The present invention relates to agglutination genes of agglutinative yeast, and to yeast which contains them.

BACKGROUND ART In the fermentation industry, yeast agglutination is an industrially important phenomenon, and the use of agglutinative yeast is being studied while much research is being undertaken to discover the cause of its agglutination. Yeast agglutination is known to be controlled by a plurality of genes, a relatively well researched example thereof, being the, agglutination gene called FL01, which is mapped on the right arm of yeast chromosome I.

Regarding the structure of the agglutination gene FL01 derived from the yeast Saccharomyces cerevisiae, it had been completely unknown, but in 1989 it was cloned for the first time by the present inventors et al., and its restriction enzyme cleavage map has been determined (Watari et al.. Agricultural and Biological Chemistry, Vol. 53, No. 3, p.901-903, 1989). (Nevertheless, it base sequence was unknown) .

We the present inventors reported that it is possible to convert non-agglutinative industrial yeasts into

agglutinative yeasts for practical use by the introduction of the agglutination gene FLOl into various industrial yeasts (Watari et al.. Agricultural and Biological Chemistry, Vol. 55, No. 6, p.1547-1552, 1991); however, it was not always possible to impart strong and stable agglutinative properties to all of the industrial yeasts.

We the present inventors thereafter diligently pursued research on the FLOl gene, and discovered that the gene that we the present inventors et al. had reported as being the FLOl gene (Watari et al.. Agricultural and Biological Chemistry, Vol. 53, No. 3, p.901-903, 1989) was not the intact FLOl gene as present on chromosome I of the yeast Saccharomyces cerevisiae strain ABXL-ID, but was the FLOl gene with a portion thereof deleted during maintenance of the plasmid containing the intact FLOl gene in Escherichia coli strain K12 (hereunder, this gene shall be referred to as FL01S). DISCLOSURE OF INVENTION

The object of the present invention is to establish the structure of the intact FLOl gene (hereunder, this gene shall be referred to as FL01L) , and to provide a technique for imparting stronger and more stable agglutinative properties to various industrial yeasts. Now, we the present inventors, as the result of varied research regarding the FLOl gene, have succeeded

in isolating the intact FLOl gene, or FL01L gene, have determined the entire base sequence of the gene, and further have discovered that by using the FL01L gene, it is possible to breed various yeasts for practical use which have stronger and more stable agglutinative ability, compared with using the FL01S gene, and thus the present invention has been completed.

In other words, the present invention relates to an agglutination gene of 4.7 ±0.2 kb in yeast which codes for a polypeptide which exhibits agglutinative activity, and specifically, it relates to the above mentioned agglutination gene which is derived from the yeast Saccharomyces cerevisiae and is defined by the restriction enzyme cleavage map in Fig. 1, and more specifically, it relates to the above mentioned agglutination gene which substantially codes for the amino acid sequence listed as Sequence No. 1. The present invention also relates to an agglutination gene of 2.6 ±0.2 kb in yeast which codes for a polypeptide which exhibits agglutinative activity, and specifically to an agglutination gene which is derived from the yeast Saccharomyces cerevisiae and is defined by the restriction enzyme cleavage map in Fig. 2, and more specifically, to an agglutination gene which substantially codes for the amino acid sequence listed as Sequence No. 2.

The present invention further relates to yeasts

containing either of the above mentioned agglutination genes and having agglutinative properties.

"Agglutination gene" as mentioned in the present specification is used to mean a gene which controls agglutination of yeast. Effect of the Invention

As described above, the agglutination genes according to the present invention are capable of imparting agglutinative properties to the non-agglutinative yeast Saccharomyces cerevisiae. Here, the significance of using agglutinative yeasts in the fermentation industry is that 1) the cells may be rapidly separated from the fermented mash after completion of fermentation, and thus the process may be simplified so that there is no need for other procedures for the separation of the yeast from the fermented mash involving use of a centrifugal separator, etc.; 2) the clarity of the fermented mash is high, and thus the burden is reduced during the final filtration of the fermented mash, and productivity is increased; 3) continuous fermentation is possible in the same manner as with immobilized cells, and no reactors or other special equipment are necessary. Furthermore, breeding of agglutinative yeast has been attempted in the past using an induction method, cross-breeding method, cell fusion method, etc. for natural or artificial mutants, but it is often reported that these methods are necessarily accompanied by a change in the genetic

properties of the original strain to be bred, also usually destroying the desirable properties of the original strain. However, according to the present invention, it is possible to improve the agglutinative properties of the strain to be grown simply by introduction thereinto of the genes according to the present invention, and the fact that they do not damage the other desirable properties of the original strain is their major advantage. BRIEF DESCRIPTION OF DRAWINGS

Fig. 1

A restriction enzyme cleavage map of the FL01L gene according to the present invention.

In the figure, the cleavage sites of each of the restriction enzymes are represented by Ac for AccI, Bg for Bglll, RV for EcoRV, K for Kpnl and Pv for PvuII. Fig. 2

A restriction enzyme cleavage map of the FL01S gene according to the present invention. In the figure, the cleavage sites of each of the restriction enzymes are represented by Ac for AccI, Bg for Bglll, RV for EcoRV, K for Kpnl and Pv for PvuII. Fig. 3

A flow chart for the preparation of plasmids YRpGLF14S and YRpGLF8L containing the agglutination genes FL01S and FL01L, for direct selection of the yeasts. Fig. 4

YCpHF19S ( 20 . 00 Kb ) . Fig . 5

YIpHFl9S (15.80 Kb). Fig. 6 5.8 kb BamHI-XhoI fragment of YCpHF19S containing the FL01S gene. Fig. 7

YCpHF19L (22.10 Kb).

Fig- 8 7.9 kb BamHI-XhoI fragment of YCpHF19L containing the FL01L gene. Fig. 9

YRpGLlO (9.70 Kb). Fig. 10 YRpGLF14S (15.50 Kb). Fig. 11

YRpGLFβL (17.66 Kb). Fig. 12

A flow chart for the preparation of plasmids pBR- ADH1-FL01S and pBR-ADHl-FLOlL containing the agglutination genes FL01S and FL01L, for incorporation onto the yeast chromosomes. Fig. 13 pAAH5 (12.60 Kb). Fig. 14

BamHI-digested pAAH5 (12.60 Kb). Fig. 15

pBR322 ( 4 . 30 Kb) . Fig . 16 pBR322-dH (4.30 Kb). Fig. 17 pBR-dEPl (2.50 Kb). Fig. 18

The open reading frame of FLOIS prepared by PCR. Fig. 19 pBR-dEPl-FLOlS (5.10 Kb). Fig. 20

The open reading frame of FL01S. Fig. 21 pBR-dH-ADHl (6.20 Kb) . Fig. 22 pBR-ADHl-FLOlS (8.80 Kb). Fig. 23

YCpHF19L (22.10 Kb). Fig. 24

EcoRV+Bglll-digested YCpHF19L. Fig. 25 pBR-dEPl-FLOlL (7.20 Kb). Fig. 26

The open reading frame of FL01L. Fig. 27 pBR-ADHl-FLOlL (10.80 Kb).

BEST MODE FOR CARRYING OUT THE INVENTION A more concrete explanation of the present invention is provided below. Agglutination gene The genes according to the present invention which impart agglutinative properties to the yeast Saccharomyces cerevisiae include a gene of 4.7 ±0.2 kb in yeast which codes for a polypeptide which exhibits agglutinative activity and an agglutination gene of 2.6 ±0.2 kb derived from the above mentioned agglutination gene, and these genes correspond respectively to the FL01L gene (also abbreviated to FL01L) and the FLOIS gene (also abbreviated to FLOIS) derived from the agglutination gene FLOl of the yeast Saccharomyces cerevisiae described above. FLOIS is the FL01L gene with a portion of the base sequence deleted. In addition, as described later, the FLOl gene also encompasses genes which are artificial or naturally occurring derivatives of the FL01L gene and have agglutinative activity, although the lengths of their open reading frames may differ. Here, the FL01L gene is the intact FLOl gene on chromosome I of the yeast Saccharomyces cerevisiae, and FLOIS is the F101L gene with a portion of the open reading frame deleted in-frame. Here, characteristically FL01L imparts a relatively strong agglutinative property to the host yeast into which it is introduced, while FLOIS imparts a weaker agglutinative property to the host

yeast in comparison to FL01L.

The agglutination genes according to the present invention are present in the yeast Saccharomyces cerevisiae in the form of plasmids which contain the genes as their constituents, and in the form, of insertions into the genome of the host. Also, for a stable expression of the agglutination genes in the yeasts, the agglutination genes according to the present invention may be placed under the control of an appropriate promotor and terminator, and be present in this form as plasmids or as insertions into the genome. The promotor and terminator used may be suitable combinations of publicly known ones, such as alcohol dehydrogenase gene (ADH1), phosphoglycerate kinase gene (PGK), etc.

Polypeptides coded for by genes

The FL01L gene according to the present invention is specified by the amino acid sequence of the polypeptide for which it codes. This polypeptide has agglutinative activity and it is one whose amino acid sequence is substantially represented by Sequence No. 1. Here, the expression "one whose amino acid sequence is substantially represented by Sequence No. 1" means that some of the amino acids may be deleted or substituted, or some amino aicds may be added thereto, so long as the polypeptide has agglutinative activity.

A typical polypeptide according to the present

invention which exhibits agglutinative activity is one which has the amino acid sequence listed as Sequence No. 1 and consists of 1,537 amino acids, and its amino acid sequence has not been known in the past. It was stated above that, according to the present invention, the expression "one whose amino acid sequence is substantially represented by Sequence No. 1" means that some of the amino acids may be deleted or substituted, or some amino acids may be added thereto, so long as the polypeptide has agglutinative activity; an example of a peptide which has such an alteration . relating to its amino acids is one in which the 329th to the 1,003rd amino acids of the amino acid sequence listed as Sequence No. 1 (FL01L sequence) are deleted (FLOIS sequence, see Sequence No. 2), and this peptide has agglutinative activity, although somewhat weaker. If the agglutinative property of a yeast during fermentation is too strong, then the number of suspended yeast cells will be lowered and this will generally tend to slow the rate of fermentation, and it is therefore desirable to breed yeast in such a way that agglutinative properties of the proper strength are imparted in each of the fermentation systems. The agglutinative properties may become too strong with introduction of the FL01L gene, and thus introduction of the FL01S gene is sometimes preferable. In that sense, although the lengths of the polypeptides according to the present invention are basically that of

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the sequence listed as Sequence No. 1, the deletion, substitution, addition, etc. of a few amino acids is highly significant for establishing agglutination activities of desired strengths in various yeasts. That is, such altered polypeptides are within the scope of the polypeptides according to the present invention which have agglutinative activity. Base sequence of agglutination gene

The DNA chain of the FL01L gene is one having the base sequence listed as Sequence No. 1 of the sequence list, or a degenerate isomer thereof, and having the base sequence which corresponds to the amino acid sequence listed as Sequence No. 1, or a degenerate isomer thereof. Here, "degenerate isomer" means a DNA chain which differs only in a degenerate codon, and is still capable of coding for the same polypeptide.

The base sequence of the DNA chain listed as Sequence No. 1 was determined for the FLOl gene obtained from Saccharomyces cerevisiae strain ABXL-ID (Yeast Genetic Stock Center, University of California, USA) using the dideoxy method. Collection of DNA chain of agglutination gene

At present there is absolutely no information available regarding the product of the FLOl gene which has agglutinative activity (the amino acid sequence of the polypeptide coded for by the FLOl gene), and therefore it is impossible to clone the FLOl gene by the

commonly employed hybridization method using an appropriate DNA probe which is chemically synthesized based on the amino acid sequence. As a result, we the present inventors constructed a gene library of the entire DNA of Saccharomyces cerevisiae strain ABXL-ID using a veast/E. coli shuttle vector plasmid, and this non-agglutinative yeast was transformed therewith to obtain an agglutinative clone, and plasmids were recovered from the transformed strain (see following Examples for details).

Introduction of agglutination gene into yeast

By introduction of the DNA chain of the agglutination gene according to the present invention which was obtained in the manner described above into yeasts which are used in the fermentation industry, for example, brewer's yeast, wine yeast, whiskey yeast, Japanese sake yeast, shochu yeast, alcohol production yeast, etc. (all of Saccharomyces cerevisiae) according to bioengineering methods, it is possible to convert them into agglutinative strains if they are non-agglutinative strains, or reinforce their agglutinative properties if they are agglutinative strains.

Yeasts

The yeasts to be transformed according to the present invention are yeasts belonging to the genus Saccharomyces cerevisiae described in The Yeasts: A Taxonomic Study, 3rd Ed. (Yarrow, D. , ed. by N.J.W. Kreger-Van Rij.

Blsevier Science Publishers B.V., Amsterdam, 1984, p.379), or their synonyms or mutants; however, in light of the object of the present invention, the various industrial yeasts belonging to the genus Saccharomyces cerevisiae, for example, brewer's yeast, wine yeast, whiskey yeast, Japanese sake yeast, shochu yeast, alcohol production yeast, etc. are preferred.

Specific examples thereof include bottom brewer's yeast: W164 (Munich Institute of Technology, Germany), W204 (Munich Institute of Technology, Germany), SMA-S

(Berlin Institute of Technology), H.H. (Berlin Institute of Technology), top brewer's yeast: obg. 160 (Berlin Institute of Technology, Germany), wine yeast: IAM 4175 (Tokyo University), whiskey yeast: AHU3200 (Hokkaido University), Japanese sake yeast: Association No. 6 (Japan Brewing Association), shochu yeast: IFO 0282 (Fermentation Research Institute Foundation), alcohol production yeast: IFO 0216 (property of Fermentation Research Institute), etc. These industrial yeasts have been selected and pure cultured over a period of many years into forms suitable for the fermentation industry, that is, forms which are capable of efficiently fermenting fermentation sources, which produce alcohol with a good flavor, and whose genetic properties are stable, etc.

Transformation

The procedures and methods of preparing the

transformant may be those commonly used in the fields of molecular biology and genetic engineering, and they may include methods other than those mentioned below according to the present invention as long as they are effected using common techniques. In order to express the agglutination gene according to the present invention in yeast, it is necessary to first insert the gene into a plasmid vector which exists stably in the yeast. The plasmid vector used here may be any of the known ones, such as YRp, YEp, YCp, Yip, etc. These plasmid vectors are not only publicly known by document, but they are also easy to prepare.

The marker to be used for selection of the desired transformant according to the present invention may be a resistance gene against a drug such as G418, etc., since there are no particularly appropriate intrinsic genetic markers requiring amino acids or nucleic acids, etc. in the case of industrial yeasts. However, using the fact that the present agglutination gene is expressed as the dominant gene, it is possible to obtain a transformant which is marked with the agglutination itself.

The insertion of the DNA chain of the agglutination gene according to the present invention into the plasmid and introduction thereof into the yeast is easily effected, but on the other hand, this type of plasmid usually cannot be stably maintained in the cells, and often escapes from the transformed cells.

In order to maintain the DNA chain of the agglutination gene according to the present invention in the yeast in a more stable manner, it may be inserted into the genome of the yeast. Particularly in the case of yeasts used in the food industry, it is more preferable to improve the yeast only with the yeast genes, without having a non-yeast DNA fragment from E. coli (contained in the plasmid vector if the plasmid was grown up in E. coli) present in the final recombinant. Here, we the present inventors chose to introduce only the yeast gene, using the co-transformation method of Penttila, et al. and the gene replacement method (Current Genetics, Vol. 12, p.413-420, 1987) by which only the yeast gene is incorporated into the genomic DNA. Also, the transformation here may be effected by any appropriate desired method which is commonly used in the fields of molecular biology or genetic engineering, such as, for example, the protoplast method of Hinnen, et al. (Proceedings of National Academy of Sciences of the United States of America, Vol. 75, p.1929-1933, 1978), the lithium acetate method of Itoh, et al. (Journal of Bacteriology, Vol. 153, p.163-168, 1983), etc. The yeast according to the present invention obtained in this manner has, except for the introduced exogenous DNA, exactly the same genetic properties as the original strain before introduction, and further, by using the chromosome introduction method wherein only the DNA chain

of the agglutination gene according to the present invention is introduced by the above mentioned co- transformation and gene replacement methods, no unnecessary vector sequences are contained therein, and thus the obtained recombinant yeast has none of the properties of the vector which is used. As a result, the superior character of the original strain is in no way impaired, and it is possible to breed industrial yeasts whose agglutination is improved in a specific manner. Production of alcoholic liquors

Fermentation of the fermentation source using a yeast transformed by an agglutination gene according to the present invention such as the one mentioned above, may be carried out to achieve the effects described above. As is obvious, the fermentation source is to be chosen depending on the object of fermentation; for example, wort is used in the production of beer and whiskey, fruit juice in the production of wine, koji in the production of Japanese sake, starch or carbohydrate sources in the production of shochu, and molasses, starch or carbohydrate sources in the production of alcohol. In addition, the conditions of fermentation may be the same conditions as conventionally used, and there is no need to modify the existing fermentation procedures or equipment when applied to the present invention.

Since the yeast which is used exhibits agglutination in the alcoholic liquor produced thereby, it rapidly

agglutinates and settles at the bottom of the fermentation vat after completion of the fermentation, and the yeast cells are readily separable from the fermentation mash. Examples

A more detailed description of the present invention is provided below with reference to the Examples. Example 1 (Collection of gene controlling agglutination of yeast) The following experiment was conducted to obtain the FLOIS gene as one of the agglutination genes according to the present invention (Watari, et al.. Agricultural and Biological Chemistry, Vol. 53, No. 3, p.901-903, 1989). The chromosomal DNA of Saccharomyces cerevisiae strain ABXL-ID (gene type: MATa FLOl, Yeast Genetic Stock Center, University of California, USA) was prepared according to the method of Cryer, et al. (Methods of Cell Biology, Vol. 12, p.39-44, 1975). The obtained chromosomal DNA was partially digested with the restriction enzyme Sau3AI, DNA fragments of over 5 kb were recovered by sucrose density gradient centrifugation, and the DNA fragments were inserted in vitro by a ligation reaction at the BamHI region of the cloning vector YCpH4 (Watari, et al.. Agricultural and Biological Chemistry, Vol. 53, No. 3, p.901-903, 1989) which contained the histidine synthesis gene HIS4 as a selection marker. Escherichia coli (E. coli) strain

MC1061 (gene type: hsdR mcrB araD139 ^* (araABC- leu)7679 ^A lacX74 galU glaK rpsL thi) was transformed with the ligation mixture, and the plasmids were extracted from the transformant to prepare a gene library for strain ABXL-ID. E. coli strain MC1061 is a strain in wide use in the field of recombinant DNA technology.

Using this gene library, the histidine-requiring non- agglutinative baker's yeast Saccharomyces cerevisiae strain YJW6 (gene type: MAT- adel ural his4 canl karl) (Agricultural and Biological Chemistry, Vol. 53, No. 3, p.901-903, 1989) was transformed. The transformation of Saccharomyces cerevisiae strain YJW6 was effected basically according to the lithium acetate method of Itoh, et al. (Journal of Bacteriology, Vol. 153, p.163- 168, 1983). That is, to 100 ml of a YPD liquid culture medium (1% yeast extract, 2% bactopeptone, 2% glucose) was inoculated one loopful of YJW6 strain and the cells were cultured at 30°C overnight, separated with a centrifuge the following morning, inoculated into a new medium of the same composition and further cultured for 3 hours at 30°C. The collected cells were washed with 40 ml of sterilized water, and then finally suspended in 20 ml of a TE solution (10 mM Tris-HCl buffer solution containing 1 mM of EDTA, pH 7.5). Of this, 5 ml was transferred to an L-shaped test tube (Monod tube), 5 ml of a 0.2 M lithium acetate solution was added thereto, and the mixture was shaken at room temperature for 1

hour, at 100 cycles/min. From the mixture 0.1 ml was taken and added to a 1.5 ml Eppendorf tube which already contained 50 μg of the recombinant plasmid (ethanol precipitated, and then air-dried), and the mixture was stirred well and allowed to stand for 30 minutes at 30°C. The Eppendorf tube was then stirred well, 0.1 ml of 70% polyethylene glycol #4,000 was added thereto, and the mixture was further stirred well and then allowed to stand for 1 hour at 30°C. This was heated at 42°C for 5 minutes (heat shock treatment), allowed to cool to room temperature, and then the cells were washed with . sterilized water. Finally, the cells were suspended in 0.5 ml of sterilized water, and the solution was applied 0.1 ml at a time to a minimal culture medium which contained no histidine (0.67% Difco yeast nitrogen base without amino acids, 2% glucose, 40 μq/ml adenine sulfate, 40 μg/ml uracil, 2% Difco bacto agar), to obtain a non-histidine-requiring transformant. This transformation experiment was repeated 10 times to obtain approximately 10,000 clones of the non-histidine- requiring transformant..

Next, the agglutinative clones were screened out of the transformants. The transformants were taken from the plate one at a time using a toothpick, inoculated into a 96-well microplate [each well containing 200 μl of a minimal liquid culture medium (above mentioned minimal medium with agar removed)], and cultured at 25° for 3

days. The agglutination was examined by vigorously shaking the microplate after culturing, using a icroplate mixer (Titech micromixer) for 60 seconds, and visually locating the agglutinative clones. One clone of relatively strong agglutinative properties was obtained out of approximately 6,000 non-histidine-requiring transformants. This strain was cultured in a non- selective YPD culture medium, upon which a clone which had become histidine-requiring, that is, which had lost the plasmid, was obtained. This clone, in becoming histidine-requiring, had also lost its agglutinative properties. In addition, when DNA was recovered from the originally obtained agglutinative transformants, the plasmid was recovered from E.coli strain MC1061, and non- histidine-requiring transformants were obtained by retransforming strain YJW6 therewith, all were agglutinative. These results led to the conclusion that the agglutination exhibited by the transformed strain was not due to any genetic mutation in the host cell, but was caused by the plasmid in the transformed strain. Here, we the present inventors named the plasmid which contained the genetic sequence controlling agglutination, YCpHF19S. The restriction enzyme map thereof is shown in Fig. 4. As may be surmised from the screening test for the agglutinative yeast using the microplate, such a plasmid which contains the agglutination gene may be used as a

marker for the selection of agglutination from yeast which do not have a marker, in order to obtain the transformants. In this experiment as well, transformants in which the present plasmid had been introduced were actually obtained from the non-agglutinative yeast. In other words, this type of agglutination gene may clearly be used for obtaining transformants of yeast belonging to Saccharomyces cerevisiae without any genetic marker. Furthermore, during the screening process, there are merits in having basically no need to prepare a special culture medium (minimal medium or medium containing antibiotics) for screening for the transformants, and in culturing in a normal culture medium. Also, there are presently few yeast-derived genetic markers for obtaining yeast transformants, and they are very useful in yeast self-cloning experiments. Example 2

(Mapping and identification of cloned agglutination gene) In order to determine whether or not the agglutination gene cloned in Example 1 was the FLOl gene on yeast chromosome I, the following physical mapping experiment was conducted with the present agglutination gene (Watari, et al.. Agricultural and Biological Chemistry, Vol. 53, No. 3, p.901-903, 1989). An EcoRV fragment of 2.6 kb taken from the region of DNA in the plasmid YCpHF19S which contained the gene controlling agglutination, was used as a probe, and

physical mapping of the present gene fragment on the chromosome was effected by chromosome DNA electrophoresis (pulse field electrophoresis). That is, chromosome electrophoresis of Saccharomyces cerevisiae strain ABXL- ID was effected by using the method of Carle, et al.

(Proceedings of the National Academy of Sciences of the United States of America, Vol. 82, p.3756-3760, 1985) to prepare a sample, and using a Biorad CHEF electrophoresis apparatus. After completion of the electrophoresis, the DNA band on the electrophoresis gel was subjected to

Southern blotting and hybridization, following the method of Maniatis, et al. (Molecular Cloning, p.382-389, Cold Spring Harbor Laboratory, 1982). As a result, the above mentioned 2.6 kb EcoRV fragment hybridized to chromosome I of strain ABXL-ID, indicating that the agglutination gene cloned in the present experiment was the gene on chromosome I.

Next, genealogical mapping of the cloned agglutination gene was attempted (Watari, et al., Agricultural and Biological Chemistry, Vol. 55, No. 6, p.1547-1552, 1991). YCpHF19S was partially digested with the restriction enzyme Xbal, and the yeast centromere gene (CEN4) and the yeast replication origin ARS1 were removed to prepare the Yip plasmid YIpHF19S (see Fig. 5) to be incorporated. After this plasmid was digested with the restriction enzyme BamHI to raise the efficiency of incorporation of the cloned agglutination gene portion

into the yeast, Saccharomyces cerevisiae strain YJW2A (gene type: MATa FLOl his4) was transformed therewith by the method described above to obtain a non-histidine- requiring transformant. The obtained strain was crossed with Saccharomyces cerevisiae strain YJW6 (gene type:

MAT- adel ural his4 canl karl) to obtain a diploid, which was sporulated and subjected to genetic analysis (tetrad analysis). As a result, genealogical linkage (parental ditype:nonparental ditype:tetratype= 22:0:7) was accomplished between the His+ characteristic (non- histidine-requiring) and ADE1 on chromosome I, clearly showing that the cloned agglutination gene portion of the YIpHF19S plasmid had been incorporated on chromosome I of strain YJW2A. From the above results of physical and genealogical mapping, we the present inventors concluded that the cloned agglutination gene was the FLOl gene on yeast chromosome I. However, at this point, it was not known that the FLOl gene obtained here was not the intact FLOl gene as present on the yeast chromosome (or, the FL01L gene), but rather the FLOIS gene lacking a portion of the

DNA sequence of FL01L, as described below.

Example 3

(Analysis of base sequence of FLOIS) We the present inventors conducted an experiment to determine the base sequence of the FLOl gene (actually the FLOIS gene) obtained above.

As a result of subcloning, it had been discovered that the region necessary for the expression of agglutination by the FLOIS gene consisted of the 4.1 kb DNA fragment between BamHI-(BamHI/Sau3AI) of the plasmid YCpHF19S. Here, the region containing this DNA fragment was subcloned at the multi-linker sites of the sequencing vectors pUCllδ and puC119 (both products of Takara Brewing Co.). Next, each of the subclones were subjected to the method of Henikoff, et al. (Gene, Vol. 28, p.351- 359, 1984) and the method of Yanisch-Perron, et al. (Gene, Vol. 33, p.103,119, 1985), by treating the insertion sections of their plasmids with exonuclease III and mangbean nuclease, resulting in the preparation of short lengths on various clones with the inserted fragment partially missing and thus differing chain lengths. During this process, a kilosequencing deletion kit (product of Takara Brewing Co.) was used. Regarding the inserted fragments of the resulting various clones, the dideoxy method of Sanger, et al. (Science, Vol. 214, p.1205-1210, 1981) was followed and an automatic DNA sequencer of Applied Biosystems Japan, Inc. was used to determine the base sequence of the above mentioned 4.1 kb DNA fragment. As a result of the analysis thereof, an open reading frame of 2,586 bp (Sequence No. 2) was found to be present which is capable of coding for a polypeptide of 862 amino acids with an estimated molecular weight of 89,368.

Example 4

(Southern hybridization experiment)

As described above, the agglutination gene obtained in Example 1 was clearly at the FLOl locus on yeast chromosome I, but in order to determine whether or not this was the intact FLOl gene, a Southern hybridization experiment such as the following was conducted. First, all of the DNA was extracted from the yeast Saccharomyces cerevisiae strain ABXL-ID from which the agglutination gene had been cloned, and was completely digested with restriction enzyme EcoRV and subjected to electrophoresis, and then to genomic Southern analysis using as a probe the 2.6 kb EcoRV DNA fragment containing the open reading frame mentioned above in Example 2. Here, the Southern blotting and hybridization were effected according to the method of Maniatis, et al. (Molecular Cloning, p.382-389, Cold Spring Harbor Laboratory, 1982).

The results were that, surprisingly, no hybridization signal was detected at the location corresponding to approximately 2.6 kb, but a hybridization signal was obtained at the location corresponding to approximately 4.7 kb. This led the present inventors to suppose that the cloned agglutination gene might not be identical to the FLOl gene of strain ABXL-ID, but rather might be the intact FLOl gene with a portion of the DNA sequence lost for some reason during the cloning process.

Example 5

(PCR (polymerase chain reaction) experiment)

Here, the present inventors conducted an experiment such as the following to confirm the structure of the FLOl gene of ABXL-ID, by the PCR (polymerase chain reaction) method. First, a DNA chain was chemically synthesized using the base sequence mentioned above in

Example 3. That is, a DNA sequence of 33 bases including the initiation codon region of the open reading frame of the present gene was chemically synthesized with a DNA synthesizer (product of ABI Co.) and used as the PCR 5' probe.

(Sequence No. 3)

Linker site (The ATG- starting at the 14th base from the 5' end of the above sequence is the 5' end sequence of FLOIS) . In addition, a DNA sequence of 33 bases was chemically synthesized in the same manner, which included a complementary strand (reverse strand) of the region containing the termination codon of the open reading frame of the present agglutination gene, and this was used as the PCR 3' probe (Sequence No. 4).

STOP (Sequence No. 4) Linker site (The TTA- starting at the 10th base from the 5' end of the above sequence is the 3' end sequence of FLOIS

(reverse strand) ) .

Next, using these 5' and 3' probes, the PCR experiment was conducted with the entire DNA of Saccharomyces cerevisiae strain ABXL-ID as the template. A zymoreactor Model AB-1800 (product of Ato Co.) was used for the PCR experiment, and Pfu DNA polymerase

(Stratagene Co.) was used as the DNA polymerase. Also, the conditions of the PCR experiment were according to the method of Inis, et al. (PCR Technology, p.3-12, Stockton Press, ed. Henry A. Erlich, 1989). Upon confirmation of the bands of the DNA amplified as a result of the PCR experiment by electrophoresis on an agarose gel, a single band was obtained in the area of approximately 4.7 kb, and it was surmised that the open reading frame of FLOl of strain ABXL-ID was approximately 4.7 kb. Further, in a control experiment in which plasmid YCpHF19 containing the agglutination gene cloned by the present inventors was used as the template, a band in the area of approximately 2.6 kb was obtained. From these results, the present inventors concluded that the intact open reading frame of FLOl gene as present in the yeast Saccharomyces cerevisiae strain ABXL-ID is approximately 4.7 kb, and not approximately 2.6 kb.

Therefore, we the present inventors concluded that the agglutination gene obtained by us was the FLOl gene with a portion thereof missing for some reason, most likely as a result of intramolecular recombination during the process of maintaining YCpHF19 in E. coli strain

MC1061.

Example 6

(Collection of FL01L gene)

Here, we the present inventors made a reexamination based on our expectation that the plasmid containing the intact FLOl gene might be contaminated in the YCpHF19S plasmid solution initially recovered from E. coli, during the cloning of FLOl. First, a portion of the plasmid solution was taken, digested with the restriction enzyme EcoRV and subjected to electrophoresis on an agarose gel, upon which an extremely faint but clearly observable band was discovered in the area of 4.7 kb, in addition to the 2.6 kb band obtained for YCpHF19S. This suggests that the plasmid solution is a mixture of two types of plasmids. Here, we the present inventors used this plasmid solution to transform E. coli strain JA221 (gene type: recAl, lacY leuB trp ^A E5 thr thi hsdR hsdM) , and upon extraction of the plasmids from the obtained ^• transformants and examination thereof, another type of plasmid was separated in addition to YCpHFl9S, which was approximately 2.1 kb larger than YCpHF19S. Here, initially cloned plasmid was named YCpHF19S, and the plasmid separated from the YCpHF19S plasmid solution which was 2.1 kb longer was named YCpHF19L. Also, as a result of analysis of the respective restriction enzyme cleavage patterns of YCpHF19S and YCpHF19L, the plasmids were found to have no differences in any DNA regions

other than the DNA region containing the open reading frame of the agglutination gene (see Figs. 4 and 7). That is, the open reading frame of the agglutination gene of YCpHF19L was shown to be 2.1 kb longer than that of YCpHF19S. This led to the conclusion that the initial cloning of the intact FLOl gene (i.e., FLOIL) was successful, but during the process of maintaining the YCpHF19L plasmid in E. coli strain MC1061, a portion of the open reading frame of the FLOIL gene had been deleted in-frame, converting FLOIL to FLOIS, or YCpHF19L to

YCpHF19S, due to intramolecular recombination in vivo. Here, it was thought that since the deletion occurred in- frame, FLOIS was still capable of coding for a polypeptide exhibiting agglutinative properties. Distinction shall hereunder be made between FLOIS as the agglutination gene on YCpHF19S, and FLOIL as the agglutination gene on YCpHF19L.

Further, the frequency of occurrence of the deletion is greatly influenced by the type of E. coli maintaining the YCpHF19L plasmid, and the present inventors discovered the phenomenon that, for example, a high rate of conversion occurs with strain MC1061 (gene type: hsdR mcrB araDl39 ^A (araABC-leu)7679 ^Λ lacX74 galU galK rpsL thi) and strain DH5- (gene type: supE44 ^A lacU169(-801acZ ^A M15) hsdR17-recAl endAl gyrA96 thi-1 relAl), whereas comparatively little conversion occurs with strain JA221 (gene type: recAl lacY leuB trp ^A B5 thr thi hsdR hsdM) .

Therefore, the present inventors mainly used strain JA221 when the plasmid was maintained in E. coli. However, at present the reason for this conversion is not clear. Example 7 (Analysis of base sequence of FLOIL)

The DNA fragment containing FLOIL was cut off from the YCpHF19L plasmid obtained above, and its entire base sequence was determined by exactly the same method as in Example 3. As a result, it was confirmed that the open reading frame of FLOIL was a base sequence of 4,611 bp which codes for a polypeptide of 1,537 amino acids with an estimated molecular weight of 160,692 (Sequence No. 1). Also, it was shown that FLOIS is FLOIL with an in- frame deletion of a DNA chain consisting of the 985th to the 3,009th bases from the initiation codon of the open reading frame (corresponding to the 329th to the 1,003rd amino acids of the amino acid sequence) (see Sequence Nos. 1 and 2) .

In addition, judging from the results of analysis of the amino acid sequence of FLOIL, a repeated sequence

(direct repeat) of 45 amino acids is found from the 278th to the 1,087th amino acids of the sequence [basically represented by the following sequence, with the amino acids separated by a "/" within the parentheses indicating alternative candidates.

ThrThrThr(Glu/Gln)ProTrp(Asn/Thr/Asp) (Gly/Asp/Ser)ThrPheT hrSerThrSer(Thr/Ala)Glu(Met/Leu/Val) (Thr/Ser)Thr(Val/Ile)

ThrGlyThrAsnGly(Leu/Val/Gln) (Pro/Arg)ThrAspGluThr(Val/Ile )IleVal(Ile/Vla) (Arg/Lys)ThrProThr(Thr/Ser) (Ala/Glu) (Thr/ Gly/Ser/Ile) (Thr/Leu/Ser) (Als/Ile/Val/Ser) (Met/Ser/Ile/Th r)], and in FLOIL there are 18 of this repeated sequence. On the other hand, in FLOIS, the major portion of the region of this repeated sequence is deleted (FLOIS has the 329th to the 1,003rd amino acids of the amino acid sequence of FLOIL deleted), and only 3 copies of the repeated sequence are present. The present inventors believe at the present time that the difference in the agglutinative capabilities of FLOIL and FLOIS (the former imparts a stronger agglutinative property to the host cell than does the latter), is connected with the number of these direct repeats. We the present inventors presume that in the future it will be possible to achieve a desired agglutinative capability for a cell, i.e. regulate agglutinative capabilities at will, by regulation of the number of direct repeats. Example 8 [Introduction of FLOIL gene into various yeast strains for practical use (1: using plasmid vectors)]

The agglutination genes FLOIS and FLOIL obtained above were introduced into various industrial yeasts (all non-agglutinative) to determine whether or not they are actually effective for the breeding of agglutinative yeast strains for practical use. First, plasmids were prepared which contained directly selectable FLOIS or

FLOIL genes, for transformation of the industrial yeasts (A flow chart is shown in Fig. 3. The numbers next to each plasmid and open reading frame (ORF) in the flow chart match the numbers next to the plasmids and open reading frames shown in detail in Figs. 4-11). A 5.8 kb BamHI-XhoI fragment (Fig. 6) containing the FLOIS gene of YCpHF19S was inserted into the gap between BamHI-Sall of the plasmid YRpGLlO to be used for direct selection (having a G418- resistant Tn903 gene as the marker gene for direct selection, and an ARS1 sequence as the replication origin within the yeasts. See Fig. 9), to prepare the YRpGLF14S plasmid (Fig. 10). In addition, a 7.9 kb BamHI-XhoI fragment (Fig. 8) from YCpHF19L was inserted into the gap between BamHI-Sall of YRpGLlO to prepare YRpGLFβL as a similar plasmid containing the FLOIL gene (Fig. 11).

The method used for transformation of the industrial yeasts by the plasmids will now be described. The method for transformation of the industrial yeasts was basically identical to the one used for the experimental yeasts described in Example 1, but the present inventors made some slight modifications as indicated below (Watari, et al.. Agricultural and Biological Chemistry, Vol. 55, No. 6, p.1547-1552, 1991). That is, to 100 ml of a YPD liquid culture medium (1% yeast extract, 2% bactopeptone, 2% glucose) was inoculated one loopful of cells, which were cultured at 30°C overnight, separated with a

centrifuge the following morning, inoculated into a new medium of the same composition and further cultured for 3 hours at 30°C. The collected cells were washed with 40 ml of sterilized water, and then finally suspended in approximately 20 ml of a TE solution (10 mM Tris-HCl buffer solution containing 1 mM of EDTA, pH 7.5). (However, a hematometer was used here to adjust the concentration of the suspension to achieve a final cell concentration of about 2 x 108 cells/ml). Of this, 5 ml was transferred to an L-shaped test tube (Monod tube), 5 ml of a 0.2 M lithium acetate solution was added thereto, and the mixture was shaken at room temperature for 1 hour, at 100 cycles/min. From the mixture, 0.1 ml was taken and added to a 1.5 ml Eppendorf tube which already contained 50 μg of the recombinant plasmid (ethanol precipitated, and then air-dried), and the mixture was stirred well and then allowed to stand for 30 minutes at 30°C. The Eppendorf tube was stirred well, 0.1 ml of 70% polyethylene glycol #4,000 was added thereto, and the mixture was stirred well and then allowed to stand for 1 hour at 30°C. Next, the mixture was heated to 42°C for 5 minutes, (heat shock treatment), allowed to cool to room temperature, and then the cells were washed with sterilized water. Finally, the cells were suspended in 1.4 ml of a YPD solution in an Eppendorf tube, and cultured while standing for 16-20 hours at 30°C. The culture solution was then applied 0.1 ml at a time to a

YPD agar medium containing 200 μg/ml of G418, and incubated at 30°C for 2-3 days to obtain the transformants.

The experiment for transformation of various industrial yeasts was carried out using this method. The results are shown in Table 1. In addition, the method of evaluating the agglutination was as follows. Each of the transformants was inoculated into an L-shaped test tube (Monod tube) which contained 10 ml of a YPD liquid medium (containing 100 μg/ml fo G418) and shaken for culturing at 28°C for 3 days (100 cycles/min), and the agglutination was evaluated by visual examination. The evaluation scale for the level of agglutination was according to the scaling method of Johnston, et al. (Yeast Genetics: Fundamental and Applied Aspects, p.205- 224, Springer Verlag, New York, ed. by J.F.T. Spencer, D.M. Spencer, A.R.W. Smith, 1983).

Table 1

Introduction of agglutination genes FLOIS and FLOIL into various yeasts for practical use and expression thereof Yeast/plasmid Bottom brewer's yeast W204 / W164 / SMA-S / H.H. Top brewer's yeast obg. 160

Whiskey yeast AHU3200

Wine yeast IAM4175

Japanese sake yeast Association No. 6 Shochu yeast IFO 0282

Alcohol yeast IFO 0216

Note:) Evaluation of agglutination shown as 6 levels, 0-5. 0: non-agglutinative, 1: very weakly agglutinative, 2: weakly agglutinative, 3: moderately agglutinative, 4: strongly agglutinative, 5: very strongly agglutinative. These results show that by introduction of the agglutination genes FL01S and FL01L, it was possible to convert all of the various non-agglutinative industrial yeasts into agglutinative yeasts, although there was some degree of difference in the agglutination. It need not be mentioned that with introduction of the vector plasmid YRpGLlO, the host cells remained non-agglutinative.

Furthermore, it was evident that introduction of the FL01L gene produced a stronger agglutinative property in the host strain than did introduction of the FL01S gene. Example 9 [(Introduction of FL01L gene into various yeast strains

for practical use (2: incorporation into yeast chromosomes) ]

In general, when exogenous genes are introduced into host cells in the form of plasmids, the plasmids escape from the cells as a result of successive culturing under non-selective pressure. Actually, the plasmids were observed to escape readily from the transformants obtained in Example 8 when selective pressure by G418 was not applied. Here, in order to stably maintain the FLOl gene in the yeasts, the present inventors attempted to incorporate the FLOl gene into the yeast chromosomes, (i) Preparation of an FLOl expression cassette for incorporation (A flow chart is shown in Fig. 12. The numbers next to each plasmid and open reading frame (ORF) in the flow chart match the numbers next to the plasmids and open reading frames shown in detail in Figs. 13-27). For the expression of the FLOl gene in the yeasts at high frequencies, a promotor was incorporated upstream from the 5' end of the open reading frame of the FLOl gene, and a terminator was incorporated downstream from the 3' end thereof, at the unit controlling transcription/translation of the yeast alcohol dehydrogenase gene. That is, the open reading frame sequence of either FLOIS or FLOIL was inserted at the Hindlll site of plasmid pBR-dH-ADHl which contained the promotor and terminator sequences for the yeast alcohol dehydrogenase gene, to obtain pBR-ADHl-FLOlS (Fig. 22)

and pBR-ADHl-FLOIL (Fig. 27), respectively. The present expression cassette was prepared at the time of preparation of the open reading frame of FLOIS using the PCR method (the PCR experiment was the same as in Example 5), and we the present inventors confirmed by the results of restriction enzyme analysis and DNA sequencing that the base sequence of the FLOIS gene prepared in this manner was exactly identical to the base sequence shown by the restriction enzyme cleavage map in Fig. 2 obtained from the results of Example 3 and listed as Sequence No. 2.

(ii) Example of incorporation of FLOl expression cassette into brewer's yeast genome by the cotransformation method The cotransformation method of Penttila, et al.

(Current Genetics, Vol. 12, p.413-420, 1987) was used to incorporate an FLOl expression cassette which contained no vector-derived sequence (sequence derived from the vector plasmid pBR322) into the chromosomal DNA of the yeast (non-agglutinative bottom brewer's yeast W204).

That is, 50 μg of either plasmid pBR-ADHl-FLOlS or pBR- ADH1-FL01L obtained above in (i) was digested with restriction enzyme BamHI and subjected to phenol/chloroform treatment, after which 50 μg of the G418-resistant plasmid YRpGLlO was added thereto and they were subjected to precipitation with ethanol. The DNA sample was air-dried, and the yeast was transformed

according to the method described above in Example 8. The transformants were selected with G418-resistance as the marker, and the obtained transformants were screened by the microplate assay method (see Example 1) to obtain the agglutinative strain. Here, the details of the microplate assay method are as follows. The resulting transformants were taken up from the plate one at a time using a toothpick, inoculated into a 96-well microplate (each well containing 200 μl of YPD liquid medium), and cultured at 25°C for 3 days. The determination of agglutination was made by vigorously shaking the microplate for 60 seconds after culturing using a microplate mixer (product of Titech Co.) and then visually locating the agglutinative clones. The agglutinative strain obtained in this manner was non-selectively cultured for 10-20 generations in a YPD medium, after which the cells were appropriately diluted, applied onto a YPD agar medium, and cultured at 30°C for 2-3 days. The colony accumulated on the plate was replicated onto one YPD agar medium which contained 200 μg/ml of G418 and another YPD agar medium which contained no G418, and the G418-resistance of the colonies was examined to recover strains which exhibited no G418- resistance. These strains had plasmid YRpGLlO missing from their cells, but it is thought that the FLOl expression cassette (i.e., the open reading frame of the FL01S or FL01L gene under control of expression of the

ADH1 promotor and ADH1 terminator) had been incorporated onto the ADH1 locus of the chromosome by in vivo gene replacement using the homologous sequence portion of the ADH1 gene. Of these, the strain into which the FLOIS expression cassette was incorporated was named W204-FL01S, and the strain into which the FLOIL expression cassette was incorporated was named W204-FL01L. When.these strains were cultured for 50 generations, they still maintained their agglutinative properties at the same levels as prior to culturing. Also, when the W204-FL01S and W204- FL01L strains were subjected to genomic Southern analysis, the present inventors confirmed that all of the FLOl expression cassettes had been incorporated into the chromosomal DNA.

(iii) Fermentation test

A beer fermentation test on as small a scale as 2 liters was conducted using the W204-FL01S and W204-FL01L strains obtained in (ii). That is, the method followed was the standard method of the European Institute of Brewing (Journal of the Institute of Brewing, EBC Analytica Microbiologica, Method 2.5.4., Tubes E.B.C., Vol. 83, p.117-118, 1977). The cells were cultured while standing in 50 ml of wort at 20°C for 3 days, and the entire amount thereof was added to 1 1 of wort and then cultured while standing at 15°C for 1 week. The grown-up cells were collected by centrifugal separation (5,000 rpm

x 10 minutes). The obtained yeast cells were added to wort (with an oxygen concentration of 9 ppm adjusted in advance) at 11°P (plateau degrees) to a concentration of 0.5% (wet v/v). Stationary fermentation was then effected at 10°C for 10 days. When the amounts of agglutination and sedimentation were compared at this point, the parent strain W204, being non-agglutinative, had a lower amount of settled yeast, and the amount of yeast recovered was roughly the same as the amount of yeast initially added (i.e., 100% recovery). However, the W204-FL01L strain into which the FL01L expression cassette had been incorporated exhibited a strong agglutinative property, and the amount of yeast recovered was twice or more the amount of yeast initially added (i.e., 200% or greater). Nevertheless, the W204-FL01S strain into which the FL01S expression cassette had been incorporated exhibited only a very weak agglutinative property, and the amount of yeast recovered was no- more than in the case of W204. It is assumed that this suggests that adequate agglutination of the host cells cannot be induced in wort with the introduction of a single copy of FL01S (With the introduction of multiple copies of FL01S obtained in Example 8, W204 exhibited agglutination even in wort). Nevertheless, the W204-FL01S strain with a single copy of FL01S introduced onto the chromosome exhibited moderate agglutination in the YPD culture medium, but the

reason this was not exhibited in the wort is not clear at the present time.

Also, after completion of the fermentation (pre- fermentation) described above, maturation (after- fermentation) of the supernatant thereof (young beer) was effected. That is, after completion of the process of after-fermentation at 5°C for 2 weeks and 0 ^β C for 1 week, the fermentate was subjected to filtration with a membrane filter and carbonation at 0°C, 2 atmospheres for 2 days, after which it was chemically analyzed and taste sampled. The results showed no difference whatsoever between W204 and W204-FL01L. Therefore, by brewing beer using the yeasts according to the present invention, it was made clear that only the agglutinative properties of the yeasts were improved without causing any modification whatsoever to the flavoring components of the control strains. Deposition

The transformed strain Escherichia coli FLOIL derived from the introduction of the plasmid pBR-dEPl-FLOlL (Fig. 25) containing the DNA chain according to the present invention (open reading frame of the FLOIL gene) into E. coli strain JA221 was deposited at the MITI National Institute of Bioscience and Human Technology as of January 13, 1993, and has been assigned the Deposit No. FERM BP-4136.

Sequence List Sequence number: 1 Sequence length: 4614 Sequence type: nucleic acid Strandedness: double Topology: linear Molecule type: Genomic DNA Original source: Saccharomyces cerevisiae ABXL-ID

Sequence 1

ATG ACA ATG CCT CAT CGC TAT ATG TTT TTG GCA GTC TTT ACA CTT CTG 4 Met Thr Met Pro His Arg Tyr Met Phe Leu Ala Val Phe Thr Leu Leu

15 GCA CTA ACT AGT GTG GCC TCA GGA GCC ACA GAG GCG TGC TTA CCA GCA 9 Ala Leu Thr Ser Val Ala Ser Gly Ala Thr Glu Ala Cys Leu Pro Ala

30 GGC CAG AGG AAA AGT GGG ATG AAT ATA AAT TTT TAC CAG TAT TCA TTG 14 Gly Gin Arg Lys Ser Gly Met Asn He Asn Phe Tyr Gin Tyr Ser Leu

45 AAA GAT TCC TCC ACA TAT TCG AAT GCA GCA TAT ATG GCT TAT GGA TAT 19 Lys Asp Ser Ser Thr Tyr Ser Asn Ala Ala Tyr Met Ala Tyr Gly Tyr

60 GCC TCA AAA ACC AAA CTA GGT TCT GTC GGA GGA CAA ACT GAT ATC TCG 24 Ala Ser Lys Thr Lys Leu Gly Ser Val Gly Gly Gin Thr Asp He Ser

75 ATT GAT TAT AAT ATT CCC TGT GTT AGT TCA TCA GGC ACA TTT CCT TGT 28 lie Asp Tyr Asn He Pro Cys Val Ser Ser Ser Gly Thr Phe Pro Cys

90 CCT CAA GAA GAT TCC TAT GGA AAC TGG GGA TGC AAA GGA ATG GGT GCT 33 Pro Gin Glu Asp Ser Tyr Gly Asn Trp Gly Cys Lys Gly Met Gly Ala

105 TGT TCT AAT AGT CAA GGA ATT GCA TAC TGG AGT ACT GAT TTA TTT GGT 38 Cys Ser Asn Ser Gin Gly He Ala Tyr Trp Ser Thr Asp Leu Phe Gly

120 TTC TAT ACT ACC CCA ACA AAC GTA ACC CTA GAA ATG ACA GGT TAT TTT 43 Phe Tyr Thr Thr Pro Thr Asn Val Thr Leu Glu Met Thr Gly Tyr Phe

135 TTA CCA CCA CAG ACG GGT TCT TAC ACA TTC AAG TTT GCT ACA GTT GAC 48 Leu Pro Pro Gin Thr Gly Ser Tyr Thr Phe Lys Phe Ala Thr Val Asp

150 GAC TCT GCA ATT CTA TCA GTA GGT GGT GCA ACC GCG TTC AAC TGT TGT 52 Asp Ser Ala He Leu Ser Val Gly Gly Ala Thr Ala Phe Asn Cys Cys

165 GCT CAA CAG CAA CCG CCG ATC ACA TCA ACG AAC TTT ACC ATT GAC GGT 57 Ala Gin Gin Gin Pro Pro He Thr Ser Thr Asn Phe Thr He Asp Gly

180 ATC AAG CCA TGG GGT GGA AGT TTG CCA CCT AAT ATC GAA GGA ACC GTC 62 He Lys Pro Trp Gly Gly Ser Leu Pro Pro Asn He Glu Gly Thr Val

195 TAT ATG TAC GCT GGC TAC TAT TAT CCA ATG AAG GTT GTT TAC TCG AAC 67 Tyr Met Tyr Ala Gly Tyr Tyr Tyr Pro Met Lys Val Val Tyr Ser Asn

210 GCT GTT TCT TGG GGT ACA CTT CCA ATT AGT GTG ACA CTT CCA GAT GGT 72 Ala Val Ser Trp Gly Thr Leu Pro He Ser Val Thr Leu Pro Asp Gly 225 240

ACC ACT GTA AGT GAT GAC TTC GAA GGG TAC GTC TAT TCC TTT GAC GAT 76 Thr Thr Val Ser Asp Asp Phe Glu Gly Tyr Val Tyr Ser Phe Asp Asp

255 GAC CTA AGT CAA TCT AAC TGT ACT GTC CCT GAC CCT TCA AAT TAT GCT 816 Asp Leu Ser Gin Ser Asn Cys Thr Val Pro Asp Pro Ser Asn Tyr Ala

270 GTC AGT ACC ACT ACA ACT ACA ACG GAA CCA TGG ACC GGT ACT TTC ACT 864 Val Ser Thr Thr Thr Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr

285 TCT ACA TCT ACT GAA ATG ACC ACC GTC ACC GGT ACC AAC GGC GTT CCA 912 Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Val Pro

300 ACT GAC GAA ACC GTC ATT GTC ATC AGA ACT CCA ACA ACT GCT AGC ACC 960 Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Thr Ala Ser Thr

315 ATC ATA ACT ACA ACT GAG CCA TGG AAC AGC ACT TTT ACC TCT ACT TCT 1008 He He Thr Thr Thr Glu Pro Trp Asn Ser Thr Phe Thr Ser Thr Ser

330 ACC GAA TTG ACC ACA GTC ACT GGC ACC AAT GGT GTA CGA ACT GAC GAA 1056 Thr Glu Leu Thr Thr Val Thr Gly Thr Asn Gly Val Arg Thr Asp Glu

345 ACC ATC ATT GTA ATC AGA ACA CCA ACA ACA GCC ACT ACT GCC ATA ACT 1104 Thr He He Val He Arg Thr Pro Thr Thr Ala Thr Thr Ala He Thr

360 ACA ACT GAG CCA TGG AAC AGC ACT TTT ACC TCT ACT TCT ACC GAA TTG 1152 Thr Thr Glu Pro Trp Asn Ser Thr Phe Thr Ser Thr Ser Thr Glu Leu

375 ACC ACA GTC ACC GGT ACC AAT GGT TTG CCA ACT GAT GAG ACC ATC ATT 1200 Thr Thr Val Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr He He

390 GTC ATC AGA ACA CCA ACA ACA GCC ACT ACT GCC ATG ACT ACA ACT CAG 1248 Val He Arg Thr Pro Thr Thr Ala Thr Thr Ala Met Thr Thr Thr Gin

405 CCA TGG AAC GAC ACT TTT ACC TCT ACA TCC ACT GAA ATG ACC ACC GTC 1296 Pro Trp Asn Asp Thr Phe Thr Ser Thr Ser Thr Glu Met Thr Thr Val

420 ACC GGT ACC AAC GGT TTG CCA ACT GAT GAA ACC ATC ATT GTC ATC AGA 1344 Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr He He Val He Arg

435 ACA CCA ACA ACA GCC ACT ACT GCT ATG ACT ACA ACT CAG CCA TGG GAC 1392 Thr Pro Thr Thr Ala Thr Thr Ala Met Thr Thr Thr Gin Pro Trp Asp

450 GAC ACT TTT ACC TCT ACA TCC ACT GAA ATG ACC ACC GTC ACC GGT ACC 1440 Asp Thr Phe Thr Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr 465 480

AAC GGT TTG CCA ACT GAT GAA ACC ATC ATT GTC ATC AGA ACA CCA ACA 1488 Asn Gly Leu Pro Thr Asp Glu Thr He He Val He Arg Thr Pro Thr

495 ACA GCC ACT ACT GCC ATG ACT ACA ACT CAG CCA TGG AAC GAC ACT TTT 1536 Thr Ala Thr Thr Ala Met Thr Thr Thr Gin Pro Trp Asn Asp Thr Phe

510

ACC TCT ACA TCC ACT GAA ATG ACC ACC GTC ACC GGT ACC AAT GGT TTG 1584 Thr Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Leu

525 CCA ACT GAT GAG ACC ATC ATT GTC ATC AGA ACA CCA ACA ACA GCC ACT 1632 Pro Thr Asp Glu Thr He He Val He Arg Thr Pro Thr Thr Ala Thr

540 ACT GCC ATG ACT ACA ACT CAG CCA TGG AAC GAC ACT TTT ACC TCT ACA 1680 Thr Ala Met Thr Thr Thr Gin Pro Trp Asn Asp Thr Phe Thr Ser Thr

555 TCC ACT GAA ATG ACC ACC GTC ACC GGT ACC AAC GGT TTG CCA ACT GAT 1728 Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Leu Pro Thr Asp

570 GAA ACC ATC ATT GTC ATC AGA ACA CCA ACA ACA GCC ACT ACT GCC ATA 1776 Glu Thr He He Val He Arg Thr Pro Thr Thr Ala Thr Thr Ala He

585 ACT ACA ACT GAG CCA TGG AAC AGC ACT TTT ACC TCT ACT TCT ACC GAA 1824 Thr Thr Thr Glu Pro Trp Asn Ser Thr Phe Thr Ser Thr Ser Thr Glu

600 TTG ACC ACA GTC ACC GGT ACC AAT GGT TTG CCA ACT GAT GAG ACC ATC 1872 Leu Thr Thr Val Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr He

615 ATT GTC ATC AGA ACA CCA ACA ACA GCC ACT ACT GCC ATG ACT ACA ACT 1920 He Val He Arg Thr Pro Thr Thr Ala Thr Thr Ala Met Thr Thr Thr

630 CAG CCA TGG AAC GAC ACT TTT ACC TCT ACA TCC ACT GAA ATG ACC ACC 1968 Gin Pro Trp Asn Asp Thr Phe Thr Ser Thr Ser Thr Glu Met Thr Thr

645 GTC ACC GGT ACC AAC GGT TTG CCA ACT GAT GAA ACC ATC ATT GTC ATC 2016 Val Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr He He Val He

660 AGA ACA CCA ACA ACA GCC ACT ACT GCC ATG ACT ACA ACT CAG CCA TGG 2064 Arg Thr Pro Thr Thr Ala Thr Thr Ala Met Thr Thr Thr Gin Pro Trp

675 AAC GAC ACT TTT ACC TCT ACA TCC ACT GAA ATG ACC ACC GTC ACC GGT 2112 Asn Asp Thr Phe Thr Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly

690 ACC AAC GGT TTG CCA ACT GAT GAG ACC ATC ATT GTC ATC AGA ACA CCA 2160 Thr Asn Gly Leu Pro Thr Asp Glu Thr He He Val He Arg Thr Pro 705 720

ACA ACA GCC ACT ACT GCC ATG ACT ACA ACT CAG CCA TGG AAC GAC ACT 2208 Thr Thr Ala Thr Thr Ala Met Thr Thr Thr Gin Pro Trp Asn Asp Thr

735 TTT ACC TCT ACA TCC ACT GAA ATG ACC ACC GTC ACC GGT ACC AAC GGC 2256 Phe Thr Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly

750 GTT CCA ACT GAC GAA ACC GTC ATT GTC ATC AGA ACT CCA ACT AGT GAA 2304 Val Pro Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Ser Glu

765 GGT CTA ATC AGC ACC ACC ACT GAA CCA TGG ACT GGT ACT TTC ACC TCT 2352

Gly Leu He Ser Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser

780 ACA TCC ACT GAG ATG ACC ACC GTC ACC GGT ACT AAC GGT CAA CCA ACT 2400 Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Gin Pro Thr

795 GAC GAA ACC GTG ATT GTT ATC AGA ACT CCA ACC AGT GAA GGT TTG GTT 2448 Asp Glu Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu Val

810 ACA ACC ACC ACT GAA CCA TGG ACT GGT ACT TTT ACT TCT ACA TCT ACT 2496 Thr Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr

825 GAA ATG ACC ACC ATT ACT GGA ACC AAC GGC GTT CCA ACT GAC GAA ACC 2544 Glu Met Thr Thr He Thr Gly Thr Asn Gly Val Pro Thr Asp Glu Thr

840 GTC ATT GTC ATC AGA ACT CCA ACC AGT GAA GGT CTA ATC AGC ACC ACC 2592 Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu He Ser Thr Thr

855 ACT GAA CCA TGG ACT GGT ACT TTT ACT TCT ACA TCT ACT GAA ATG ACC 2640 Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met Thr

870 ACC ATT ACT GGA ACC AAT GGT CAA CCA ACT GAC GAA ACC GTT ATT GTT 2688 Thr He Thr Gly Thr Asn Gly Gin Pro Thr Asp Glu Thr Val He Val

885 ATC AGA ACT CCA ACT AGT GAA GGT CTA ATC AGC ACC ACC ACT GAA CCA 2736 He Arg Thr Pro Thr Ser Glu Gly Leu He Ser Thr Thr Thr Glu Pro

900 TGG ACT GGT ACT TTC ACT TCT ACA TCT ACT GAA ATG ACC ACC GTC ACC 2784 Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met Thr Thr Val Thr

915 GGT ACC AAC GGC GTT CCA ACT GAC GAA ACC GTC ATT GTC ATC AGA ACT 2832 Gly Thr Asn Gly Val Pro Thr Asp Glu Thr Val He Val He Arg Thr

930 CCA ACC AGT GAA GGT CTA ATC AGC ACC ACC ACT GAA CCA TGG ACT GGC 2880 Pro Thr Ser Glu Gly Leu He Ser Thr Thr Thr Glu Pro Trp Thr Gly 945 960

ACT TTC ACT TCG ACT TCC ACT GAG GTT ACC ACC ATC ACT GGA ACC AAC 2928 Thr Phe Thr Ser Thr Ser Thr Glu Val Thr Thr He Thr Gly Thr Asn

975 GGT CAA CCA ACT GAC GAA ACT GTG ATT GTT ATC AGA ACT CCA ACC AGT 2976 Gly Gin Pro Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Ser

990 GAA GGT CTA ATC AGC ACC ACC ACT GAA CCA TGG ACT GGT ACT TTC ACT 3024 Glu Gly Leu He Ser Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr

1005 TCT ACA TCT GCT GAA ATG ACC ACC GTC ACC GGT ACT AAC GGT CAA CCA 3072 Ser Thr Ser Ala Glu Met Thr Thr Val Thr Gly Thr Asn Gly Gin Pro

1020 ACT GAC GAA ACC GTG ATT GTT ATC AGA ACT CCA ACC AGT GAA GGT TTG 3120 Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu

1035 GTT ACA ACC ACC ACT GAA CCA TGG ACT GGT ACT TTT ACT TCG ACT TCC 3168 Val Thr Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser

1050 ACT GAA ATG TCT ACT GTC ACT GGA ACC AAT GGC TTG CCA ACT GAT GAA 3216 Thr Glu Met Ser Thr Val Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu

1065 ACT GTC ATT GTT GTC AAA ACT CCA ACT ACT GCC ATC TCA TCC AGT TTG 3264 Thr Val He Val Val Lys Thr Pro Thr Thr Ala He Ser Ser Ser Leu

1080 TCA TCA TCA TCT TCA GGA CAA ATC ACC AGC TCT ATC ACG TCT TCG CGT 3312 Ser Ser Ser Ser Ser Gly Gin He Thr Ser Ser He Thr Ser Ser Arg

1095 CCA ATT ATT ACC CCA TTC TAT CCT AGC AAT GGA ACT TCT GTG ATT TCT 3360 Pro He He Thr Pro Phe Tyr Pro Ser Asn Gly Thr Ser Val He Ser

1110 TCC TCA GTA ATT TCT TCC TCA GTC ACT TCT TCT CTA TTC ACT TCT TCT 3408 Ser Ser Val He Ser Ser Ser Val Thr Ser Ser Leu Phe Thr Ser Ser

1125 CCA GTC ATT TCT TCC TCA GTC ATT TCT TCT TCT ACA ACA ACC TCC ACT 3456 Pro Val He Ser Ser Ser Val He Ser Ser Ser Thr Thr Thr Ser Thr

1140 TCT ATA TTT TCT GAA TCA TCT AAA TCA TCC GTC ATT CCA ACC AGT AGT 3504 Ser He Phe Ser Glu Ser Ser Lys Ser Ser Val He Pro Thr Ser Ser

1155 TCC ACC TCT GGT TCT TCT GAG AGC GAA ACG AGT TCA GCT GGT TCT GTC 3552 Ser Thr Ser Gly Ser Ser Glu Ser Glu Thr Ser Ser Ala Gly Ser Val

1170 TCT TCT TCC TCT TTT ATC TCT TCT GAA TCA TCA AAA TCT CCT ACA TAT 3600 Ser Ser Ser Ser Phe He Ser Ser Glu Ser Ser Lys Ser Pro Thr Tyr 1185 1200

TCT TCT TCA TCA TTA CCA CTT GTT ACC AGT GCG ACA ACA AGC CAG GAA 3648 Ser Ser Ser Ser Leu Pro Leu Val Thr Ser Ala Thr Thr Ser Gin Glu

1215 ACT GCT TCT TCA TTA CCA CCT GCT ACC ACT ACA AAA ACG AGC GAA CAA 3696 Thr Ala Ser Ser Leu Pro Pro Ala Thr Thr Thr Lys Thr Ser Glu Gin

1230 ACC ACT TTG GTT ACC GTG ACA TCC TGC GAG TCT CAT GTG TGC ACT GAA 3744 Thr Thr Leu Val Thr Val Thr Ser Cys Glu Ser His Val Cys Thr Glu

1245 TCC ATC TCC CCT GCG ATT GTT TCC ACA GCT ACT GTT ACT GTT AGC GGC 3792 Ser He Ser Pro Ala He Val Ser Thr Ala Thr Val Thr Val Ser Gly

1260 GTC ACA ACA GAG TAT ACC ACA TGG TGC CCT ATT TCT ACT ACA GAG ACA 3840 Val Thr Thr Glu Tyr Thr Thr Trp Cys Pro He Ser Thr Thr Glu Thr

1275 ACA AAG CAA ACC AAA GGG ACA ACA GAG CAA ACC ACA GAA ACA ACA AAA 3888 Thr Lys Gin Thr Lys Gly Thr Thr Glu Gin Thr Thr Glu Thr Thr Lys

1290

CAA ACC ACG GTA GTT ACA ATT TCT TCT TGT GAA TCT GAC GTA TGC TCT 3936 Gin Thr Thr Val Val Thr He Ser Ser Cys Glu Ser Asp Val Cys Ser

1305 AAG ACT GCT TCT CCA GCC ATT GTA TCT ACA AGC ACT GCT ACT ATT AAC 3984 Lys Thr Ala Ser Pro Ala He Val Ser Thr Ser Thr Ala Thr He Asn

1320 GGC GTT ACT ACA GAA TAC ACA ACA TGG TGT CCT ATT TCC ACC ACA GAA 4032 Gly Val Thr Thr Glu Tyr Thr Thr Trp Cys Pro He Ser Thr Thr Glu

1335 TCG AGG CAA CAA ACA ACG CTA GTT ACT GTT ACT TCC TGC GAA TCT GGT 4080 Ser Arg Gin Gin Thr Thr Leu Val Thr Val Thr Ser Cys Glu Ser Gly

1350 GTG TGT TCC GAA ACT GCT TCA CCT GCC ATT GTT TCG ACG GCC ACG GCT 4128 Val Cys Ser Glu Thr Ala Ser Pro Ala He Val Ser Thr Ala Thr Ala

1365 ACT GTG AAT GAT GTT GTT ACG GTC TAT CCT ACA TGG AGG CCA CAG ACT 4176 Thr Val Asn Asp Val Val Thr Val Tyr Pro Thr Trp Arg Pro Gin Thr

1380 GCG AAT GAA GAG TCT GTC AGC TCT AAA ATG AAC AGT GCT ACC GGT GAG 4224 Ala Asn Glu Glu Ser Val Ser Ser Lys Met Asn Ser Ala Thr Gly Glu

1395 ACA ACA ACC AAT ACT TTA GCT GCT GAA ACG ACT ACC AAT ACT GTA GCT 4272 Thr Thr Thr Asn Thr Leu Ala Ala Glu Thr Thr Thr Asn Thr Val Ala

1410 GCT GAG ACG ATT ACC AAT ACT GGA GCT GCT GAG ACG AAA ACA GTA GTC 4320 Ala Glu Thr He Thr Asn Thr Gly Ala Ala Glu Thr Lys Thr Val Val 1425 1440

ACC TCT TCG CTT TCA AGA TCT AAT CAC GCT GAA ACA CAG ACG GCT TCC 4368 Thr Ser Ser Leu Ser Arg Ser Asn His Ala Glu Thr Gin Thr Ala Ser

1455 GCG ACC GAT GTG ATT GGT CAC AGC AGT AGT GTT GTT TCT GTA TCC GAA 4416 Ala Thr Asp Val He Gly His Ser Ser Ser Val Val Ser Val Ser- Glu

1470 ACT GGC AAC ACC AAG AGT CTA ACA AGT TCC GGG TTG AGT ACT ATG TCG 4464 Thr Gly Asn Thr Lys Ser Leu Thr Ser Ser Gly Leu Ser Thr Met Ser

1485 CAA CAG CCT CGT AGC ACA CCA GCA AGC AGC ATG GTA GGA TAT AGT ACA 4512 Gin Gin Pro Arg Ser Thr Pro Ala Ser Ser Met Val Gly Tyr Ser Thr

1500 GCT TCT TTA GAA ATT TCA ACG TAT GCT GGC AGT GCC AAC AGC TTA CTG 4560 Ala Ser Leu Glu He Ser Thr Tyr Ala Gly Ser Ala Asn Ser Leu Leu

1515 GCC GGT AGT GGT TTA AGT GTC TTC ATT GCG TCC TTA TTG CTG GCA ATT 4608 Ala Gly Ser Gly Leu Ser Val Phe He Ala Ser Leu Leu Leu Ala He

1530 ATT TAA 4614

He ***

Sequence number: 2 Sequence length: 2589 Sequence type: nucleic acid Strandedness: double Topology: linear

Molecule type: Genomic DNA

Original source: Saccharomyces cerevisiae ABXL-ID

Sequence 2

ATG ACA ATG CCT CAT CGC TAT ATG TTT TTG GCA GTC TTT ACA CTT CTG 48 Met Thr Met Pro His Arg Tyr Met Phe Leu Ala Val Phe Thr Leu Leu

15 GCA CTA ACT AGT GTG GCC TCA GGA GCC ACA GAG GCG TGC TTA CCA GCA 96 Ala Leu Thr Ser Val Ala Ser Gly Ala Thr Glu Ala Cys Leu Pro Ala

30 GGC CAG AGG AAA AGT GGG ATG AAT ATA AAT TTT TAC CAG TAT TCA TTG 144 Gly Gin Arg Lys Ser Gly Met Asn He Asn Phe Tyr Gin Tyr Ser Leu

45 AAA GAT TCC TCC ACA TAT TCG AAT GCA GCA TAT ATG GCT TAT GGA TAT 192 Lys Asp Ser Ser Thr Tyr Ser Asn Ala Ala Tyr Met Ala Tyr Gly Tyr

60 GCC- TCA AAA ACC AAA CTA GGT TCT GTC GGA GGA CAA ACT GAT ATC TCG 240 Ala Ser Lys Thr Lys Leu Gly Ser Val Gly Gly Gin Thr Asp He Ser

75 ATT GAT TAT AAT ATT CCC TGT GTT AGT TCA TCA GGC ACA TTT CCT TGT 288 He Asp Tyr Asn He Pro Cys Val Ser Ser Ser Gly Thr Phe Pro Cys

90 CCT CAA GAA GAT TCC TAT GGA AAC TGG GGA TGC AAA GGA ATG GGT GCT 336 Pro Gin Glu Asp Ser Tyr Gly Asn Trp Gly Cys Lys Gly Met Gly Ala

105 TGT TCT AAT AGT CAA GGA ATT GCA TAC TGG AGT ACT GAT TTA TTT GGT 384 Cys Ser Asn Ser Gin Gly He Ala Tyr Trp Ser Thr Asp Leu Phe Gly

120 TTC TAT ACT ACC CCA ACA AAC GTA ACC CTA GAA ATG ACA GGT TAT TTT 432 Phe Tyr Thr Thr Pro Thr Asn Val Thr Leu Glu Met Thr Gly Tyr Phe

135 TTA CCA CCA CAG ACG GGT TCT TAC ACA TTC AAG TTT GCT ACA GTT GAC 480 Leu Pro Pro Gin Thr Gly Ser Tyr Thr Phe Lys Phe Ala Thr Val Asp

150 GAC TCT GCA ATT CTA TCA GTA GGT GGT GCA ACC GCG TTC AAC TGT TGT 528 Asp Ser Ala He Leu Ser Val Gly Gly Ala Thr Ala Phe Asn Cys Cys

165 GCT CAA CAG CAA CCG CCG ATC ACA TCA ACG AAC TTT ACC ATT GAC GGT 576 Ala Gin Gin Gin Pro Pro He Thr Ser Thr Asn Phe Thr He Asp Gly

180 ATC AAG CCA TGG GGT GGA AGT TTG CCA CCT AAT ATC GAA GGA ACC GTC 624 He Lys Pro Trp Gly Gly Ser Leu Pro Pro Asn He Glu Gly Thr Val

195 TAT ATG TAC GCT GGC TAC TAT TAT CCA ATG AAG GTT GTT TAC TCG AAC 672 Tyr Met Tyr Ala Gly Tyr Tyr Tyr Pro Met Lys Val Val Tyr Ser Asn

210 GCT GTT TCT TGG GGT ACA CTT CCA ATT AGT GTG ACA CTT CCA GAT GGT 720

Ala Val Ser Trp Gly Thr Leu Pro He Ser Val Thr Leu Pro Asp Gly 225 240

ACC ACT GTA AGT GAT GAC TTC GAA GGG TAC GTC TAT TCC TTT GAC GAT 768 Thr Thr Val Ser Asp Asp Phe Glu Gly Tyr Val Tyr Ser Phe Asp Asp

255 GAC CTA AGT CAA TCT AAC TGT ACT GTC CCT GAC CCT TCA AAT TAT GCT 816 Asp Leu Ser Gin Ser Asn Cys Thr Val Pro Asp Pro Ser Asn Tyr Ala

270 GTC AGT ACC ACT ACA ACT ACA ACG GAA CCA TGG ACC GGT ACT TTC ACT 864 Val Ser Thr Thr Thr Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr

285 TCT ACA TCT ACT GAA ATG ACC ACC GTC ACC GGT ACC AAC GGC GTT CCA 912 Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Val Pro

300 ACT GAC GAA ACC GTC ATT GTC ATC AGA ACT CCA ACA ACT GCT AGC ACC 960 Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Thr Ala Ser Thr

315 ATC ATA ACT ACA ACT GAG CCA TGG ACT GGT ACT TTC ACT TCT ACA TCT 1008 He He Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser

330 ACT GAA ATG ACC ACC GTC ACC GGT ACT AAC GGT CAA CCA ACT GAC GAA 1056 Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Gin Pro Thr Asp Glu

345 ACC GTG ATT GTT ATC AGA ACT CCA ACC AGT GAA GGT TTG GTT ACA ACC 1104 Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu Val Thr Thr

360 ACC ACT GAA CCA TGG ACT GGT ACT TTT ACT TCG ACT TCC ACT GAA ATG 1152 Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met

375 TCT ACT GTC ACT GGA ACC AAT GGC TTG CCA ACT GAT GAA ACT GTC ATT 1200 Ser Thr Val Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr Val He

390 GTT GTC AAA ACT CCA ACT ACT GCC ATC TCA TCC AGT TTG TCA TCA TCA 1248 Val Val Lys Thr Pro Thr Thr Ala He Ser Ser Ser Leu Ser Ser Ser

405 TCT TCA GGA CAA ATC ACC AGC TCT ATC ACG TCT TCG CGT CCA ATT ATT 1296 Ser Ser Gly Gin He Thr Ser Ser He Thr Ser Ser Arg Pro He He

420 ACC CCA TTC TAT CCT AGC AAT GGA ACT TCT GTG ATT TCT TCC TCA GTA 1344 Thr Pro Phe Tyr Pro Ser Asn Gly Thr Ser Val He Ser Ser Ser Val

435 ATT TCT TCC TCA GTC ACT TCT TCT CTA TTC ACT TCT TCT CCA GTC ATT 1392 He Ser Ser Ser Val Thr Ser Ser Leu Phe Thr Ser Ser Pro Val He

450 TCT TCC TCA GTC ATT TCT TCT TCT ACA ACA ACC TCC ACT TCT ATA TTT 1440 Ser Ser Ser Val He Ser Ser Ser Thr Thr Thr Ser Thr Ser He Phe 465 480

TCT GAA TCA TCT AAA TCA TCC GTC ATT CCA ACC AGT AGT TCC ACC TCT 1488 Ser Glu Ser Ser Lys Ser Ser Val He Pro Thr Ser Ser Ser Thr Ser

495 GGT TCT TCT GAG AGC GAA ACG AGT TCA GCT GGT TCT GTC TCT TCT TCC 1536 Gly Ser Ser Glu Ser Glu Thr Ser Ser Ala Gly Ser Val Ser Ser Ser

510 TCT TTT ATC TCT TCT GAA TCA TCA AAA TCT CCT ACA TAT TCT TCT TCA 1584 Ser Phe He Ser Ser Glu Ser Ser Lys Ser Pro Thr Tyr Ser Ser Ser

525 TCA TTA CCA CTT GTT ACC AGT GCG ACA ACA AGC CAG GAA ACT GCT TCT 1632 Ser Leu Pro Leu Val Thr Ser Ala Thr Thr Ser Gin Glu Thr Ala Ser

540 TCA TTA CCA CCT GCT ACC ACT ACA AAA ACG AGC GAA CAA ACC ACT TTG 1680 Ser Leu Pro Pro Ala Thr Thr Thr Lys Thr Ser Glu Gin Thr Thr Leu

555 GTT ACC GTG ACA TCC TGC GAG TCT CAT GTG TGC ACT GAA TCC ATC TCC 1728 Val Thr Val Thr Ser Cys Glu Ser His Val Cys Thr Glu Ser He Ser

570 CCT GCG ATT GTT TCC ACA GCT ACT GTT ACT GTT AGC GGC GTC ACA ACA 1776 Pro Ala He Val Ser Thr Ala Thr Val Thr Val Ser Gly Val Thr Thr

585 GAG TAT ACC ACA TGG TGC CCT ATT TCT ACT ACA GAG ACA ACA AAG CAA 1824 Glu Tyr Thr Thr Trp Cys Pro He Ser Thr Thr Glu Thr Thr Lys Gin

600 ACC AAA GGG ACA ACA GAG CAA ACC ACA GAA ACA ACA AAA CAA ACC ACG 1872 Thr Lys Gly Thr Thr Glu Gin Thr Thr Glu Thr Thr Lys Gin Thr Thr

615 GTA GTT ACA ATT TCT TCT TGT GAA TCT GAC GTA TGC TCT AAG ACT GCT 1920 Val Val Thr He Ser Ser Cys Glu Ser Asp Val Cys Ser Lys Thr Ala

630 TCT CCA GCC ATT GTA TCT ACA AGC ACT GCT ACT ATT AAC GGC GTT ACT 1968 Ser Pro Ala He Val Ser Thr Ser Thr Ala Thr He Asn Gly Val Thr

645 ACA GAA TAC ACA ACA TGG TGT CCT ATT TCC ACC ACA GAA TCG AGG CAA 2016 Thr Glu Tyr Thr Thr Trp Cys Pro He Ser Thr Thr Glu Ser Arg Gin

660 CAA ACA ACG CTA GTT ACT GTT ACT TCC TGC GAA TCT GGT GTG TGT TCC 2064 Gin Thr Thr Leu Val Thr Val Thr Ser Cys Glu Ser Gly Val Cys Ser

675 GAA ACT GCT TCA CCT GCC ATT GTT TCG ACG GCC ACG GCT ACT GTG AAT 2112 Glu Thr Ala Ser Pro Ala He Val Ser Thr Ala Thr Ala Thr Val Asn

690 GAT GTT GTT ACG GTC TAT CCT ACA TGG AGG CCA CAG ACT GCG AAT GAA 2160 Asp Val Val Thr Val Tyr Pro Thr Trp Arg Pro Gin Thr Ala Asn Glu 705 720

GAG TCT GTC AGC TCT AAA ATG AAC AGT GCT ACC GGT GAG ACA ACA ACC 2208 Glu Ser Val Ser Ser Lys Met Asn Ser Ala Thr Gly Glu Thr Thr Thr

735 AAT ACT TTA GCT GCT GAA ACG ACT ACC AAT ACT GTA GCT GCT GAG ACG 2256 Asn Thr Leu Ala Ala Glu Thr Thr Thr Asn Thr Val Ala Ala Glu Thr

750

ATT ACC AAT ACT GGA GCT GCT GAG ACG AAA ACA GTA GTC ACC TCT TCG 230 He Thr Asn Thr Gly Ala Ala Glu Thr Lys Thr Val Val Thr Ser Ser

765 CTT TCA AGA TCT AAT CAC GCT GAA ACA CAG ACG GCT TCC GCG ACC GAT 235 Leu Ser Arg Ser Asn His Ala Glu Thr Gin Thr Ala Ser Ala Thr Asp

780 GTG ATT GGT CAC AGC AGT AGT GTT GTT TCT GTA TCC GAA ACT GGC AAC 2400 Val He Gly His Ser Ser Ser Val Val Ser Val Ser Glu Thr Gly Asn

795 ACC AAG AGT CTA ACA AGT TCC GGG TTG AGT ACT ATG TCG CAA CAG CCT 2448 Thr Lys Ser Leu Thr Ser Ser Gly Leu Ser Thr Met Ser Gin Gin Pro

810 CGT AGC ACA CCA GCA AGC AGC ATG GTA GGA TAT AGT ACA GCT TCT TTA 2496 Arg Ser Thr Pro Ala Ser Ser Met Val Gly Tyr Ser Thr Ala Ser Leu

825 GAA ATT TCA ACG TAT GCT GGC AGT GCC AAC AGC TTA CTG GCC GGT AGT 2544 Glu He Ser Thr Tyr Ala Gly Ser Ala Asn Ser Leu Leu Ala Gly Ser

840 GGT TTA AGT GTC TTC ATT GCG TCC TTA TTG CTG GCA ATT ATT TAA 2589 Gly Leu Ser Val Phe He Ala Ser Leu Leu Leu Ala He He ***

855

Sequence number: 3 Sequence length: 33 Sequence type: nucleic acid Strandedness: single Topology: linear

Molecule type: other nucleic acid/synthetic DNA Sequence 3

CCCAAGCTTA AAAATGACAA TGCCTCATCG CTA

Sequence number: 4 Sequence length: 33 Sequence type: nucleic acid Strandedness: single Topology: linear

Molecule type: other nucleic acid/synthetic DNA Sequence 4

CCCAAGCTTT TAAATAATTG CCAGCAATAA GGA