leu Phe

405 410 415

Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser

420 425 430

Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln

435 440 445

Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser Val His

450 455 460

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

465 470 475 480

Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys Val Val Val

485 490 495

Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu

500 505 510

Pro Ala Glu Lys Gln Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr

515 520 525

Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly

530 535 540

Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg

545 550 555 560

Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met

565 570 575

Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg

580 585 590

Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly

595 600 605

Ala Gln Val Pro Glu Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys

610 615 620

Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp

625 630 635 640

Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His

645 650 655

Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly

660 665 670

Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu

675 680 685

Val Pro Ala Ala

690

20

1464

Unknown

KtrAPAO translational fusion with barley

alpha amylase

20

atg gcc aac aag cac ctg agc ctc tcc ctc ttc ctc gtg ctc ctc ggc 48

Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly

1 5 10 15

ctc tcc gcc tcc ctc gcc agc ggc aaa gac aac gtt gcg gac gtg gta 96

Leu Ser Ala Ser Leu Ala Ser Gly Lys Asp Asn Val Ala Asp Val Val

20 25 30

gtg gtg ggc gct ggc ttg agc ggt ttg gag acg gca cgc aaa gtc cag 144

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

35 40 45

gcc gcc ggt ctg tcc tgc ctc gtt ctt gag gcg atg gat cgt gta ggg 192

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

50 55 60

gga aag act ctg agc gta caa tcg ggt ccc ggc agg acg act atc aac 240

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

65 70 75 80

gac ctc ggc gct gcg tgg atc aat gac agc aac caa agc gaa gta tcc 288

Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser

85 90 95

aga ttg ttt gaa aga ttt cat ttg gag ggc gag ctc cag agg acg act 336

Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr

100 105 110

gga aat tca atc cat caa gca caa gac ggt aca acc act aca gct cct 384

Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro

115 120 125

tat ggt gac tcc ttg ctg agc gag gag gtt gca agt gca ctt gcg gaa 432

Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu

130 135 140

ctc ctc ccc gta tgg tct cag ctg atc gaa gag cat agc ctt caa gac 480

Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp

145 150 155 160

ctc aag gcg agc cct cag gcg aag cgg ctc gac agt gtg agc ttc gcg 528

Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala

165 170 175

cac tac tgt gag aag gaa cta aac ttg cct gct gtt ctc ggc gta gca 576

His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Ala

180 185 190

aac cag atc aca cgc gct ctg ctc ggt gtg gaa gcc cac gag atc agc 624

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

195 200 205

atg ctt ttt ctc acc gac tac atc aag agt gcc acc ggt ctc agt aat 672

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

210 215 220

att ttc tcg gac aag aaa gac ggc ggg cag tat atg cga tgc aaa aca 720

Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr

225 230 235 240

ggt atg cag tcg att tgc cat gcc atg tca aag gaa ctt gtt cca ggc 768

Gly Met Gln Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly

245 250 255

tca gtg cac ctc aac acc ccc gtc gct gaa att gag cag tcg gca tcc 816

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

260 265 270

ggc tgt aca gta cga tcg gcc tcg ggc gcc gtg ttc cga agc aaa aag 864

Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys

275 280 285

gtg gtg gtt tcg tta ccg aca acc ttg tat ccc acc ttg aca ttt tca 912

Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser

290 295 300

cca cct ctt ccc gcc gag aag caa gca ttg gcg gaa aat tct atc ctg 960

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

305 310 315 320

ggc tac tat agc aag ata gtc ttc gta tgg gac aag ccg tgg tgg cgc 1008

Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg

325 330 335

gaa caa ggc ttc tcg ggc gtc ctc caa tcg agc tgt gac ccc atc tca 1056

Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser

340 345 350

ttt gcc aga gat acc agc atc gac gtc gat cga caa tgg tcc att acc 1104

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

355 360 365

tgt ttc atg gtc gga gac ccg gga cgg aag tgg tcc caa cag tcc aag 1152

Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys

370 375 380

cag gta cga caa aag tct gtc tgg gac caa ctc cgc gca gcc tac gag 1200

Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu

385 390 395 400

aac gcc ggg gcc caa gtc cca gag ccg gcc aac gtg ctc gaa atc gag 1248

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

405 410 415

tgg tcg aag cag cag tat ttc caa gga gct ccg agc gcc gtc tat ggg 1296

Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly

420 425 430

ctg aac gat ctc atc aca ctg ggt tcg gcg ctc aga acg ccg ttc aag 1344

Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys

435 440 445

agt gtt cat ttc gtt gga acg gag acg tct tta gtt tgg aaa ggg tat 1392

Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr

450 455 460

atg gaa ggg gcc ata cga tcg ggt caa cga ggt gct gca gaa gtt gtg 1440

Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val

465 470 475 480

gct agc ctg gtg cca gca gca tag 1464

Ala Ser Leu Val Pro Ala Ala

485

21

487

Unknown

KtrAPAO translational fusion with barley alpha

amylase

21

Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly

1 5 10 15

Leu Ser Ala Ser Leu Ala Ser Gly Lys Asp Asn Val Ala Asp Val Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser

85 90 95

Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr

100 105 110

Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro

115 120 125

Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu

130 135 140

Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp

145 150 155 160

Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala

165 170 175

His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Ala

180 185 190

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

195 200 205

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

210 215 220

Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr

225 230 235 240

Gly Met Gln Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly

245 250 255

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

260 265 270

Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys

275 280 285

Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser

290 295 300

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

305 310 315 320

Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg

325 330 335

Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser

340 345 350

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

355 360 365

Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys

370 375 380

Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu

385 390 395 400

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

405 410 415

Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly

420 425 430

Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys

435 440 445

Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr

450 455 460

Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val

465 470 475 480

Ala Ser Leu Val Pro Ala Ala

22

atg gca ctt gca ccg agc tac atc aat ccc cca aac gtc gcc tcc cca 48

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

gca ggg tat tct cac gtc ggc gta ggc cca gac gga ggg agg tat gtg 96

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

20 25 30

aca ata gct gga cag att gga caa gac gct tcg ggc gtg aca gac cct 144

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Gly Val Thr Asp Pro

35 40 45

gcc tac gag aaa cag gtt gcc caa gca ttc gcc aat ctg cga gct tgc 192

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

ctt gct gca gtt gga gcc act tca aac gac gtc acc aag ctc aat tac 240

Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

tac atc gtc gac tac gcc ccg agc aaa ctc acc gca att gga gat ggg 288

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

85 90 95

ctg aag gct acc ttt gcc ctt gac agg ctc cct cct tgc acg ctg gtg 336

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

cca gtg tcg gcc ttg tct tca cct gaa tac ctc ttt gag gtt gat gcc 384

Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

acg gcg ctg gtg ccg gga cac acg acc cca gac aac gtt gcg gac gtg 432

Thr Ala Leu Val Pro Gly His Thr Thr Pro Asp Asn Val Ala Asp Val

130 135 140

gta gtg gtg ggc gct ggc ttg agc ggt ttg gag acg gca cgc aaa gtc 480

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

145 150 155 160

cag gcc gcc ggt ctg tcc tgc ctc gtt ctt gag gcg atg gat cgt gta 528

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

ggg gga aag act ctg agc gta caa tcg ggt ccc ggc agg acg act atc 576

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

180 185 190

aac gac ctc ggc gct gcg tgg atc aat gac agc aac caa agc gaa gta 624

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

195 200 205

tcc aga ttg ttt gaa aga ttt cat ttg gag ggc gag ctc cag agg acg 672

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

act gga aat tca atc cat caa gca caa gac ggt aca acc act aca gct 720

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

cct tat ggt gac tcc ttg ctg agc gag gag gtt gca agt gca ctt gcg 768

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

gaa ctc ctc ccc gta tgg tct cag ctg atc gaa gag cat agc ctt caa 816

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

260 265 270

gac ctc aag gcg agc cct cag gcg aag cgg ctc gac agt gtg agc ttc 864

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

gcg cac tac tgt gag aag gaa cta aac ttg cct gct gtt ctc ggc gta 912

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

290 295 300

gca aac cag atc aca cgc gct ctg ctc ggt gtg gaa gcc cac gag atc 960

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

305 310 315 320

agc atg ctt ttt ctc acc gac tac atc aag agt gcc acc ggt ctc agt 1008

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

325 330 335

aat att ttc tcg gac aag aaa gac ggc ggg cag tat atg cga tgc aaa 1056

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

aca ggt atg cag tcg att tgc cat gcc atg tca aag gaa ctt gtt cca 1104

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

355 360 365

ggc tca gtg cac ctc aac acc ccc gtc gct gaa att gag cag tcg gca 1152

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

370 375 380

tcc ggc tgt aca gta cga tcg gcc tcg ggc gcc gtg ttc cga agc aaa 1200

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

aag gtg gtg gtt tcg tta ccg aca acc ttg tat ccc acc ttg aca ttt 1248

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

tca cca cct ctt ccc gcc gag aag caa gca ttg gcg gaa aat tct atc 1296

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

420 425 430

ctg ggc tac tat agc aag ata gtc ttc gta tgg gac aag ccg tgg tgg 1344

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

435 440 445

cgc gaa caa ggc ttc tcg ggc gtc ctc caa tcg agc tgt gac ccc atc 1392

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

tca ttt gcc aga gat acc agc atc gac gtc gat cga caa tgg tcc att 1440

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

465 470 475 480

acc tgt ttc atg gtc gga gac ccg gga cgg aag tgg tcc caa cag tcc 1488

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

aag cag gta cga caa aag tct gtc tgg gac caa ctc cgc gca gcc tac 1536

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

gag aac gcc ggg gcc caa gtc cca gag ccg gcc aac gtg ctc gaa atc 1584

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

515 520 525

gag tgg tcg aag cag cag tat ttc caa gga gct ccg agc gcc gtc tat 1632

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

ggg ctg aac gat ctc atc aca ctg ggt tcg gcg ctc aga acg ccg ttc 1680

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

aag agt gtt cat ttc gtt gga acg gag acg tct tta gtt tgg aaa ggg 1728

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

tat atg gaa ggg gcc ata cga tcg ggt caa cga ggt gct gca gaa gtt 1776

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

gtg gct agc ctg gtg cca gca gca tag 1803

Val Ala Ser Leu Val Pro Ala Ala

595 600

23

600

Exophiala spinifera

23

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

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

20 25 30

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Gly Val Thr Asp Pro

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Thr Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

195 200 205

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

260 265 270

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

290 295 300

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

305 310 315 320

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

325 330 335

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

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

355 360 365

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

370 375 380

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

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

420 425 430

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

435 440 445

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

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

465 470 475 480

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

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

515 520 525

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

Val Ala Ser Leu Val Pro Ala Ala

595 600

24

3003

unknown

KtrAPAO

24

atg gcc aac aag cac ctg agc ctc tcc ctc ttc ctc gtg ctc ctc ggc 48

Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly

1 5 10 15

ctc tcc gcc tcc ctc gcc agc ggc gct cct act gtc aag att gat gct 96

Leu Ser Ala Ser Leu Ala Ser Gly Ala Pro Thr Val Lys Ile Asp Ala

20 25 30

ggg atg gtg gtc ggc acg act act act gtc ccc ggc acc act gcg acc 144

Gly Met Val Val Gly Thr Thr Thr Thr Val Pro Gly Thr Thr Ala Thr

35 40 45

gtc agc gag ttc ttg ggc gtt cct ttt gcc gcc tct ccg aca cga ttt 192

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

50 55 60

gcg cct cct act cgt ccc gtg cct tgg tca acg cct ttg caa gcc act 240

Ala Pro Pro Thr Arg Pro Val Pro Trp Ser Thr Pro Leu Gln Ala Thr

65 70 75 80

gca tat ggt cca gca tgc cct caa caa ttc aat tac ccc gaa gaa ctc 288

Ala Tyr Gly Pro Ala Cys Pro Gln Gln Phe Asn Tyr Pro Glu Glu Leu

85 90 95

cgt gag att acg atg gcc tgg ttc aat aca ccg ccc ccg tca gct ggt 336

Arg Glu Ile Thr Met Ala Trp Phe Asn Thr Pro Pro Pro Ser Ala Gly

100 105 110

gaa agt gag gac tgc ctg aac ctc aac atc tac gtc cca gga act gag 384

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

115 120 125

aac aca aac aaa gcc gtc atg gtt tgg ata tac ggt gga gcg ctg gaa 432

Asn Thr Asn Lys Ala Val Met Val Trp Ile Tyr Gly Gly Ala Leu Glu

130 135 140

tat ggt tgg aat tca ttc cac ctt tac gac ggg gct agt ttc gca gcc 480

Tyr Gly Trp Asn Ser Phe His Leu Tyr Asp Gly Ala Ser Phe Ala Ala

145 150 155 160

aat cag gat gtc atc gcc gtg acc atc aac tac aga acg aac att ctg 528

Asn Gln Asp Val Ile Ala Val Thr Ile Asn Tyr Arg Thr Asn Ile Leu

165 170 175

ggg ttc cct gct gcc cct cag ctt cca ata aca cag cga aat ctg ggg 576

Gly Phe Pro Ala Ala Pro Gln Leu Pro Ile Thr Gln Arg Asn Leu Gly

180 185 190

ttc cta gac caa agg ttt gct ttg gat tgg gta cag cgg aac atc gca 624

Phe Leu Asp Gln Arg Phe Ala Leu Asp Trp Val Gln Arg Asn Ile Ala

195 200 205

gcc ttt ggc ggt gat cct cga aag gtc aca ata ttt ggg cag agt gcg 672

Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Ile Phe Gly Gln Ser Ala

210 215 220

ggg ggc aga agt gtc gac gtc ctc ttg acg tct atg cca cac aac cca 720

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

225 230 235 240

ccc ttc cga gca gca atc atg gag tcc ggt gtg gct aac tac aac ttc 768

Pro Phe Arg Ala Ala Ile Met Glu Ser Gly Val Ala Asn Tyr Asn Phe

245 250 255

ccc aag gga gat ttg tcc gaa cct tgg aac acc act gtt caa gct ctc 816

Pro Lys Gly Asp Leu Ser Glu Pro Trp Asn Thr Thr Val Gln Ala Leu

260 265 270

aac tgt acc acc agt atc gac atc ttg agt tgt atg aga aga gtc gat 864

Asn Cys Thr Thr Ser Ile Asp Ile Leu Ser Cys Met Arg Arg Val Asp

275 280 285

ctc gcc act ctg atg aac acg atc gag caa ctc gga ctt ggg ttt gag 912

Leu Ala Thr Leu Met Asn Thr Ile Glu Gln Leu Gly Leu Gly Phe Glu

290 295 300

tac acg ttg gac aac gta acg gct gtg tac cgt tct gaa acg gct cgc 960

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

305 310 315 320

acg act ggt gac att gct cgt gta cct gtt ctc gtc ggg acg gtg gcc 1008

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

325 330 335

aac gac gga ctt ctc ttt gtc ctc ggg gag aat gac acc caa gca tat 1056

Asn Asp Gly Leu Leu Phe Val Leu Gly Glu Asn Asp Thr Gln Ala Tyr

340 345 350

ctc gag gag gca atc ccg aat cag ccc gac ctt tac cag act ctc ctt 1104

Leu Glu Glu Ala Ile Pro Asn Gln Pro Asp Leu Tyr Gln Thr Leu Leu

355 360 365

gga gca tat ccc att gga tcc cca ggg atc gga tcg cct caa gat cag 1152

Gly Ala Tyr Pro Ile Gly Ser Pro Gly Ile Gly Ser Pro Gln Asp Gln

370 375 380

att gcc gcc att gag acc gag gta aga ttc cag tgt cct tct gcc atc 1200

Ile Ala Ala Ile Glu Thr Glu Val Arg Phe Gln Cys Pro Ser Ala Ile

385 390 395 400

gtg gct cag gac tcc cgg aat cgg ggt atc cct tct tgg cgc tac tac 1248

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

405 410 415

tac aat gcg acc ttt gag aat ctg gag ctt ttc cct ggg tcc gaa gtg 1296

Tyr Asn Ala Thr Phe Glu Asn Leu Glu Leu Phe Pro Gly Ser Glu Val

420 425 430

tac cac agc tct gaa gtc ggg atg gtg ttt ggc acg tat cct gtc gca 1344

Tyr His Ser Ser Glu Val Gly Met Val Phe Gly Thr Tyr Pro Val Ala

435 440 445

agt gcg acc gcc ttg gag gcc cag acg agc aaa tac atg cag ggt gcc 1392

Ser Ala Thr Ala Leu Glu Ala Gln Thr Ser Lys Tyr Met Gln Gly Ala

450 455 460

tgg gcg gcc ttt gcc aaa aac ccc atg aat ggg cct ggg tgg aaa caa 1440

Trp Ala Ala Phe Ala Lys Asn Pro Met Asn Gly Pro Gly Trp Lys Gln

465 470 475 480

gtg ccg aat gtc gcg gcg ctt ggc tca cca ggc aaa gcc atc cag gtt 1488

Val Pro Asn Val Ala Ala Leu Gly Ser Pro Gly Lys Ala Ile Gln Val

485 490 495

gac gtc tct cca gcg aca ata gac caa cga tgt gcc ttg tac acg cgt 1536

Asp Val Ser Pro Ala Thr Ile Asp Gln Arg Cys Ala Leu Tyr Thr Arg

500 505 510

tat tat act gag ttg ggc aca atc gcg ccg agg aca ttt ggc gga ggc 1584

Tyr Tyr Thr Glu Leu Gly Thr Ile Ala Pro Arg Thr Phe Gly Gly Gly

515 520 525

agc ggc gga ggc agc ggc gga ggc agc aaa gac aac gtt gcg gac gtg 1632

Ser Gly Gly Gly Ser Gly Gly Gly Ser Lys Asp Asn Val Ala Asp Val

530 535 540

gta gtg gtg ggc gct ggc ttg agc ggt ttg gag acg gca cgc aaa gtc 1680

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

545 550 555 560

cag gcc gcc ggt ctg tcc tgc ctc gtt ctt gag gcg atg gat cgt gta 1728

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

565 570 575

ggg gga aag act ctg agc gta caa tcg ggt ccc ggc agg acg act atc 1776

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

580 585 590

aac gac ctc ggc gct gcg tgg atc aat gac agc aac caa agc gaa gta 1824

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

595 600 605

tcc aga ttg ttt gaa aga ttt cat ttg gag ggc gag ctc cag agg acg 1872

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

610 615 620

act gga aat tca atc cat caa gca caa gac ggt aca acc act aca gct 1920

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

625 630 635 640

cct tat ggt gac tcc ttg ctg agc gag gag gtt gca agt gca ctt gcg 1968

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

645 650 655

gaa ctc ctc ccc gta tgg tct cag ctg atc gaa gag cat agc ctt caa 2016

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

660 665 670

gac ctc aag gcg agc cct cag gcg aag cgg ctc gac agt gtg agc ttc 2064

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

675 680 685

gcg cac tac tgt gag aag gaa cta aac ttg cct gct gtt ctc ggc gta 2112

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

690 695 700

gca aac cag atc aca cgc gct ctg ctc ggt gtg gaa gcc cac gag atc 2160

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

705 710 715 720

agc atg ctt ttt ctc acc gac tac atc aag agt gcc acc ggt ctc agt 2208

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

725 730 735

aat att ttc tcg gac aag aaa gac ggc ggg cag tat atg cga tgc aaa 2256

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

740 745 750

aca ggt atg cag tcg att tgc cat gcc atg tca aag gaa ctt gtt cca 2304

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

755 760 765

ggc tca gtg cac ctc aac acc ccc gtc gct gaa att gag cag tcg gca 2352

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

770 775 780

tcc ggc tgt aca gta cga tcg gcc tcg ggc gcc gtg ttc cga agc aaa 2400

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

785 790 795 800

aag gtg gtg gtt tcg tta ccg aca acc ttg tat ccc acc ttg aca ttt 2448

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

805 810 815

tca cca cct ctt ccc gcc gag aag caa gca ttg gcg gaa aat tct atc 2496

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

820 825 830

ctg ggc tac tat agc aag ata gtc ttc gta tgg gac aag ccg tgg tgg 2544

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

835 840 845

cgc gaa caa ggc ttc tcg ggc gtc ctc caa tcg agc tgt gac ccc atc 2592

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

850 855 860

tca ttt gcc aga gat acc agc atc gac gtc gat cga caa tgg tcc att 2640

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

865 870 875 880

acc tgt ttc atg gtc gga gac ccg gga cgg aag tgg tcc caa cag tcc 2688

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

885 890 895

aag cag gta cga caa aag tct gtc tgg gac caa ctc cgc gca gcc tac 2736

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

900 905 910

gag aac gcc ggg gcc caa gtc cca gag ccg gcc aac gtg ctc gaa atc 2784

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

915 920 925

gag tgg tcg aag cag cag tat ttc caa gga gct ccg agc gcc gtc tat 2832

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

930 935 940

ggg ctg aac gat ctc atc aca ctg ggt tcg gcg ctc aga acg ccg ttc 2880

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

945 950 955 960

aag agt gtt cat ttc gtt gga acg gag acg tct tta gtt tgg aaa ggg 2928

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

965 970 975

tat atg gaa ggg gcc ata cga tcg ggt caa cga ggt gct gca gaa gtt 2976

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

980 985 990

gtg gct agc ctg gtg cca gca gca tag 3003

Val Ala Ser Leu Val Pro Ala Ala

995 1000

25

1000

unknown

KtrAPAO

25

Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly

1 5 10 15

Leu Ser Ala Ser Leu Ala Ser Gly Ala Pro Thr Val Lys Ile Asp Ala

20 25 30

Gly Met Val Val Gly Thr Thr Thr Thr Val Pro Gly Thr Thr Ala Thr

35 40 45

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

50 55 60

Ala Pro Pro Thr Arg Pro Val Pro Trp Ser Thr Pro Leu Gln Ala Thr

65 70 75 80

Ala Tyr Gly Pro Ala Cys Pro Gln Gln Phe Asn Tyr Pro Glu Glu Leu

85 90 95

Arg Glu Ile Thr Met Ala Trp Phe Asn Thr Pro Pro Pro Ser Ala Gly

100 105 110

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

115 120 125

Asn Thr Asn Lys Ala Val Met Val Trp Ile Tyr Gly Gly Ala Leu Glu

130 135 140

Tyr Gly Trp Asn Ser Phe His Leu Tyr Asp Gly Ala Ser Phe Ala Ala

145 150 155 160

Asn Gln Asp Val Ile Ala Val Thr Ile Asn Tyr Arg Thr Asn Ile Leu

165 170 175

Gly Phe Pro Ala Ala Pro Gln Leu Pro Ile Thr Gln Arg Asn Leu Gly

180 185 190

Phe Leu Asp Gln Arg Phe Ala Leu Asp Trp Val Gln Arg Asn Ile Ala

195 200 205

Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Ile Phe Gly Gln Ser Ala

210 215 220

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

225 230 235 240

Pro Phe Arg Ala Ala Ile Met Glu Ser Gly Val Ala Asn Tyr Asn Phe

245 250 255

Pro Lys Gly Asp Leu Ser Glu Pro Trp Asn Thr Thr Val Gln Ala Leu

260 265 270

Asn Cys Thr Thr Ser Ile Asp Ile Leu Ser Cys Met Arg Arg Val Asp

275 280 285

Leu Ala Thr Leu Met Asn Thr Ile Glu Gln Leu Gly Leu Gly Phe Glu

290 295 300

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

305 310 315 320

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

325 330 335

Asn Asp Gly Leu Leu Phe Val Leu Gly Glu Asn Asp Thr Gln Ala Tyr

340 345 350

Leu Glu Glu Ala Ile Pro Asn Gln Pro Asp Leu Tyr Gln Thr Leu Leu

355 360 365

Gly Ala Tyr Pro Ile Gly Ser Pro Gly Ile Gly Ser Pro Gln Asp Gln

370 375 380

Ile Ala Ala Ile Glu Thr Glu Val Arg Phe Gln Cys Pro Ser Ala Ile

385 390 395 400

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

405 410 415

Tyr Asn Ala Thr Phe Glu Asn Leu Glu Leu Phe Pro Gly Ser Glu Val

420 425 430

Tyr His Ser Ser Glu Val Gly Met Val Phe Gly Thr Tyr Pro Val Ala

435 440 445

Ser Ala Thr Ala Leu Glu Ala Gln Thr Ser Lys Tyr Met Gln Gly Ala

450 455 460

Trp Ala Ala Phe Ala Lys Asn Pro Met Asn Gly Pro Gly Trp Lys Gln

465 470 475 480

Val Pro Asn Val Ala Ala Leu Gly Ser Pro Gly Lys Ala Ile Gln Val

485 490 495

Asp Val Ser Pro Ala Thr Ile Asp Gln Arg Cys Ala Leu Tyr Thr Arg

500 505 510

Tyr Tyr Thr Glu Leu Gly Thr Ile Ala Pro Arg Thr Phe Gly Gly Gly

515 520 525

Ser Gly Gly Gly Ser Gly Gly Gly Ser Lys Asp Asn Val Ala Asp Val

530 535 540

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

545 550 555 560

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

565 570 575

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

580 585 590

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

595 600 605

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

610 615 620

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

625 630 635 640

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

645 650 655

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

660 665 670

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

675 680 685

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

690 695 700

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

705 710 715 720

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

725 730 735

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

740 745 750

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

755 760 765

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

770 775 780

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

785 790 795 800

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

805 810 815

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

820 825 830

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

835 840 845

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

850 855 860

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

865 870 875 880

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

885 890 895

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

900 905 910

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

915 920 925

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

930 935 940

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

945 950 955 960

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

965 970 975

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

980 985 990

Val Ala Ser Leu Val Pro Ala Ala

995 1000

26

2976

Unknown

Barley alpha amylase signal sequenceBEST1

matureartificial spacerKtrAPAO

26

atg gcc aac aag cac ctg agc ctc tcc ctc ttc ctc gtg ctc ctc ggc 48

Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly

-20 -15 -10

ctc tcc gcc tcc ctc gcc agc ggc acg gat ttt ccg gtc cgc agg acc 96

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

-5 -1 1 5

gat ctg ggc cag gtt cag gga ctg gcc ggg gac gtg atg agc ttt cgc 144

Asp Leu Gly Gln Val Gln Gly Leu Ala Gly Asp Val Met Ser Phe Arg

10 15 20

gga ata ccc tat gca gcg ccg ccg gtg ggc ggg ctg cgt tgg aag ccg 192

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

25 30 35 40

ccc caa cac gcc cgg ccc tgg gcg ggc gtt cgc ccc gcc acc caa ttt 240

Pro Gln His Ala Arg Pro Trp Ala Gly Val Arg Pro Ala Thr Gln Phe

45 50 55

ggc tcc gac tgc ttc ggc gcg gcc tat ctt cgc aaa ggc agc ctc gcc 288

Gly Ser Asp Cys Phe Gly Ala Ala Tyr Leu Arg Lys Gly Ser Leu Ala

60 65 70

ccc ggc gtg agc gag gac tgt ctt tac ctc aac gta tgg gcg ccg tca 336

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

75 80 85

ggc gct aaa ccc ggc cag tac ccc gtc atg gtc tgg gtc tac ggc ggc 384

Gly Ala Lys Pro Gly Gln Tyr Pro Val Met Val Trp Val Tyr Gly Gly

90 95 100

ggc ttc gcc ggc ggc acg gcc gcc atg ccc tac tac gac ggc gag gcg 432

Gly Phe Ala Gly Gly Thr Ala Ala Met Pro Tyr Tyr Asp Gly Glu Ala

105 110 115 120

ctt gcg cga cag ggc gtc gtc gtg gtg acg ttt aac tat cgg acg aac 480

Leu Ala Arg Gln Gly Val Val Val Val Thr Phe Asn Tyr Arg Thr Asn

125 130 135

atc ctg ggc ttt ttc gcc cat cct ggt ctc tcg cgc gag agc ccc acc 528

Ile Leu Gly Phe Phe Ala His Pro Gly Leu Ser Arg Glu Ser Pro Thr

140 145 150

gga act tcg ggc aac tac ggc cta ctc gac att ctc gcc gct ctt cgg 576

Gly Thr Ser Gly Asn Tyr Gly Leu Leu Asp Ile Leu Ala Ala Leu Arg

155 160 165

tgg gtg cag agc aac gcc cgc gcc ttc gga ggg gac ccc ggc cga gtg 624

Trp Val Gln Ser Asn Ala Arg Ala Phe Gly Gly Asp Pro Gly Arg Val

170 175 180

acg gtc ttt ggt gaa tcg gcc gga gcg agc gcg atc gga ctt ctg ctc 672

Thr Val Phe Gly Glu Ser Ala Gly Ala Ser Ala Ile Gly Leu Leu Leu

185 190 195 200

acc tcg ccg ctg agc aag ggt ctc ttc cgt ggc gct atc ctc gaa agt 720

Thr Ser Pro Leu Ser Lys Gly Leu Phe Arg Gly Ala Ile Leu Glu Ser

205 210 215

cca ggg ctg acg cga ccg ctc gcg acg ctc gcc gac agc gcc gcc tcg 768

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

220 225 230

ggc gag cgc ctc gac gcc gat ctt tcg cga ctg cgc tcg acc gac cca 816

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

235 240 245

gcc acc ctg atg gcg cgc gcc gac gcg gcc cgc ccg gca tcg cgg gac 864

Ala Thr Leu Met Ala Arg Ala Asp Ala Ala Arg Pro Ala Ser Arg Asp

250 255 260

ctg cgc agg ccg cgt ccg acc gga ccg atc gtc gat ggc cat gtg ctg 912

Leu Arg Arg Pro Arg Pro Thr Gly Pro Ile Val Asp Gly His Val Leu

265 270 275 280

ccg cag acc gac agc gcg gcg atc gcg gcg ggg cag ctg gcg ccg gtt 960

Pro Gln Thr Asp Ser Ala Ala Ile Ala Ala Gly Gln Leu Ala Pro Val

285 290 295

cgg gtc ctg atc gga acc aat gcc gac gaa ggc cgc gcc ttc ctc ggg 1008

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

300 305 310

cgc gcg ccg atg gag acg cca gcg gac tac caa gcc tat ctg gag gcg 1056

Arg Ala Pro Met Glu Thr Pro Ala Asp Tyr Gln Ala Tyr Leu Glu Ala

315 320 325

cag ttt ggc gac caa gcc gcc gcc gtg gcg gcg tgc tat ccc ctc gac 1104

Gln Phe Gly Asp Gln Ala Ala Ala Val Ala Ala Cys Tyr Pro Leu Asp

330 335 340

ggc cgg gcc acg ccc aag gaa atg gtc gcg cgc atc ttc ggc gac aat 1152

Gly Arg Ala Thr Pro Lys Glu Met Val Ala Arg Ile Phe Gly Asp Asn

345 350 355 360

cag ttc aat cgg ggg gtc tcg gcc ttc tcg gaa gcg ctt gtg cgc cag 1200

Gln Phe Asn Arg Gly Val Ser Ala Phe Ser Glu Ala Leu Val Arg Gln

365 370 375

ggc gcg ccc gtg tgg cgt tat cag ttc aac ggt aat acc gag ggt gga 1248

Gly Ala Pro Val Trp Arg Tyr Gln Phe Asn Gly Asn Thr Glu Gly Gly

380 385 390

aga gcg ccg gct acc cac gga gcc gaa att ccc tac gtt ttc ggg gtg 1296

Arg Ala Pro Ala Thr His Gly Ala Glu Ile Pro Tyr Val Phe Gly Val

395 400 405

ttc aag ctc gac gag ttg ggt ctg ttc gat tgg ccg ccc gag ggg ccc 1344

Phe Lys Leu Asp Glu Leu Gly Leu Phe Asp Trp Pro Pro Glu Gly Pro

410 415 420

acg ccc gcc gac cgt gcg ctg ggc caa ctg atg tcc tcc gcc tgg gtc 1392

Thr Pro Ala Asp Arg Ala Leu Gly Gln Leu Met Ser Ser Ala Trp Val

425 430 435 440

cgg ttc gcc aag aat ggc gac ccc gcc ggg gac gcc ctt acc tgg cct 1440

Arg Phe Ala Lys Asn Gly Asp Pro Ala Gly Asp Ala Leu Thr Trp Pro

445 450 455

gcc tat tct acg ggc aag tcg acc atg aca ttc ggt ccc gag ggc cgc 1488

Ala Tyr Ser Thr Gly Lys Ser Thr Met Thr Phe Gly Pro Glu Gly Arg

460 465 470

gcg gcg gtg gtg tcg ccc gga cct tcc atc ccc cct tgc gcg gat ggc 1536

Ala Ala Val Val Ser Pro Gly Pro Ser Ile Pro Pro Cys Ala Asp Gly

475 480 485

gcc aag gcg ggg ggc gga ggc agc ggc gga ggc agc ggc gga ggc agc 1584

Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

490 495 500

aaa gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt 1632

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

505 510 515 520

ttg gag acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt 1680

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

525 530 535

ctt gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg 1728

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

540 545 550

ggt ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat 1776

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

555 560 565

gac agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg 1824

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

570 575 580

gag ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa 1872

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

585 590 595 600

gac ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag 1920

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

605 610 615

gag gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg 1968

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

620 625 630

atc gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag 2016

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

635 640 645

cgg ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac 2064

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

650 655 660

ttg cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc 2112

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

665 670 675 680

ggt gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc 2160

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

685 690 695

aag agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc 2208

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

700 705 710

ggg cag tat atg cga tgc aaa aca ggt atg cag tcg att tgc cat gcc 2256

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala

715 720 725

atg tca aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc 2304

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

730 735 740

gct gaa att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg 2352

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

745 750 755 760

ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc 2400

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

765 770 775

ttg tat ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa 2448

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

780 785 790

gca ttg gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc 2496

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

795 800 805

gta tgg gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc 2544

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

810 815 820

caa tcg agc tgt gac ccc atc tca ttt gcc aga gat acc agc atc gac 2592

Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

825 830 835 840

gtc gat cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga 2640

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

845 850 855

cgg aag tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg 2688

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

860 865 870

gac caa ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag 2736

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

875 880 885

ccg gcc aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa 2784

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

890 895 900

gga gct ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt 2832

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

905 910 915 920

tcg gcg ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag 2880

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

925 930 935

acg tct tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt 2928

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

940 945 950

caa cga ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 2976

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

955 960 965

27

991

Unknown

Barley alpha amylase signal sequenceBEST1

matureartificial spacerKtrAPAO

27

Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly

-20 -15 -10

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

-5 -1 1 5

Asp Leu Gly Gln Val Gln Gly Leu Ala Gly Asp Val Met Ser Phe Arg

10 15 20

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

25 30 35 40

Pro Gln His Ala Arg Pro Trp Ala Gly Val Arg Pro Ala Thr Gln Phe

45 50 55

Gly Ser Asp Cys Phe Gly Ala Ala Tyr Leu Arg Lys Gly Ser Leu Ala

60 65 70

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

75 80 85

Gly Ala Lys Pro Gly Gln Tyr Pro Val Met Val Trp Val Tyr Gly Gly

90 95 100

Gly Phe Ala Gly Gly Thr Ala Ala Met Pro Tyr Tyr Asp Gly Glu Ala

105 110 115 120

Leu Ala Arg Gln Gly Val Val Val Val Thr Phe Asn Tyr Arg Thr Asn

125 130 135

Ile Leu Gly Phe Phe Ala His Pro Gly Leu Ser Arg Glu Ser Pro Thr

140 145 150

Gly Thr Ser Gly Asn Tyr Gly Leu Leu Asp Ile Leu Ala Ala Leu Arg

155 160 165

Trp Val Gln Ser Asn Ala Arg Ala Phe Gly Gly Asp Pro Gly Arg Val

170 175 180

Thr Val Phe Gly Glu Ser Ala Gly Ala Ser Ala Ile Gly Leu Leu Leu

185 190 195 200

Thr Ser Pro Leu Ser Lys Gly Leu Phe Arg Gly Ala Ile Leu Glu Ser

205 210 215

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

220 225 230

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

235 240 245

Ala Thr Leu Met Ala Arg Ala Asp Ala Ala Arg Pro Ala Ser Arg Asp

250 255 260

Leu Arg Arg Pro Arg Pro Thr Gly Pro Ile Val Asp Gly His Val Leu

265 270 275 280

Pro Gln Thr Asp Ser Ala Ala Ile Ala Ala Gly Gln Leu Ala Pro Val

285 290 295

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

300 305 310

Arg Ala Pro Met Glu Thr Pro Ala Asp Tyr Gln Ala Tyr Leu Glu Ala

315 320 325

Gln Phe Gly Asp Gln Ala Ala Ala Val Ala Ala Cys Tyr Pro Leu Asp

330 335 340

Gly Arg Ala Thr Pro Lys Glu Met Val Ala Arg Ile Phe Gly Asp Asn

345 350 355 360

Gln Phe Asn Arg Gly Val Ser Ala Phe Ser Glu Ala Leu Val Arg Gln

365 370 375

Gly Ala Pro Val Trp Arg Tyr Gln Phe Asn Gly Asn Thr Glu Gly Gly

380 385 390

Arg Ala Pro Ala Thr His Gly Ala Glu Ile Pro Tyr Val Phe Gly Val

395 400 405

Phe Lys Leu Asp Glu Leu Gly Leu Phe Asp Trp Pro Pro Glu Gly Pro

410 415 420

Thr Pro Ala Asp Arg Ala Leu Gly Gln Leu Met Ser Ser Ala Trp Val

425 430 435 440

Arg Phe Ala Lys Asn Gly Asp Pro Ala Gly Asp Ala Leu Thr Trp Pro

445 450 455

Ala Tyr Ser Thr Gly Lys Ser Thr Met Thr Phe Gly Pro Glu Gly Arg

460 465 470

Ala Ala Val Val Ser Pro Gly Pro Ser Ile Pro Pro Cys Ala Asp Gly

475 480 485

Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

490 495 500

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

505 510 515 520

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

525 530 535

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

540 545 550

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

555 560 565

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

570 575 580

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

585 590 595 600

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

605 610 615

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

620 625 630

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

635 640 645

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

650 655 660

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

665 670 675 680

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

685 690 695

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

700 705 710

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala

715 720 725

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

730 735 740

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

745 750 755 760

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

765 770 775

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

780 785 790

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

795 800 805

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

810 815 820

Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

825 830 835 840

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

845 850 855

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

860 865 870

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

875 880 885

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

890 895 900

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

905 910 915 920

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

925 930 935

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

940 945 950

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

955 960 965

28

3618

Unknown

gstesp1spKtrAPAO

28

atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa 45

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln

-225 -220 -215

ccc act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag 90

Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu

-210 -205 -200

cat ttg tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag 135

His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys

-195 -190 -185

ttt gaa ttg ggt ttg gag ttt ccc aat ctt cct tat tat att gat 180

Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp

-180 -175 -170

ggt gat gtt aaa tta aca cag tct atg gcc atc ata cgt tat ata 225

Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile

-165 -160 -155

gct gac aag cac aac atg ttg ggt ggt tgt cca aaa gag cgt gca 270

Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala

-150 -145 -140

gag att tca atg ctt gaa gga gcg gtt ttg gat att aga tac ggt 315

Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly

-135 -130 -125

gtt tcg aga att gca tat agt aaa gac ttt gaa act ctc aaa gtt 360

Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val

-120 -115 -110

gat ttt ctt agc aag cta cct gaa atg ctg aaa atg ttc gaa gat cgt 408

Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg

-105 -100 -95

tta tgt cat aaa aca tat tta aat ggt gat cat gta acc cat cct gac 456

Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp

-90 -85 -80

ttc atg ttg tat gac gct ctt gat gtt gtt tta tac atg gac cca atg 504

Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met

-75 -70 -65

tgc ctg gat gcg ttc cca aaa tta gtt tgt ttt aaa aaa cgt att gaa 552

Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu

-60 -55 -50

gct atc cca caa att gat aag tac ttg aaa tcc agc aag tat ata gca 600

Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala

-45 -40 -35 -30

tgg cct ttg cag ggc tgg caa gcc acg ttt ggt ggt ggc gac cat cct 648

Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro

-25 -20 -15

cca aaa tcg gat ctg gtt ccg cgt gga tcc ccg gaa ttc gct cct act 696

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

-10 -5 -1 1

gtc aag att gat gct ggg atg gtg gtc ggc acg act act act gtc ccc 744

Val Lys Ile Asp Ala Gly Met Val Val Gly Thr Thr Thr Thr Val Pro

5 10 15

ggc acc act gcg acc gtc agc gag ttc ttg ggc gtt cct ttt gcc gcc 792

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

20 25 30 35

tct ccg aca cga ttt gcg cct cct act cgt ccc gtg cct tgg tca acg 840

Ser Pro Thr Arg Phe Ala Pro Pro Thr Arg Pro Val Pro Trp Ser Thr

40 45 50

cct ttg caa gcc act gca tat ggt cca gca tgc cct caa caa ttc aat 888

Pro Leu Gln Ala Thr Ala Tyr Gly Pro Ala Cys Pro Gln Gln Phe Asn

55 60 65

tac ccc gaa gaa ctc cgt gag att acg atg gcc tgg ttc aat aca ccg 936

Tyr Pro Glu Glu Leu Arg Glu Ile Thr Met Ala Trp Phe Asn Thr Pro

70 75 80

ccc ccg tca gct ggt gaa agt gag gac tgc ctg aac ctc aac atc tac 984

Pro Pro Ser Ala Gly Glu Ser Glu Asp Cys Leu Asn Leu Asn Ile Tyr

85 90 95

gtc cca gga act gag aac aca aac aaa gcc gtc atg gtt tgg ata tac 1032

Val Pro Gly Thr Glu Asn Thr Asn Lys Ala Val Met Val Trp Ile Tyr

100 105 110 115

ggt gga gcg ctg gaa tat ggt tgg aat tca ttc cac ctt tac gac ggg 1080

Gly Gly Ala Leu Glu Tyr Gly Trp Asn Ser Phe His Leu Tyr Asp Gly

120 125 130

gct agt ttc gca gcc aat cag gat gtc atc gcc gtg acc atc aac tac 1128

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

135 140 145

aga acg aac att ctg ggg ttc cct gct gcc cct cag ctt cca ata aca 1176

Arg Thr Asn Ile Leu Gly Phe Pro Ala Ala Pro Gln Leu Pro Ile Thr

150 155 160

cag cga aat ctg ggg ttc cta gac caa agg ttt gct ttg gat tgg gta 1224

Gln Arg Asn Leu Gly Phe Leu Asp Gln Arg Phe Ala Leu Asp Trp Val

165 170 175

cag cgg aac atc gca gcc ttt ggc ggt gat cct cga aag gtc aca ata 1272

Gln Arg Asn Ile Ala Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Ile

180 185 190 195

ttt ggg cag agt gcg ggg ggc aga agt gtc gac gtc ctc ttg acg tct 1320

Phe Gly Gln Ser Ala Gly Gly Arg Ser Val Asp Val Leu Leu Thr Ser

200 205 210

atg cca cac aac cca ccc ttc cga gca gca atc atg gag tcc ggt gtg 1368

Met Pro His Asn Pro Pro Phe Arg Ala Ala Ile Met Glu Ser Gly Val

215 220 225

gct aac tac aac ttc ccc aag gga gat ttg tcc gaa cct tgg aac acc 1416

Ala Asn Tyr Asn Phe Pro Lys Gly Asp Leu Ser Glu Pro Trp Asn Thr

230 235 240

act gtt caa gct ctc aac tgt acc acc agt atc gac atc ttg agt tgt 1464

Thr Val Gln Ala Leu Asn Cys Thr Thr Ser Ile Asp Ile Leu Ser Cys

245 250 255

atg aga aga gtc gat ctc gcc act ctg atg aac acg atc gag caa ctc 1512

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

260 265 270 275

gga ctt ggg ttt gag tac acg ttg gac aac gta acg gct gtg tac cgt 1560

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

280 285 290

tct gaa acg gct cgc acg act ggt gac att gct cgt gta cct gtt ctc 1608

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

295 300 305

gtc ggg acg gtg gcc aac gac gga ctt ctc ttt gtc ctc ggg gag aat 1656

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

310 315 320

gac acc caa gca tat ctc gag gag gca atc ccg aat cag ccc gac ctt 1704

Asp Thr Gln Ala Tyr Leu Glu Glu Ala Ile Pro Asn Gln Pro Asp Leu

325 330 335

tac cag act ctc ctt gga gca tat ccc att gga tcc cca ggg atc gga 1752

Tyr Gln Thr Leu Leu Gly Ala Tyr Pro Ile Gly Ser Pro Gly Ile Gly

340 345 350 355

tcg cct caa gat cag att gcc gcc att gag acc gag gta aga ttc cag 1800

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

360 365 370

tgt cct tct gcc atc gtg gct cag gac tcc cgg aat cgg ggt atc cct 1848

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

375 380 385

tct tgg cgc tac tac tac aat gcg acc ttt gag aat ctg gag ctt ttc 1896

Ser Trp Arg Tyr Tyr Tyr Asn Ala Thr Phe Glu Asn Leu Glu Leu Phe

390 395 400

cct ggg tcc gaa gtg tac cac agc tct gaa gtc ggg atg gtg ttt ggc 1944

Pro Gly Ser Glu Val Tyr His Ser Ser Glu Val Gly Met Val Phe Gly

405 410 415

acg tat cct gtc gca agt gcg acc gcc ttg gag gcc cag acg agc aaa 1992

Thr Tyr Pro Val Ala Ser Ala Thr Ala Leu Glu Ala Gln Thr Ser Lys

420 425 430 435

tac atg cag ggt gcc tgg gcg gcc ttt gcc aaa aac ccc atg aat ggg 2040

Tyr Met Gln Gly Ala Trp Ala Ala Phe Ala Lys Asn Pro Met Asn Gly

440 445 450

cct ggg tgg aaa caa gtg ccg aat gtc gcg gcg ctt ggc tca cca ggc 2088

Pro Gly Trp Lys Gln Val Pro Asn Val Ala Ala Leu Gly Ser Pro Gly

455 460 465

aaa gcc atc cag gtt gac gtc tct cca gcg aca ata gac caa cga tgt 2136

Lys Ala Ile Gln Val Asp Val Ser Pro Ala Thr Ile Asp Gln Arg Cys

470 475 480

gcc ttg tac acg cgt tat tat act gag ttg ggc aca atc gcg ccg agg 2184

Ala Leu Tyr Thr Arg Tyr Tyr Thr Glu Leu Gly Thr Ile Ala Pro Arg

485 490 495

aca ttt ggc gga ggc agc ggc gga ggc agc ggc gga ggc agc aaa gac 2232

Thr Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Lys Asp

500 505 510 515

aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt ttg gag 2280

Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu Glu

520 525 530

acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt ctt gag 2328

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

535 540 545

gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg ggt ccc 2376

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

550 555 560

ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat gac agc 2424

Gly Arg Thr Thr Ile Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser

565 570 575

aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg gag ggc 2472

Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly

580 585 590 595

gag ctc cag agg acg act gga aat tca atc cat caa gca caa gac ggt 2520

Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly

600 605 610

aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag gag gtt 2568

Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val

615 620 625

gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg atc gaa 2616

Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu

630 635 640

gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag cgg ctc 2664

Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu

645 650 655

gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac ttg cct 2712

Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro

660 665 670 675

gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc ggt gtg 2760

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

680 685 690

gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc aag agt 2808

Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys Ser

695 700 705

gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc ggg cag 2856

Ala Thr Gly Leu Ser Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln

710 715 720

tat atg cga tgc aaa aca ggt atg cag tcg att tgc cat gcc atg tca 2904

Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met Ser

725 730 735

aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc gct gaa 2952

Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val Ala Glu

740 745 750 755

att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg ggc gcc 3000

Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala

760 765 770

gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc ttg tat 3048

Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr

775 780 785

ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa gca ttg 3096

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

790 795 800

gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc gta tgg 3144

Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp

805 810 815

gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc caa tcg 3192

Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser

820 825 830 835

agc tgt gac ccc atc tca ttt gcc aga gat acc agc atc gac gtc gat 3240

Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val Asp

840 845 850

cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga cgg aag 3288

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

855 860 865

tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg gac caa 3336

Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln

870 875 880

ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag ccg gcc 3384

Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Ala

885 890 895

aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa gga gct 3432

Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala

900 905 910 915

ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt tcg gcg 3480

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

920 925 930

ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag acg tct 3528

Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr Ser

935 940 945

tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt caa cga 3576

Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg

950 955 960

ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 3618

Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

965 970 975

29

1205

Unknown

gstesp1spKtrAPAO

29

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln

-225 -220 -215

Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu

-210 -205 -200

His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys

-195 -190 -185

Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp

-180 -175 -170

Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile

-165 -160 -155

Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala

-150 -145 -140

Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly

-135 -130 -125

Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val

-120 -115 -110

Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg

-105 -100 -95

Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp

-90 -85 -80

Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met

-75 -70 -65

Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu

-60 -55 -50

Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala

-45 -40 -35 -30

Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro

-25 -20 -15

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

-10 -5 -1 1

Val Lys Ile Asp Ala Gly Met Val Val Gly Thr Thr Thr Thr Val Pro

5 10 15

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

20 25 30 35

Ser Pro Thr Arg Phe Ala Pro Pro Thr Arg Pro Val Pro Trp Ser Thr

40 45 50

Pro Leu Gln Ala Thr Ala Tyr Gly Pro Ala Cys Pro Gln Gln Phe Asn

55 60 65

Tyr Pro Glu Glu Leu Arg Glu Ile Thr Met Ala Trp Phe Asn Thr Pro

70 75 80

Pro Pro Ser Ala Gly Glu Ser Glu Asp Cys Leu Asn Leu Asn Ile Tyr

85 90 95

Val Pro Gly Thr Glu Asn Thr Asn Lys Ala Val Met Val Trp Ile Tyr

100 105 110 115

Gly Gly Ala Leu Glu Tyr Gly Trp Asn Ser Phe His Leu Tyr Asp Gly

120 125 130

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

135 140 145

Arg Thr Asn Ile Leu Gly Phe Pro Ala Ala Pro Gln Leu Pro Ile Thr

150 155 160

Gln Arg Asn Leu Gly Phe Leu Asp Gln Arg Phe Ala Leu Asp Trp Val

165 170 175

Gln Arg Asn Ile Ala Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Ile

180 185 190 195

Phe Gly Gln Ser Ala Gly Gly Arg Ser Val Asp Val Leu Leu Thr Ser

200 205 210

Met Pro His Asn Pro Pro Phe Arg Ala Ala Ile Met Glu Ser Gly Val

215 220 225

Ala Asn Tyr Asn Phe Pro Lys Gly Asp Leu Ser Glu Pro Trp Asn Thr

230 235 240

Thr Val Gln Ala Leu Asn Cys Thr Thr Ser Ile Asp Ile Leu Ser Cys

245 250 255

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

260 265 270 275

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

280 285 290

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

295 300 305

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

310 315 320

Asp Thr Gln Ala Tyr Leu Glu Glu Ala Ile Pro Asn Gln Pro Asp Leu

325 330 335

Tyr Gln Thr Leu Leu Gly Ala Tyr Pro Ile Gly Ser Pro Gly Ile Gly

340 345 350 355

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

360 365 370

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

375 380 385

Ser Trp Arg Tyr Tyr Tyr Asn Ala Thr Phe Glu Asn Leu Glu Leu Phe

390 395 400

Pro Gly Ser Glu Val Tyr His Ser Ser Glu Val Gly Met Val Phe Gly

405 410 415

Thr Tyr Pro Val Ala Ser Ala Thr Ala Leu Glu Ala Gln Thr Ser Lys

420 425 430 435

Tyr Met Gln Gly Ala Trp Ala Ala Phe Ala Lys Asn Pro Met Asn Gly

440 445 450

Pro Gly Trp Lys Gln Val Pro Asn Val Ala Ala Leu Gly Ser Pro Gly

455 460 465

Lys Ala Ile Gln Val Asp Val Ser Pro Ala Thr Ile Asp Gln Arg Cys

470 475 480

Ala Leu Tyr Thr Arg Tyr Tyr Thr Glu Leu Gly Thr Ile Ala Pro Arg

485 490 495

Thr Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Lys Asp

500 505 510 515

Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly Leu Glu

520 525 530

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

535 540 545

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

550 555 560

Gly Arg Thr Thr Ile Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser

565 570 575

Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly

580 585 590 595

Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly

600 605 610

Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val

615 620 625

Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu

630 635 640

Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu

645 650 655

Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro

660 665 670 675

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

680 685 690

Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys Ser

695 700 705

Ala Thr Gly Leu Ser Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln

710 715 720

Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala Met Ser

725 730 735

Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val Ala Glu

740 745 750 755

Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala

760 765 770

Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr

775 780 785

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

790 795 800

Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp

805 810 815

Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser

820 825 830 835

Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp Val Asp

840 845 850

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

855 860 865

Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln

870 875 880

Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Ala

885 890 895

Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala

900 905 910 915

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

920 925 930

Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu Thr Ser

935 940 945

Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg

950 955 960

Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

965 970 975

30

3591

Unknown

Open reading frame of BEST1KtrAPAO fusion for

bacterial exression vector pGEX-4T-1

30

atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa 45

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln

-225 -220 -215

ccc act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag 90

Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu

-210 -205 -200

cat ttg tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag 135

His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys

-195 -190 -185

ttt gaa ttg ggt ttg gag ttt ccc aat ctt cct tat tat att gat 180

Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp

-180 -175 -170

ggt gat gtt aaa tta aca cag tct atg gcc atc ata cgt tat ata 225

Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile

-165 -160 -155

gct gac aag cac aac atg ttg ggt ggt tgt cca aaa gag cgt gca 270

Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala

-150 -145 -140

gag att tca atg ctt gaa gga gcg gtt ttg gat att aga tac ggt 315

Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly

-135 -130 -125

gtt tcg aga att gca tat agt aaa gac ttt gaa act ctc aaa gtt 360

Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val

-120 -115 -110

gat ttt ctt agc aag cta cct gaa atg ctg aaa atg ttc gaa gat cgt 408

Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg

-105 -100 -95

tta tgt cat aaa aca tat tta aat ggt gat cat gta acc cat cct gac 456

Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp

-90 -85 -80

ttc atg ttg tat gac gct ctt gat gtt gtt tta tac atg gac cca atg 504

Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met

-75 -70 -65

tgc ctg gat gcg ttc cca aaa tta gtt tgt ttt aaa aaa cgt att gaa 552

Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu

-60 -55 -50

gct atc cca caa att gat aag tac ttg aaa tcc agc aag tat ata gca 600

Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala

-45 -40 -35 -30

tgg cct ttg cag ggc tgg caa gcc acg ttt ggt ggt ggc gac cat cct 648

Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro

-25 -20 -15

cca aaa tcg gat ctg gtt ccg cgt gga tcc ccg gaa ttc acg gat ttt 696

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

-10 -5 -1 1

ccg gtc cgc agg acc gat ctg ggc cag gtt cag gga ctg gcc ggg gac 744

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

5 10 15

gtg atg agc ttt cgc gga ata ccc tat gca gcg ccg ccg gtg ggc ggg 792

Val Met Ser Phe Arg Gly Ile Pro Tyr Ala Ala Pro Pro Val Gly Gly

20 25 30 35

ctg cgt tgg aag ccg ccc caa cac gcc cgg ccc tgg gcg ggc gtt cgc 840

Leu Arg Trp Lys Pro Pro Gln His Ala Arg Pro Trp Ala Gly Val Arg

40 45 50

ccc gcc acc caa ttt ggc tcc gac tgc ttc ggc gcg gcc tat ctt cgc 888

Pro Ala Thr Gln Phe Gly Ser Asp Cys Phe Gly Ala Ala Tyr Leu Arg

55 60 65

aaa ggc agc ctc gcc ccc ggc gtg agc gag gac tgt ctt tac ctc aac 936

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

70 75 80

gta tgg gcg ccg tca ggc gct aaa ccc ggc cag tac ccc gtc atg gtc 984

Val Trp Ala Pro Ser Gly Ala Lys Pro Gly Gln Tyr Pro Val Met Val

85 90 95

tgg gtc tac ggc ggc ggc ttc gcc ggc ggc acg gcc gcc atg ccc tac 1032

Trp Val Tyr Gly Gly Gly Phe Ala Gly Gly Thr Ala Ala Met Pro Tyr

100 105 110 115

tac gac ggc gag gcg ctt gcg cga cag ggc gtc gtc gtg gtg acg ttt 1080

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

120 125 130

aac tat cgg acg aac atc ctg ggc ttt ttc gcc cat cct ggt ctc tcg 1128

Asn Tyr Arg Thr Asn Ile Leu Gly Phe Phe Ala His Pro Gly Leu Ser

135 140 145

cgc gag agc ccc acc gga act tcg ggc aac tac ggc cta ctc gac att 1176

Arg Glu Ser Pro Thr Gly Thr Ser Gly Asn Tyr Gly Leu Leu Asp Ile

150 155 160

ctc gcc gct ctt cgg tgg gtg cag agc aac gcc cgc gcc ttc gga ggg 1224

Leu Ala Ala Leu Arg Trp Val Gln Ser Asn Ala Arg Ala Phe Gly Gly

165 170 175

gac ccc ggc cga gtg acg gtc ttt ggt gaa tcg gcc gga gcg agc gcg 1272

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

180 185 190 195

atc gga ctt ctg ctc acc tcg ccg ctg agc aag ggt ctc ttc cgt ggc 1320

Ile Gly Leu Leu Leu Thr Ser Pro Leu Ser Lys Gly Leu Phe Arg Gly

200 205 210

gct atc ctc gaa agt cca ggg ctg acg cga ccg ctc gcg acg ctc gcc 1368

Ala Ile Leu Glu Ser Pro Gly Leu Thr Arg Pro Leu Ala Thr Leu Ala

215 220 225

gac agc gcc gcc tcg ggc gag cgc ctc gac gcc gat ctt tcg cga ctg 1416

Asp Ser Ala Ala Ser Gly Glu Arg Leu Asp Ala Asp Leu Ser Arg Leu

230 235 240

cgc tcg acc gac cca gcc acc ctg atg gcg cgc gcc gac gcg gcc cgc 1464

Arg Ser Thr Asp Pro Ala Thr Leu Met Ala Arg Ala Asp Ala Ala Arg

245 250 255

ccg gca tcg cgg gac ctg cgc agg ccg cgt ccg acc gga ccg atc gtc 1512

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

260 265 270 275

gat ggc cat gtg ctg ccg cag acc gac agc gcg gcg atc gcg gcg ggg 1560

Asp Gly His Val Leu Pro Gln Thr Asp Ser Ala Ala Ile Ala Ala Gly

280 285 290

cag ctg gcg ccg gtt cgg gtc ctg atc gga acc aat gcc gac gaa ggc 1608

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

295 300 305

cgc gcc ttc ctc ggg cgc gcg ccg atg gag acg cca gcg gac tac caa 1656

Arg Ala Phe Leu Gly Arg Ala Pro Met Glu Thr Pro Ala Asp Tyr Gln

310 315 320

gcc tat ctg gag gcg cag ttt ggc gac caa gcc gcc gcc gtg gcg gcg 1704

Ala Tyr Leu Glu Ala Gln Phe Gly Asp Gln Ala Ala Ala Val Ala Ala

325 330 335

tgc tat ccc ctc gac ggc cgg gcc acg ccc aag gaa atg gtc gcg cgc 1752

Cys Tyr Pro Leu Asp Gly Arg Ala Thr Pro Lys Glu Met Val Ala Arg

340 345 350 355

atc ttc ggc gac aat cag ttc aat cgg ggg gtc tcg gcc ttc tcg gaa 1800

Ile Phe Gly Asp Asn Gln Phe Asn Arg Gly Val Ser Ala Phe Ser Glu

360 365 370

gcg ctt gtg cgc cag ggc gcg ccc gtg tgg cgt tat cag ttc aac ggt 1848

Ala Leu Val Arg Gln Gly Ala Pro Val Trp Arg Tyr Gln Phe Asn Gly

375 380 385

aat acc gag ggt gga aga gcg ccg gct acc cac gga gcc gaa att ccc 1896

Asn Thr Glu Gly Gly Arg Ala Pro Ala Thr His Gly Ala Glu Ile Pro

390 395 400

tac gtt ttc ggg gtg ttc aag ctc gac gag ttg ggt ctg ttc gat tgg 1944

Tyr Val Phe Gly Val Phe Lys Leu Asp Glu Leu Gly Leu Phe Asp Trp

405 410 415

ccg ccc gag ggg ccc acg ccc gcc gac cgt gcg ctg ggc caa ctg atg 1992

Pro Pro Glu Gly Pro Thr Pro Ala Asp Arg Ala Leu Gly Gln Leu Met

420 425 430 435

tcc tcc gcc tgg gtc cgg ttc gcc aag aat ggc gac ccc gcc ggg gac 2040

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

440 445 450

gcc ctt acc tgg cct gcc tat tct acg ggc aag tcg acc atg aca ttc 2088

Ala Leu Thr Trp Pro Ala Tyr Ser Thr Gly Lys Ser Thr Met Thr Phe

455 460 465

ggt ccc gag ggc cgc gcg gcg gtg gtg tcg ccc gga cct tcc atc ccc 2136

Gly Pro Glu Gly Arg Ala Ala Val Val Ser Pro Gly Pro Ser Ile Pro

470 475 480

cct tgc gcg gat ggc gcc aag gcg ggg ggc gga ggc agc ggc gga ggc 2184

Pro Cys Ala Asp Gly Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly

485 490 495

agc ggc gga ggc agc aaa gac aac gtt gcg gac gtg gta gtg gtg ggc 2232

Ser Gly Gly Gly Ser Lys Asp Asn Val Ala Asp Val Val Val Val Gly

500 505 510 515

gct ggc ttg agc ggt ttg gag acg gca cgc aaa gtc cag gcc gcc ggt 2280

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

520 525 530

ctg tcc tgc ctc gtt ctt gag gcg atg gat cgt gta ggg gga aag act 2328

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

535 540 545

ctg agc gta caa tcg ggt ccc ggc agg acg act atc aac gac ctc ggc 2376

Leu Ser Val Gln Ser Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly

550 555 560

gct gcg tgg atc aat gac agc aac caa agc gaa gta tcc aga ttg ttt 2424

Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe

565 570 575

gaa aga ttt cat ttg gag ggc gag ctc cag agg acg act gga aat tca 2472

Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser

580 585 590 595

atc cat caa gca caa gac ggt aca acc act aca gct cct tat ggt gac 2520

Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp

600 605 610

tcc ttg ctg agc gag gag gtt gca agt gca ctt gcg gaa ctc ctc ccc 2568

Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro

615 620 625

gta tgg tct cag ctg atc gaa gag cat agc ctt caa gac ctc aag gcg 2616

Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala

630 635 640

agc cct cag gcg aag cgg ctc gac agt gtg agc ttc gcg cac tac tgt 2664

Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys

645 650 655

gag aag gaa cta aac ttg cct gct gtt ctc ggc gta gca aac cag atc 2712

Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Ala Asn Gln Ile

660 665 670 675

aca cgc gct ctg ctc ggt gtg gaa gcc cac gag atc agc atg ctt ttt 2760

Thr Arg Ala Leu Leu Gly Val Glu Ala His Glu Ile Ser Met Leu Phe

680 685 690

ctc acc gac tac atc aag agt gcc acc ggt ctc agt aat att ttc tcg 2808

Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser

695 700 705

gac aag aaa gac ggc ggg cag tat atg cga tgc aaa aca ggt atg cag 2856

Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln

710 715 720

tcg att tgc cat gcc atg tca aag gaa ctt gtt cca ggc tca gtg cac 2904

Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser Val His

725 730 735

ctc aac acc ccc gtc gct gaa att gag cag tcg gca tcc ggc tgt aca 2952

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

740 745 750 755

gta cga tcg gcc tcg ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt 3000

Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys Val Val Val

760 765 770

tcg tta ccg aca acc ttg tat ccc acc ttg aca ttt tca cca cct ctt 3048

Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu

775 780 785

ccc gcc gag aag caa gca ttg gcg gaa aat tct atc ctg ggc tac tat 3096

Pro Ala Glu Lys Gln Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr

790 795 800

agc aag ata gtc ttc gta tgg gac aag ccg tgg tgg cgc gaa caa ggc 3144

Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly

805 810 815

ttc tcg ggc gtc ctc caa tcg agc tgt gac ccc atc tca ttt gcc aga 3192

Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg

820 825 830 835

gat acc agc atc gac gtc gat cga caa tgg tcc att acc tgt ttc atg 3240

Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met

840 845 850

gtc gga gac ccg gga cgg aag tgg tcc caa cag tcc aag cag gta cga 3288

Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg

855 860 865

caa aag tct gtc tgg gac caa ctc cgc gca gcc tac gag aac gcc ggg 3336

Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly

870 875 880

gcc caa gtc cca gag ccg gcc aac gtg ctc gaa atc gag tgg tcg aag 3384

Ala Gln Val Pro Glu Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys

885 890 895

cag cag tat ttc caa gga gct ccg agc gcc gtc tat ggg ctg aac gat 3432

Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp

900 905 910 915

ctc atc aca ctg ggt tcg gcg ctc aga acg ccg ttc aag agt gtt cat 3480

Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His

920 925 930

ttc gtt gga acg gag acg tct tta gtt tgg aaa ggg tat atg gaa ggg 3528

Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly

935 940 945

gcc ata cga tcg ggt caa cga ggt gct gca gaa gtt gtg gct agc ctg 3576

Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu

950 955 960

gtg cca gca gca tag 3591

Val Pro Ala Ala

965

31

1196

Unknown

Open reading frame of BEST1KtrAPAO fusion for

bacterial exression vector pGEX-4T-1

31

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln

-225 -220 -215

Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu

-210 -205 -200

His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys

-195 -190 -185

Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp

-180 -175 -170

Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile

-165 -160 -155

Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala

-150 -145 -140

Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly

-135 -130 -125

Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val

-120 -115 -110

Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg

-105 -100 -95

Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp

-90 -85 -80

Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met

-75 -70 -65

Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu

-60 -55 -50

Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala

-45 -40 -35 -30

Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro

-25 -20 -15

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

-10 -5 -1 1

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

5 10 15

Val Met Ser Phe Arg Gly Ile Pro Tyr Ala Ala Pro Pro Val Gly Gly

20 25 30 35

Leu Arg Trp Lys Pro Pro Gln His Ala Arg Pro Trp Ala Gly Val Arg

40 45 50

Pro Ala Thr Gln Phe Gly Ser Asp Cys Phe Gly Ala Ala Tyr Leu Arg

55 60 65

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

70 75 80

Val Trp Ala Pro Ser Gly Ala Lys Pro Gly Gln Tyr Pro Val Met Val

85 90 95

Trp Val Tyr Gly Gly Gly Phe Ala Gly Gly Thr Ala Ala Met Pro Tyr

100 105 110 115

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

120 125 130

Asn Tyr Arg Thr Asn Ile Leu Gly Phe Phe Ala His Pro Gly Leu Ser

135 140 145

Arg Glu Ser Pro Thr Gly Thr Ser Gly Asn Tyr Gly Leu Leu Asp Ile

150 155 160

Leu Ala Ala Leu Arg Trp Val Gln Ser Asn Ala Arg Ala Phe Gly Gly

165 170 175

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

180 185 190 195

Ile Gly Leu Leu Leu Thr Ser Pro Leu Ser Lys Gly Leu Phe Arg Gly

200 205 210

Ala Ile Leu Glu Ser Pro Gly Leu Thr Arg Pro Leu Ala Thr Leu Ala

215 220 225

Asp Ser Ala Ala Ser Gly Glu Arg Leu Asp Ala Asp Leu Ser Arg Leu

230 235 240

Arg Ser Thr Asp Pro Ala Thr Leu Met Ala Arg Ala Asp Ala Ala Arg

245 250 255

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

260 265 270 275

Asp Gly His Val Leu Pro Gln Thr Asp Ser Ala Ala Ile Ala Ala Gly

280 285 290

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

295 300 305

Arg Ala Phe Leu Gly Arg Ala Pro Met Glu Thr Pro Ala Asp Tyr Gln

310 315 320

Ala Tyr Leu Glu Ala Gln Phe Gly Asp Gln Ala Ala Ala Val Ala Ala

325 330 335

Cys Tyr Pro Leu Asp Gly Arg Ala Thr Pro Lys Glu Met Val Ala Arg

340 345 350 355

Ile Phe Gly Asp Asn Gln Phe Asn Arg Gly Val Ser Ala Phe Ser Glu

360 365 370

Ala Leu Val Arg Gln Gly Ala Pro Val Trp Arg Tyr Gln Phe Asn Gly

375 380 385

Asn Thr Glu Gly Gly Arg Ala Pro Ala Thr His Gly Ala Glu Ile Pro

390 395 400

Tyr Val Phe Gly Val Phe Lys Leu Asp Glu Leu Gly Leu Phe Asp Trp

405 410 415

Pro Pro Glu Gly Pro Thr Pro Ala Asp Arg Ala Leu Gly Gln Leu Met

420 425 430 435

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

440 445 450

Ala Leu Thr Trp Pro Ala Tyr Ser Thr Gly Lys Ser Thr Met Thr Phe

455 460 465

Gly Pro Glu Gly Arg Ala Ala Val Val Ser Pro Gly Pro Ser Ile Pro

470 475 480

Pro Cys Ala Asp Gly Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly

485 490 495

Ser Gly Gly Gly Ser Lys Asp Asn Val Ala Asp Val Val Val Val Gly

500 505 510 515

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

520 525 530

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

535 540 545

Leu Ser Val Gln Ser Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly

550 555 560

Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe

565 570 575

Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser

580 585 590 595

Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp

600 605 610

Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro

615 620 625

Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala

630 635 640

Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys

645 650 655

Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Ala Asn Gln Ile

660 665 670 675

Thr Arg Ala Leu Leu Gly Val Glu Ala His Glu Ile Ser Met Leu Phe

680 685 690

Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser

695 700 705

Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln

710 715 720

Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser Val His

725 730 735

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

740 745 750 755

Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys Val Val Val

760 765 770

Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu

775 780 785

Pro Ala Glu Lys Gln Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr

790 795 800

Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly

805 810 815

Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg

820 825 830 835

Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met

840 845 850

Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg

855 860 865

Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly

870 875 880

Ala Gln Val Pro Glu Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys

885 890 895

Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp

900 905 910 915

Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His

920 925 930

Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly

935 940 945

Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu

950 955 960

Val Pro Ala Ala

965

32

1803

Unknown

Glyc(-) APAO coding sequence; mutation in

putative glycosylation sites

32

atg gca ctt gca ccg agc tac atc aat ccc cca aac gtc gcc tcc cca 48

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

gca ggg tat tct cac gtc ggc gta ggc cca gac gga ggg agg tat gtg 96

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

20 25 30

aca ata gct gga cag att gga caa gac gct tcg ggc gtg aca gac cct 144

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Gly Val Thr Asp Pro

35 40 45

gcc tac gag aaa cag gtt gcc caa gca ttc gcc aat ctg cga gct tgc 192

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

ctt gct gca gtt gga gcc act tca aac gac gtc acc aag ctc aat tac 240

Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

tac atc gtc gac tac gcc ccg agc aaa ctc acc gca att gga gat ggg 288

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

85 90 95

ctg aag gct acc ttt gcc ctt gac agg ctc cct cct tgc acg ctg gtg 336

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

cca gtg tcg gcc ttg tct tca cct gaa tac ctc ttt gag gtt gat gcc 384

Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

acg gcg ctg gtg ccg gga cac acg acc cca gac aac gtt gcg gac gtg 432

Thr Ala Leu Val Pro Gly His Thr Thr Pro Asp Asn Val Ala Asp Val

130 135 140

gta gtg gtg ggc gct ggc ttg agc ggt ttg gag acg gca cgc aaa gtc 480

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

145 150 155 160

cag gcc gcc ggt ctg tcc tgc ctc gtt ctt gag gcg atg gat cgt gta 528

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

ggg gga aag act ctg agc gta caa tcg ggt ccc ggc agg acg act atc 576

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

180 185 190

aac gac ctc ggc gct gcg tgg atc aat gat agc aat cag gcc gaa gta 624

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ala Glu Val

195 200 205

tcc aga ttg ttt gaa aga ttt cat ttg gag ggc gag ctc cag agg acg 672

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

act gga aat tca atc cat caa gca caa gac ggt aca acc act aca gct 720

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

cct tat ggt gac tcc ttg ctg agc gag gag gtt gca agt gca ctt gcg 768

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

gaa ctc ctc ccc gta tgg tct cag ctg atc gaa gag cat agc ctt caa 816

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

260 265 270

gac ctc aag gcg agc cct cag gcg aag cgg ctc gac agt gtg agc ttc 864

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

gcg cac tac tgt gag aag gaa cta aac ttg cct gct gtt ctc ggc gta 912

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

290 295 300

gca aac cag atc aca cgc gct ctg ctc ggt gtg gaa gcc cac gag atc 960

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

305 310 315 320

agc atg ctt ttt ctc acc gac tac atc aag agt gcc acc ggt ctc agt 1008

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

325 330 335

aat att ttc tcg gac aag aaa gac ggc ggg cag tat atg cga tgc aaa 1056

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

aca ggt atg cag tcg att tgc cat gcc atg tca aag gaa ctt gtt cca 1104

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

355 360 365

ggc tca gtg cac ctc aac acc ccc gtc gct gaa att gag cag tcg gca 1152

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

370 375 380

tcc ggc tgt aca gta cga tcg gcc tcg ggc gcc gtg ttc cga agc aaa 1200

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

aag gtg gtg gtt tcg tta ccg aca acc ttg tat ccc acc ttg aca ttt 1248

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

tca cca cct ctt ccc gcc gag aag caa gca ttg gcg gaa aat tct atc 1296

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

420 425 430

ctg ggc tac tat agc aag ata gtc ttc gta tgg gac aag ccg tgg tgg 1344

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

435 440 445

cgc gaa caa ggc ttc tcg ggc gtc ctc caa tcg agc tgt gac ccc atc 1392

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

tca ttt gcc aga gat acc agc atc gac gtc gat cga caa tgg tcc att 1440

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

465 470 475 480

acc tgt ttc atg gtc gga gac ccg gga cgg aag tgg tcc caa cag tcc 1488

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

aag cag gta cga caa aag tct gtc tgg gac caa ctc cgc gca gcc tac 1536

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

gag aac gcc ggg gcc caa gtc cca gag ccg gcc aac gtg ctc gaa atc 1584

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

515 520 525

gag tgg tcg aag cag cag tat ttc caa gga gct ccg agc gcc gtc tat 1632

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

ggg ctg aac gat ctc atc aca ctg ggt tcg gcg ctc aga acg ccg ttc 1680

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

aag agt gtt cat ttc gtt gga acg gag acg tct tta gtt tgg aaa ggg 1728

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

tat atg gaa ggg gcc ata cga tcg ggt caa cga ggt gct gca gaa gtt 1776

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

gtg gct agc ctg gtg cca gca gca tag 1803

Val Ala Ser Leu Val Pro Ala Ala

595 600

33

600

Unknown

Glyc(-) APAO coding sequence; mutation in

putative glycosylation sites

33

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

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

20 25 30

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Gly Val Thr Asp Pro

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Thr Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ala Glu Val

195 200 205

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

260 265 270

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

290 295 300

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

305 310 315 320

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

325 330 335

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

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

355 360 365

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

370 375 380

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

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

420 425 430

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

435 440 445

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

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

465 470 475 480

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

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

515 520 525

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

Val Ala Ser Leu Val Pro Ala Ala

595 600

34

37

Artificial Sequence

37-mer oligonucleotide

34

ggggaattca tggcacttgc accgagctac atcaatc 37

35

atggcacttg caccgagcta catcaatccc ccaaacgtcg cctccccagc agggtattcc 60

cacatcggcg taggcccaaa cgaagcgagg tatgtgacaa tagctggaca gattggacaa 120

gacgctttgg gcgtgacaga cccagcctac gagaaacagg ttgcccaagc attcgccaat 180

ctgcgagctt gccttgctgc agttggagcc tcttcaaacg acgtcaccaa gctcaattac 240

tacatcgtcg actacgcccc gagcaaactc accgcaattg gagatgggct gaagtctacc 300

tttgcccttg acaggctccc tccttgcacg ctggtgccag taccggcctt ggcttcacct 360

gaatacctct ttgaggttga tgccacggcg ctggtgccag gacactcgac cccagacaac 420

gttgcggacg tggtagtggt gggcgctggc ttgagcggtt tggagacggc acgcaaagtc 480

caggccgccg gtctgtcctg cctcgttctt gaggcgatgg atcgtgtagg gggaaagact 540

ctgagcgtac aatcgggtcc cggcaggacg actatcaacg acctcggcgc tgcgtggatc 600

aatgacagca accaaagcga agtatccaga ttgtttgaaa gatttcattt ggagggcgag 660

ctccagagga cgaccggaaa ttcaatccat caagcacaag acggtacaac cactacagct 720

ccttatggtg actccccggt aagcacaatc ccactttgtg atgagacctc tgtcgagtgt 780

agaatacagt cactgactcc acttcgtcca gctgagcgag gaggttgcaa gtgcacttgc 840

ggaactcctc cccgtatggt ctcagctgat cgaagagtat agccttgaag accccaaggc 900

gagccctcag gcgaagcggc tcgacagtgt gagcttcgcg cactactgtg agaaggacct 960

aaacttgcct gctgttctca gcgtggcaaa ccagatcaca cgcgctctgc tcggtgtgga 1020

agcccacgag atcagcatgc tttttctcac cgactacatc aagagtgcca ccggtctcag 1080

taatattgtc tcggacaaga aagacggcgg gcagtatatg cgatgcaaaa caggtgcgtg 1140

cggtgtcctc tcaggtaggg gactcgtttc ttagtggtca ttccaggtat gcagtcgatt 1200

tgccatgcca tgtcaaagga acttgttcca ggctcagtgc acctcaacac ccccgtcgct 1260

ggaattgagc agtcggcgtc cggctgtata gtacgatcgg cctcgggcgc cgtgttccga 1320

agcaaaaagg tggtggtttc gttaccgaca acattgtatc ccaccttgac attttcacca 1380

cctcttcccg ccgagaagca agcattggcg gaaaaatcta tcctcggcta ctatagcaag 1440

atagtcttcg tatgggacaa cccgtggtgg cgcgaacaag gcttctcggg cgtcctccaa 1500

tcgagctgtg accccatctc atttgccaga gataccagca tcgaagtcga tcggcaatgg 1560

tccattacct gtttcatggt cggagacccg ggacggaagt ggtcccaaca gtccaagcag 1620

gtacgacaaa agtctgtctg ggaccaactc cgcgcagcct acgagaacgc cggggcccaa 1680

gtcccagagc cggccaacgt gctcgaaatc gagtggtcga agcagcagta tttccaagga 1740

gctccgagcg ccgtctatgg gctgaacgat ctcatcacac tgggttcggc gctcagaacg 1800

ccgttcaagt gtgttcattt cgttggaacg gagacgtctt tagtttggaa agggtatatg 1860

gaaggggcca tacgatcggg tcaacgaggt gctgcagaag ttgtggctag cctggtgcca 1920

gcagcatag 1929

36

600

Exophilia spinifera

36

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

Ala Gly Tyr Ser His Ile Gly Val Gly Pro Asn Glu Ala Arg Tyr Val

20 25 30

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

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

Leu Ala Ala Val Gly Ala Ser Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Leu Lys Ser Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Pro Ala Leu Ala Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Ser Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

195 200 205

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

Pro Tyr Gly Asp Ser Pro Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu Tyr Ser Leu Glu

260 265 270

Asp Pro Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

Ala His Tyr Cys Glu Lys Asp Leu Asn Leu Pro Ala Val Leu Ser Val

290 295 300

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

305 310 315 320

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

325 330 335

Asn Ile Val Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

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

355 360 365

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

370 375 380

Ser Gly Cys Ile Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

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

420 425 430

Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp Asp Asn Pro Trp Trp

435 440 445

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

Ser Phe Ala Arg Asp Thr Ser Ile Glu Val Asp Arg Gln Trp Ser Ile

465 470 475 480

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

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

515 520 525

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

Lys Cys Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

Val Ala Ser Leu Val Pro Ala Ala

595 600

37

atggcacttg caccgagcta catcaatccc ccaaacgtcg cctccccagc agggtattcc 60

cacatcggcg taggcccaaa cgaagcgagg tatgtgacaa tagctggaca gattggacaa 120

gacgctttgg gcgtgacaga cccagcctac gagaaacagg ttgcccaagc attcgccaat 180

ctgcgagctt gccttgctgc agttggagcc tcttcaaacg acgtcaccaa gctcaattac 240

tacatcgtcg actacgcccc gagcaaactc accgcaattg gagatgggct gaagtctacc 300

tttgcccttg acaggctccc tccttgcacg ctggtgccag taccggcctt ggcttcacct 360

gaatacctct ttgaggttga cgccacggcg ctggtgccag gacactcgac cccagacaac 420

gttgcggacg tggtagtggt gggcgctggc ttgagcggct tggagacggc acgcaaagtc 480

caggccgccg gtctgtcctg cctcgttctt gaggcgatgg atcgtgtagg gggaaagact 540

ctgagcgtac aatcgggtcc cggcaggacg actatcaacg acctcggcgc tgcgtggatc 600

aatgacagca accaaagcga agtatccaga ttgtttgaaa gatttcattt ggagggcgag 660

ctccagagga cgaccggaaa ttcaatccat caagcacaag acggtacaac cactacagct 720

ccttatggtg actccccggt aagcacaatc ccactttgtg atgagacctc tgtcgagtgt 780

agaatacagt cactgactcc acttcgtcca gctgagcgag gaggttgcaa gtgcacttgc 840

ggaactcctc cccgtatggt ctcagctgat cgaagagtat agccttgaag accccaaggc 900

gagccctcag gcgaagcggc tcgacagtgt gagcttcgcg cactactgtg agaaggacct 960

aaacttgcct gctgttctca gcgtggcaaa ccagatcaca cgcgctctgc tcggtgtgga 1020

agcccacgag atcagcatgc tttttctcac cgactacatc aagagtgcca ccggtctcag 1080

taatattgtc tcggacaaga aagacggcgg gcagtatatg cgatgcaaaa caggtgcgtg 1140

cggtgtcctc tcaggtaggg gactcgtttc ttagtggtca ttccaggtat gcagtcgatt 1200

tgccatgcca tgtcaaagga acttgttcca ggctcagtgc acctcaacac ccccgtcgct 1260

ggaattgagc agtcggcgtc cggctgtata gtacgatcgg cctcgggcgc cgtgttccga 1320

agcaaaaagg tggtggtttc gttaccgaca acattgtatc ccaccttgac attttcacca 1380

cctcttcccg ccgagaagca agcattggcg gaaaaatcta tcctcggcta ctatagcaag 1440

atagtcttcg tatgggacaa cccgtggtgg cgcgaacaag gcttctcggg cgtcctccaa 1500

tcgagctgtg accccatctc atttgccaga gataccagca tcgaagtcga tcggcaatgg 1560

tccattacct gtttcatggt cggagacccg ggacggaagt ggtcccaaca gtccaagcag 1620

gtacgacaaa agtctgtctg ggaccaactc cgcgcagcct acgagaacgc cggggcccaa 1680

gtcccagagc cggccaacgt gctcgaaatc gagtggtcga agcagcagta tttccaagga 1740

gctccgagcg ccgtctatgg gctgaacgat ctcatcacac tgggttcggc gctcagaacg 1800

ccgttcaagt gtgttcattt cgttggaacg gagacgtctt tagtttggaa agggtatatg 1860

gaaggggcca tacgatcggg tcaacgaggt gctgcagaag ttgtggctag cctggtgcca 1920

gcagcatag 1929

38

600

Exophiala spinifera

38

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

Ala Gly Tyr Ser His Ile Gly Val Gly Pro Asn Glu Ala Arg Tyr Val

20 25 30

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

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

Leu Ala Ala Val Gly Ala Ser Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Leu Lys Ser Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Pro Ala Leu Ala Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Ser Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

195 200 205

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

Pro Tyr Gly Asp Ser Pro Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu Tyr Ser Leu Glu

260 265 270

Asp Pro Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

Ala His Tyr Cys Glu Lys Asp Leu Asn Leu Pro Ala Val Leu Ser Val

290 295 300

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

305 310 315 320

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

325 330 335

Asn Ile Val Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

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

355 360 365

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

370 375 380

Ser Gly Cys Ile Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

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

420 425 430

Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp Asp Asn Pro Trp Trp

435 440 445

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

Ser Phe Ala Arg Asp Thr Ser Ile Glu Val Asp Arg Gln Trp Ser Ile

465 470 475 480

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

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

515 520 525

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

Lys Cys Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

Val Ala Ser Leu Val Pro Ala Ala

595 600

39

atggcacttg caccgagcta catcaatccc ccaaacgtcg cctccccagc agggtattct 60

cacgtcggcg taggcccaga cggagggagg tatgtgacaa tagctggaca gattggacaa 120

gacgcttcgg gcgtgacaga ccctgcctac gagaaacagg ttgcccaagc attcgccaat 180

ctgcgagctt gccttgctgc agttggagcc acttcaaacg acgtcaccaa gctcaattac 240

tacatcgtcg actacgcccc gagcaaactc accgcaattg gagatgggct gaaggctacc 300

tttgcccttg acaggctccc tccttgcacg ctggtgccag tgtcggcctt gtcttcacct 360

gaatacctct ttgaggttga tgccacggcg ctggtgccgg gacacacgac cccagacaac 420

gttgcggacg tggtagtggt gggcgctggc ttgagcggtt tggagacggc acgcaaagtc 480

caggccgccg gtctgtcctg cctcgttctt gaggcgatgg atcgtgtagg gggaaagact 540

ctgagcgtac aatcgggtcc cggcaggacg actatcaacg acctcggcgc tgcgtggatc 600

aatgacagca accaaagcga agtatccaga ttgtttgaaa gatttcatnt ggagggcgag 660

ctccagagga cgactggaaa ttcaatccat caagcacaag acggtacaac cactacagct 720

ccttatggtg actccttggt aagcacaatc ccactttgtg atgagacctc tgtcgagtgt 780

agaatacagt cactgattcc acttcgtcca gctgagcgag gaggttgcaa gtgcacttgc 840

ggaactcctc cccgtatggt ctcagctgat cgaagagcat agccttcaag acctcaaggc 900

gagccctcag gcgaagcggc tcgacagtgt gagcttcgcg cactactgtg agaaggaact 960

aaacttgcct gctgttctcg gcgtagcaaa ccagatcaca cgcgctctgc tcggtgtgga 1020

agcccacgag atcagcatgc tttttctcac cgactacatc aagagtgcca ccggtctcag 1080

taatattttc tcggacaaga aagacggcgg gcagtatatg cgatgcaaaa caggtgcgtg 1140

tggtgtcgtc tcaggtgggg gactcgtttc tcaagtggtc atttcaggta tgcagtcgat 1200

ttgccatgcc atgtcaaagg aacttgttcc aggctcagtg cacctcaaca cccccgtcgc 1260

tgaaattgag cagtcggcat ccggctgtac agtacgatcg gcctcgggcg ccgtgttccg 1320

aagcaaaaag gtggtggttt cgttaccgac aaccttgtat cccaccttga cattttcacc 1380

acctctcccc gccgagaagc aagcattggc ggaaaattct atcctgggct actatagcaa 1440

gatagtcttc gtatgggaca agccgtggtg gcgcgaacaa ggcttctcgg gcgtcctcca 1500

atcgagctgt gaccccatct catttgccag agataccagc atcgacgtcg atcgacaatg 1560

gtccattacc tgtttcatgg tcggagaccc gggacggaag tggtcccaac agtccaagca 1620

ggtacgacaa aagtctgtct gggaccaact ccgcgcagcc tacgagaacg ccggggccca 1680

agtcccagag ccggccaacg tgctcgaaat cgagtggtcg aagcagcagt atttccaagg 1740

agctccgagc gccgtctatg ggctgaacga tctcatcaca ctgggttcgg cgctcagaac 1800

gccgttcaag agtgttcatt tcgttggaac ggagacgtct ttagtttgga aagggtatat 1860

ggaaggggcc atacgatcgg gtcaacgagg tgctgcagaa gttgtggcta gcctggtgcc 1920

agcagcatag 1930

40

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

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

20 25 30

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Gly Val Thr Asp Pro

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Thr Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Ser

195 200 205

Arg Leu Phe Glu Arg Phe His Xaa Glu Gly Glu Leu Gln Arg Thr Thr

210 215 220

Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro

225 230 235 240

Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu

245 250 255

Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln Asp

260 265 270

Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe Ala

275 280 285

His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val Asn

290 295 300

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

305 310 315 320

Leu Phe Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Leu Ser Asn Ile

325 330 335

Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly

340 345 350

Met Gln Ser Ile Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser

355 360 365

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

370 375 380

Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys Lys Val

385 390 395 400

Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe Ser Pro

405 410 415

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

420 425 430

Tyr Tyr Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg Glu

435 440 445

Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser Phe

450 455 460

Ala Arg Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Ser Ile Thr Cys

465 470 475 480

Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys Gln

485 490 495

Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr Glu Asn

500 505 510

Ala Gly Ala Gln Val Pro Glu Pro Ala Asn Val Leu Glu Ile Glu Trp

515 520 525

Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr Gly Leu

530 535 540

Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys Ser

545 550 555 560

Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr Met

565 570 575

Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val Ala

580 585 590

Ser Leu Val Pro Ala Ala

595

41

atggcacttg caccgagcta catcaatccc ccaaacctcg cctccccagc agggtattcc 60

cacgtcggcg taggcccaaa cggagggagg tatgcgacaa tagctggaca gattggacaa 120

gacgcttcgg ccgtgacaga ccctgcctac gagaaacagg ttgcccaagc attcgccaac 180

ctgcgagctt gtcttgctgc agttggagcc acttcaaacg acattaccaa gctcaattac 240

tacatcgtcg actacaaccc gagcaaactc accgcaattg gagatgggct gaaggctacc 300

tttgcccttg acaggctccc tccttgcacg ctggtgccag tgccggccct ggcttcacct 360

gaatacccct ttgaggttga tgccacggcg ctggttccag gacactcaac cccagacaat 420

gttgcggacg tggtcgtggt gggcgctggc ttgagcggtt tggagacggc acgcaaagtc 480

caggctgccg ggctgtcctg cctcgttctt gaggcgatgg atcgtgtggg gggaaagact 540

ctgagcgtac aatcgggtcc cggcaggacg gctatcaatg acctcggcgc tgcgtggatc 600

aatgacagca accaaagcga agtattcaaa ttatttgaaa gatttcattt ggagggcgag 660

ctccagagga cgaccggaaa ttcaatccat caagcacaag acggtacaac cactacagct 720

ccttatggtg attccctggt aagcacaatt ccatcttgtg atgagacctc tgtcgtgtgt 780

agaatacagt cgctgactcc acatcgtcca gctgagcgag gaggttgcaa gtgcactcgc 840

ggaactcctt cccgcatggt ctcagctgat cgaagagcat agtcttgaag accccaaggc 900

gagccctcaa gcgaagcagc tcgacagtgt gagcttcgca cactactgtg agaaggatct 960

aagcttgcct gctgttctcg gcgtggcaaa ccagatcaca cgcgctctgc tcggtgtgga 1020

agcccacgag atcagcatgc tttttctcac cgactacatc aagagtgcca ccggtctcag 1080

taatattgtc tcggataaga aagacggtgg gcagtatatg cgatgcaaaa caggtgcgtg 1140

tggtgttctc tcagtgggag actcgtttct tagtggtcat tccaggtatg cagtcgcttt 1200

gccatgccat gtcaaaggaa cttgttccag gctcagtgca cctcaacacc cccgtcgccg 1260

aaattgagca gtcggcatcc ggctgtacag tacgatcggc ctcgggcggc gtgttccgaa 1320

gtaaaaaggt ggtggtttcg ttaccgacaa ccttgtatcc caccttgata ttttcaccac 1380

ctcttcccgc cgagaagcaa gcattggctg aaaaatccat cctgggctac tatagcaaga 1440

tagtcttcgt atgggacaag ccgtggtggc gcgaacaagg cttctcgggc gtcctccaat 1500

cgagctgtga ccccatctca tttgccagag ataccagcat cgaagtcgat cggcaatggt 1560

ccattacctg tttcatggtc ggagacccgg gacggaagtg gtcccaacag tccaagcagg 1620

tacgacagaa gtctgtctgg aaccaactcc gcgcagccta cgagaacgcc ggggcccaag 1680

tcccagagcc ggccaacgtg ctcgagatcg agtggtcgaa gcagcagtat ttccaaggag 1740

cgccgagcgt cgtctatggg ctgaactgtc tcaacacact gggttcggcg ctcagaacgc 1800

cgttcaaggg tgttcatttc gttggaacgg agacgtcttt ggtttggaaa gggtatatgg 1860

aaggggccat acgatcgggt cagcgaggcg ctgcagaagt tgtggctagc ctggtgccag 1920

cagcatag 1928

42

598

Rhinocladiella atrovirens

42

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Leu Ala Ser Pro

1 5 10 15

Ala Gly Tyr Ser His Val Gly Val Gly Pro Asn Gly Gly Arg Tyr Ala

20 25 30

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Ala Val Thr Asp Pro

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

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

65 70 75 80

Tyr Ile Val Asp Tyr Asn Pro Ser Lys Leu Thr Ala Ile Gly Asp Gly

85 90 95

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Pro Ala Leu Ala Ser Pro Glu Tyr Pro Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Ser Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu Leu

245 250 255

Leu Pro Ala Trp Ser Gln Leu Ile Glu Glu His Ser Leu Glu Asp Pro

260 265 270

Lys Ala Ser Pro Gln Ala Lys Gln Leu Asp Ser Val Ser Phe Ala His

275 280 285

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

290 295 300

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

305 310 315 320

Leu Phe Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Leu Ser Asn Ile

325 330 335

Val Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly

340 345 350

Met Gln Ser Leu Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser

355 360 365

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

370 375 380

Cys Thr Val Arg Ser Ala Ser Gly Gly Val Phe Arg Ser Lys Lys Val

385 390 395 400

Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Ile Phe Ser Pro

405 410 415

Pro Leu Pro Ala Glu Lys Gln Ala Leu Ala Glu Lys Ser Ile Leu Gly

420 425 430

Tyr Tyr Ser Lys Ile Val Phe Val Trp Asp Lys Pro Trp Trp Arg Glu

435 440 445

Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile Ser Phe

450 455 460

Ala Arg Asp Thr Ser Ile Glu Val Asp Arg Gln Trp Ser Ile Thr Cys

465 470 475 480

Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser Lys Gln

485 490 495

Val Arg Gln Lys Ser Val Trp Asn Gln Leu Arg Ala Ala Tyr Glu Asn

500 505 510

Ala Gly Ala Gln Val Pro Glu Pro Ala Asn Val Leu Glu Ile Glu Trp

515 520 525

Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Val Val Tyr Gly Leu

530 535 540

Asn Cys Leu Asn Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe Lys Gly

545 550 555 560

Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly Tyr Met

565 570 575

Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val Val Ala

580 585 590

Ser Leu Val Pro Ala Ala

595

43

atggcacttg caccgagcta catcaatccc ccaaacctcg cctccccagc agggtattcc 60

tacgtcggcg taggcccaaa cggagggagg tatgtgacaa tagctggaca gattggacaa 120

gacgcttcgg ccgtgacaga ccctgcctac gagaaacagg ttgcccaagc attcgccaac 180

ctgcgagctt gtcttgctgc agttggagcc acttcaaacg acattaccaa gctcaattac 240

tacatcgtcg actacaaccc gagcaaactc accgcaattg gagatgggct gaaggctacc 300

tttgcccttg acaggctccc tccttgcacg ctggtgccag tgccggccct ggcttcacct 360

gaatacctct ttgaggttga tgccacggcg ctggttccag gacactcaac cccagacaat 420

gttgcggacg tggtcgtggt gggcgctggc ttgagcggtt tggagacggc acgcaaagtc 480

caggctgccg ggctgtcctg cctcgttctt gaggcgatgg atcgtgtggg gggaaagact 540

ctgagcgtac aatcgggtcc cggcaggacg actatcaatg acctcggcgc tgcgtggatc 600

aatgacagca accaaagcga agtattcaaa ttatttgaaa gatttcattt ggagggcgag 660

ctccagagga cgaccggaaa ttcaatccat caagcacaag acggtacaac cactacagct 720

ccttatggtg attccctggt aagcacaatt ccatcttgtg atgagacctc tgtcgtgtgt 780

agaatacagt cgctgactcc acatcgtcca gctgagcgag gaggttgcaa gtgcactcgc 840

ggaactcctt cccgcatggt ctcagctgat cgaagagcat agtcttgaag accccaaggc 900

gagccctcaa gcgaagcagc tcgacagtgt gagcttcgca cactactgtg agaaggatct 960

aaacttgcct gctgttctcg gcgtggcaaa ccagatcaca cgcgctctgc tcggtgtgga 1020

agcccacgag atcagcatgt tttttctcac cgactacatc aagagtgcca ccggtctcag 1080

taatattgtc tcggataaga aagacggtgg gcagtatatg cgatgcaaaa caggtgcgtg 1140

tggtgttctc tcagtgggag actcgtttct tagtggtcat tccaggtatg cagtcgcttt 1200

gccatgccat gtcaaaggaa cttgttccag gctcagtgca cctcaacacc cccgtcgccg 1260

aaattgagca gtcggcatcc ggctgtacag tacgatcggc ctcgggcggc gtgttccgaa 1320

gtaaaaaggt ggtggtttcg ttaccgacaa ccttgtatcc caccttgata ttttcaccac 1380

ctcttcccgc cgagaagcaa gcattggctg aaaaatccat cctgggctac tatagcaaga 1440

tagtcttcgt atgggacaag ccgtggtggc gcgaacaagg cttctcgggc gtcctccaat 1500

cgagctgtga ccccatctca tttgccagag ataccagcat cgaagtcgat cggcaatggt 1560

ccattacctg tttcatggtc ggagacccgg gacggaagtg gtcccaacag tccaagcagg 1620

tacgacagaa gtctgtctgg aaccaactcc gcgcagccta cgagaacgcc ggggcccaag 1680

tcccagagcc ggccaacgtg ctcgagatcg agtggtcgaa gcagcagtat ttccaaggag 1740

cgccgagcgc cgtctatggg ctgaactgtc tcaacacact gggttcggcg ctcagaacgc 1800

cgttcaaggg tgttcatttc gttggaacgg agacgtcttt ggtttggaaa gggtatatgg 1860

aaggggccat acgatcgggt cagcgaggcg ctgcagaagt tgtggctagc ctggtgccag 1920

cagcatag 1928

44

591

Rhinocladiella atrovirens

44

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Leu Ala Ser Pro

1 5 10 15

Ala Gly Tyr Ser Tyr Val Gly Val Gly Pro Asn Gly Gly Arg Tyr Val

20 25 30

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Ala Val Thr Asp Pro

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

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

65 70 75 80

Tyr Ile Val Asp Tyr Asn Pro Ser Lys Leu Thr Ala Ile Gly Asp Gly

85 90 95

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Pro Ala Leu Ala Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Ser Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Lys

195 200 205

Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr Gly

210 215 220

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

225 230 235 240

Gly Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu Leu Leu

245 250 255

Pro Ala Ser Gln Leu Ile Glu Glu His Ser Leu Glu Asp Pro Lys Ala

260 265 270

Ser Pro Gln Ala Lys Gln Leu Asp Ser Val Ser Phe Ala His Tyr Cys

275 280 285

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

290 295 300

Ala Leu Leu Gly Val Glu Ala His Glu Ile Ser Met Phe Phe Leu Thr

305 310 315 320

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

325 330 335

Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Leu

340 345 350

Cys His Ala Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn

355 360 365

Thr Pro Val Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg

370 375 380

Ser Ala Ser Gly Gly Val Phe Arg Ser Lys Lys Val Val Leu Pro Thr

385 390 395 400

Leu Tyr Pro Thr Leu Ile Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

405 410 415

Ala Leu Ala Glu Lys Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

420 425 430

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

435 440 445

Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Glu

450 455 460

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

465 470 475 480

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

485 490 495

Asn Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Glu

500 505 510

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

515 520 525

Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn Cys Leu Asn Thr Leu Gly

530 535 540

Ser Ala Leu Arg Thr Pro Phe Lys Gly Val His Phe Val Gly Thr Glu

545 550 555 560

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

565 570 575

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

580 585 590

45

atggcacttg caccgagcta catcaatccc ccaaacctcg cctccccagc agggtattcc 60

cacgtcggcg taggcccaaa cggagggagg tatgtgacaa tagctggaca gattggacaa 120

gacgcttcgg ccgtgacaga ccctgcctac gagaaacagg ttgcccaagc attcgccaac 180

ctgcgagctt gtcttgctgc agttggagcc acttcaaacg acattaccaa gctcaattac 240

tacatcgtcg actacaaccc gagcaaactc accgcaattg gagatgggct gaaggctacc 300

tttgcccttg acaggctccc tccttgcacg ctggtgccag tgccggccct ggcttcacct 360

gaatacctct ttgaggttga tgctacggcg ctggttccag gacactcaac cccagacaat 420

gttgcggacg tggtcgtggt gggcgctggc ttgagcggtt tggagacggc acgcaaagtc 480

caggctgccg ggctgtcctg cctcgttctt gaggcgatgg atcgtgtggg gggaaagact 540

ctgagcgtac aatcgggtcc cggcaggacg actatcaatg acctcggcgc tgcgtggatc 600

aatgacagca accaaagcga agtattcaaa ttatttgaaa gatttcattt ggagggcgag 660

ctccagagga cgaccggaaa ttcaatccat caagcacaag acggtacaac cactacagct 720

ccttatggtg attccctggt aggcacaatt ccatcttgtg atgagacctc tgtcgtgtgt 780

agaatacagt cgctgactcc acatcgtcca gctgagcgag gaggttgcaa gtgcactcgc 840

ggaactcctt cccgcatggt ctcagctgat cgaagagcat agtcttgaag accccaaggc 900

gagccctcaa gcgaagcagc tcgacagtgt gagcttcgca cactactgtg agaaggatct 960

aaacttgcct gctgttctcg gcgtggcaaa ccagatcaca cgcgctctgc tcggtgtgga 1020

agcccacgag atcagcatgc tttttctcac cgactacatc aagagtgcca ccggtctcag 1080

taatattgtc tcggataaga aagacggtgg gcagtatatg cgatgcaaaa caggtgcgtg 1140

tggtgttctc tcagtgggag actcgtttct tagtggtcat tccaggtatg cagtcgcttt 1200

gccatgccat gtcaaaggaa cttgttccag gctcagtgca cctcaacacc cccgtcgccg 1260

aaattgagca gtcggcatcc ggctgtacag tacgatcggc ctcgggcggc gtgttccgaa 1320

gtaaaaaggt ggtggtttcg ttaccgacaa ccttgtatcc caccttgata ttttcaccac 1380

ctcttcccgc cgagaagcaa gcattggctg aaaaatccat cctgggctac tatagcaaga 1440

tagtcttcgt atgggacaag ctgtggtggc gcgaacaagg cttctcgggc gtcctccaat 1500

cgagctgtga ccccatctca tttgccagag ataccagcat cgaagtcgat cggcaatggt 1560

ccattacctg tttcatggtc ggagacccgg gacggaagtg gtcccaacag tccaagcagg 1620

tacgacagaa gtctgtctgg aaccaactcc gcgcagccta cgagaacgcc ggggcccaag 1680

tcccagagcc ggccaacgtg ctcgagatcg agtggtcgaa gcagcagtat ttccaaggag 1740

cgccgagcgc cgtctatggg ctgaactgtc tcaacacact gggttcggcg ctcagaacgc 1800

cgttcaaggg tgttcatttc gttggaacgg agacgtcttt ggtttggaaa gggtatatgg 1860

aaggggccat acgatcgggt cagcgaggcg ctgcagaagt tgtgcctagc ctggtgccag 1920

cagcatag 1928

46

591

Rhinocladiella atrovirens

46

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Ala Ser Pro Ala

1 5 10 15

Gly Tyr Ser His Val Gly Val Gly Pro Asn Gly Gly Arg Tyr Val Thr

20 25 30

Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Ala Val Thr Asp Pro Ala

35 40 45

Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys Leu

50 55 60

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

65 70 75 80

Ile Val Asp Tyr Asn Pro Ser Lys Leu Thr Ala Ile Gly Asp Gly Leu

85 90 95

Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val Pro

100 105 110

Val Pro Ala Leu Ala Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala Thr

115 120 125

Ala Leu Val Pro Gly His Ser Thr Pro Asp Asn Val Ala Asp Val Val

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val Phe

195 200 205

Lys Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr Thr

210 215 220

Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala Pro

225 230 235 240

Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala Glu

245 250 255

Leu Leu Pro Ala Trp Ser Gln Leu Ile Glu Glu His Ser Leu Glu Asp

260 265 270

Pro Lys Ala Ser Pro Gln Ala Lys Gln Leu Asp Ser Val Ser Phe Ala

275 280 285

His Tyr Cys Glu Lys Asp Leu Asn Leu Pro Ala Val Leu Gly Val Ala

290 295 300

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

305 310 315 320

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

325 330 335

Ile Val Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys Thr

340 345 350

Gly Met Gln Ser Leu Cys His Ala Met Ser Lys Glu Leu Val Pro Gly

355 360 365

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

370 375 380

Gly Cys Thr Val Arg Ser Ala Ser Gly Gly Val Phe Arg Ser Lys Lys

385 390 395 400

Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Ile Phe Ser Pro Leu

405 410 415

Pro Ala Glu Lys Gln Ala Leu Ala Glu Lys Ser Ile Gly Tyr Tyr Ser

420 425 430

Lys Ile Val Phe Val Asp Lys Leu Trp Trp Arg Glu Gln Gly Phe Ser

435 440 445

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

450 455 460

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

465 470 475 480

Pro Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val

485 490 495

Trp Asn Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro

500 505 510

Glu Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe

515 520 525

Gln Ala Pro Ser Ala Val Tyr Gly Leu Asn Cys Leu Asn Thr Leu Gly

530 535 540

Ser Ala Leu Arg Thr Pro Phe Lys Gly Val His Phe Val Gly Thr Glu

545 550 555 560

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

565 570 575

Gln Arg Gly Ala Ala Glu Val Val Pro Ser Leu Val Pro Ala Ala

580 585 590

47

600

Exophiala spinifera

47

Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pro Asn Val Ala Ser Pro

1 5 10 15

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

20 25 30

Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Ser Gly Val Thr Asp Pro

35 40 45

Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Ala Asn Leu Arg Ala Cys

50 55 60

Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Val Thr Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pro Pro Cys Thr Leu Val

100 105 110

Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Leu Phe Glu Val Asp Ala

115 120 125

Thr Ala Leu Val Pro Gly His Thr Thr Pro Asp Asn Val Ala Asp Val

130 135 140

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

145 150 155 160

Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Ala Met Asp Arg Val

165 170 175

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

180 185 190

Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Glu Val

195 200 205

Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gln Arg Thr

210 215 220

Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gly Thr Thr Thr Thr Ala

225 230 235 240

Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Leu Ala

245 250 255

Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Glu His Ser Leu Gln

260 265 270

Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Ser Phe

275 280 285

Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gly Val

290 295 300

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

305 310 315 320

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

325 330 335

Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cys Lys

340 345 350

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

355 360 365

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

370 375 380

Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Val Phe Arg Ser Lys

385 390 395 400

Lys Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Thr Phe

405 410 415

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

420 425 430

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

435 440 445

Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pro Ile

450 455 460

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

465 470 475 480

Thr Cys Phe Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gln Ser

485 490 495

Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Ala Tyr

500 505 510

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

515 520 525

Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Val Tyr

530 535 540

Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pro Phe

545 550 555 560

Lys Ser Val His Phe Val Gly Thr Glu Thr Ser Leu Val Trp Lys Gly

565 570 575

Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Glu Val

580 585 590

Val Ala Ser Leu Val Pro Ala Ala

595 600

48

1392

Unknown

Cys (-) APAO; removal of cysteine 461

48

aaa gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt 48

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

ttg gag acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt 96

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

20 25 30

ctt gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg 144

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

ggt ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat 192

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

50 55 60

gac agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg 240

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

gag ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa 288

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

gac ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag 336

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

gag gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg 384

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

atc gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag 432

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

cgg ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac 480

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

ttg cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc 528

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

165 170 175

ggt gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc 576

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

aag agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc 624

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

195 200 205

ggg cag tat atg cga tgc aaa aca ggt atg cag tcg att tgc cat gcc 672

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala

210 215 220

atg tca aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc 720

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

gct gaa att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg 768

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc 816

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

ttg tat ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa 864

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

gca ttg gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc 912

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

gta tgg gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc 960

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

caa tcg agc tcc gac ccc atc tca ttt gcc aga gat acc agc atc gac 1008

Gln Ser Ser Ser Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

gtc gat cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga 1056

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

cgg aag tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg 1104

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

gac caa ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag 1152

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

370 375 380

ccg gcc aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa 1200

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

gga gct ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt 1248

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

405 410 415

tcg gcg ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag 1296

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

acg tct tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt 1344

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

caa cga ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 1392

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

49

463

Unknown

Cys (-) APAO; removal of cysteine 461

49

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

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

20 25 30

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

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

50 55 60

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

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

165 170 175

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

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

195 200 205

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cys His Ala

210 215 220

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

Gln Ser Ser Ser Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

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

370 375 380

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

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

405 410 415

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

50

1392

Unknown

Cys (-) APAO; removal of cysteines 359 and 461

50

aaa gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt 48

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

ttg gag acg gca cgc aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gtt 96

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

20 25 30

ctt gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg 144

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

ggt ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat 192

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

50 55 60

gac agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg 240

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

gag ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa 288

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

gac ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag 336

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

gag gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg 384

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

atc gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag 432

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

cgg ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac 480

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

ttg cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc 528

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

165 170 175

ggt gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc 576

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

aag agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc 624

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

195 200 205

ggg cag tat atg cga tgc aaa aca ggt atg cag tcg att tcg cat gcc 672

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Ser His Ala

210 215 220

atg tca aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc 720

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

gct gaa att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg 768

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc 816

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

ttg tat ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa 864

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

gca ttg gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc 912

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

gta tgg gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc 960

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

caa tcg agc tcc gac ccc atc tca ttt gcc aga gat acc agc atc gac 1008

Gln Ser Ser Ser Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

gtc gat cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga 1056

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

cgg aag tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg 1104

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

gac caa ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag 1152

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

370 375 380

ccg gcc aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa 1200

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

gga gct ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt 1248

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

405 410 415

tcg gcg ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag 1296

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

acg tct tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt 1344

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

caa cga ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 1392

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

51

463

Unknown

Cys (-) APAO; removal of cysteines 359 and 461

51

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

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

20 25 30

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

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

50 55 60

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

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

165 170 175

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

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

195 200 205

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Ser His Ala

210 215 220

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

Gln Ser Ser Ser Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

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

370 375 380

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

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

405 410 415

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

52

1392

Unknown

Cys (-) APAO; removal of cysteines 169, 359,

and 461

52

aaa gac aac gtt gcg gac gtg gta gtg gtg ggc gct ggc ttg agc ggt 48

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

ttg gag acg gca cgc aaa gtc cag gcc gcc ggt ctg agc tcc ctc gtt 96

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

20 25 30

ctt gag gcg atg gat cgt gta ggg gga aag act ctg agc gta caa tcg 144

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

ggt ccc ggc agg acg act atc aac gac ctc ggc gct gcg tgg atc aat 192

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

50 55 60

gac agc aac caa agc gaa gta tcc aga ttg ttt gaa aga ttt cat ttg 240

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

gag ggc gag ctc cag agg acg act gga aat tca atc cat caa gca caa 288

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

gac ggt aca acc act aca gct cct tat ggt gac tcc ttg ctg agc gag 336

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

gag gtt gca agt gca ctt gcg gaa ctc ctc ccc gta tgg tct cag ctg 384

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

atc gaa gag cat agc ctt caa gac ctc aag gcg agc cct cag gcg aag 432

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

cgg ctc gac agt gtg agc ttc gcg cac tac tgt gag aag gaa cta aac 480

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

ttg cct gct gtt ctc ggc gta gca aac cag atc aca cgc gct ctg ctc 528

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

165 170 175

ggt gtg gaa gcc cac gag atc agc atg ctt ttt ctc acc gac tac atc 576

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

aag agt gcc acc ggt ctc agt aat att ttc tcg gac aag aaa gac ggc 624

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

195 200 205

ggg cag tat atg cga tgc aaa aca ggt atg cag tcg att tcg cat gcc 672

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Ser His Ala

210 215 220

atg tca aag gaa ctt gtt cca ggc tca gtg cac ctc aac acc ccc gtc 720

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

gct gaa att gag cag tcg gca tcc ggc tgt aca gta cga tcg gcc tcg 768

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

ggc gcc gtg ttc cga agc aaa aag gtg gtg gtt tcg tta ccg aca acc 816

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

ttg tat ccc acc ttg aca ttt tca cca cct ctt ccc gcc gag aag caa 864

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

gca ttg gcg gaa aat tct atc ctg ggc tac tat agc aag ata gtc ttc 912

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

gta tgg gac aag ccg tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ctc 960

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

caa tcg agc tcc gac ccc atc tca ttt gcc aga gat acc agc atc gac 1008

Gln Ser Ser Ser Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

gtc gat cga caa tgg tcc att acc tgt ttc atg gtc gga gac ccg gga 1056

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

cgg aag tgg tcc caa cag tcc aag cag gta cga caa aag tct gtc tgg 1104

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

gac caa ctc cgc gca gcc tac gag aac gcc ggg gcc caa gtc cca gag 1152

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

370 375 380

ccg gcc aac gtg ctc gaa atc gag tgg tcg aag cag cag tat ttc caa 1200

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

gga gct ccg agc gcc gtc tat ggg ctg aac gat ctc atc aca ctg ggt 1248

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

405 410 415

tcg gcg ctc aga acg ccg ttc aag agt gtt cat ttc gtt gga acg gag 1296

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

acg tct tta gtt tgg aaa ggg tat atg gaa ggg gcc ata cga tcg ggt 1344

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

caa cga ggt gct gca gaa gtt gtg gct agc ctg gtg cca gca gca tag 1392

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

53

463

Unknown

Cys (-) APAO; removal of cysteines 169, 359,

and 461

53

Lys Asp Asn Val Ala Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10 15

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

20 25 30

Leu Glu Ala Met Asp Arg Val Gly Gly Lys Thr Leu Ser Val Gln Ser

35 40 45

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

50 55 60

Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Phe His Leu

65 70 75 80

Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gln

85 90 95

Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Glu

100 105 110

Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Leu

115 120 125

Ile Glu Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Lys

130 135 140

Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cys Glu Lys Glu Leu Asn

145 150 155 160

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

165 170 175

Gly Val Glu Ala His Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Ile

180 185 190

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

195 200 205

Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Ile Ser His Ala

210 215 220

Met Ser Lys Glu Leu Val Pro Gly Ser Val His Leu Asn Thr Pro Val

225 230 235 240

Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Ser

245 250 255

Gly Ala Val Phe Arg Ser Lys Lys Val Val Val Ser Leu Pro Thr Thr

260 265 270

Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gln

275 280 285

Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Phe

290 295 300

Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Leu

305 310 315 320

Gln Ser Ser Ser Asp Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile Asp

325 330 335

Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gly

340 345 350

Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Ser Val Trp

355 360 365

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

370 375 380

Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gln

385 390 395 400

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

405 410 415

Ser Ala Leu Arg Thr Pro Phe Lys Ser Val His Phe Val Gly Thr Glu

420 425 430

Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gly

435 440 445

Gln Arg Gly Ala Ala Glu Val Val Ala Ser Leu Val Pro Ala Ala

450 455 460

54

11

Exophiala spinifera

54

Asp Val Val Val Val Gly Ala Gly Leu Ser Gly

1 5 10