JPH0767664A - New ho gene and method for raising conjugative type yeast using yeast having inactivated the same gene - Google Patents

Abstract

PURPOSE:To obtain a new HO gene containing a base sequence encoding an endonuclease capable of cleaving a conjugative type gene locus and sufficiently suppressing transfer of yeast to homotalism and smoothly multiplying excellent yeast. CONSTITUTION:Saccharomyces carlsbergens is KBY003 as a beer fermenting yeast is cultured, the yeast is collected, the cells are suspended in a buffer solution, made into a protoplast by a conventional method, mixed with a surfactant, maintained at 65 deg.C for 15 minutes, subjected to complete bacteriolysis, slowly added to a density gradient tube of 10-40 % sucrose and ultracentrifuged to give a chromosomeal DNA. Then the chromosomeal DNA is treated with a restriction enzyme, each fragment is linked to a vector, inserted into Escherichia coli, transformed and screened by a probe having a homothalism (HO) gene sequence to select a positive clone. Its DNA is recovered and treated with a restriction enzyme to give a new HO gene containing a base sequence encoding an endonuclease capable of cleaving a conjugative type gene locus.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel homothalism (HO) gene, an inactivated fragment of the HO gene, a transformed yeast characterized by being transformed with the inactivated fragment, and the transformed yeast. The present invention relates to a yeast breeding method.

[0002]

2. Description of the Related Art Alcohol production in alcoholic beverages such as sake, wine and beer, and fermented foods such as bread, miso and soy sauce is all produced by yeast. That is, yeast has been deeply involved in human daily life since history. Furthermore, due to the recent development of biotechnology, it has become involved in the manufacture of pharmaceuticals.

For effective utilization of yeast, it is essential to breed excellent yeast. As a traditional method for breeding such yeast, a conventional breeding method by selection for many years can be mentioned, but the probability of obtaining a desired trait is low. In addition to this, in recent years, artificial mutation methods and cell fusion methods using drugs and radiation, and methods that positively utilize the mating phenomenon between yeasts can be mentioned.

However, in the above-mentioned mutation method, it is generally difficult to identify the gene site causing the mutation, and even if a yeast having an excellent trait can be obtained by the mutation method, There are many problems in applying this directly to foods and the like. In addition, since yeast is a high-order polyploidy, it is difficult to obtain a mutant having a desired property at a high frequency, like bacteria such as Escherichia coli, even if it is mutated.

Further, rearrangement (rearrangement) of the chromosome of the fusion produced by the cell fusion method has been reported (1992, Agricultural Chemistry Society, Yonehana et al .: Fukuyama University, Biotechnology), and in the cell fusion method, this method was used. It is difficult to predict what kind of trait yeast can be obtained. Therefore, in the breeding of yeasts, it has been widely attempted to carry out cross breeding by positively utilizing the mating phenomenon between yeasts, but such a method is not without drawbacks.

[0006] This method has the advantages that the conjugation frequency is high, that it can be carried out without a marker, and that backcrossing is possible, as compared with fusion. That is, by breeding yeasts (heterotalism strains) having a stable zygote having an excellent trait for breeding useful yeasts, 2
The ideal is to obtain a descendant of a polyploid or a higher polyploid (heterotalism). However, it is a useful yeast,
Saccha myces (Saccha myces), which is currently applied in many fields
In yeast belonging to the genus romyces, there is a gene called homotalism (HO) gene that induces non-zygotic diploid or higher polyploid offspring (homotarhythmism) through a zygotic spore transiently. To do.

Due to the presence of such HO gene, spores having a desired trait are self-multiplied in the process of growth, which makes it difficult to isolate mating yeast having a desired phenotype. There is. Therefore, when conjugating, a method of giving a genetic marker to the yeast to be conjugated and using forcible conjugation at low frequency, or a method of contacting spores and cells isolated under a microscope or spores and conjugating them is used. Has been.

However, practical yeasts usually lack genetic markers. In addition, conjugation under a microscope allows spores to be joined without characterizing them, and the qualities of the spores are not known until the zygote is separated. For this reason, brewing yeast, which has a low bonding frequency, undergoes enormous work under a microscope,
It requires screening of an extremely large number of yeasts and is not practical for breeding.

Therefore, the location of the HO gene and the nucleotide sequence of the gene are further clarified, and a drug resistance marker or the like is introduced into the HO gene to use a HO gene fragment in which its original function is lost, Attempts have been made to transform the yeast to be bred to inactivate the function of the HO gene of the yeast. Certainly, such an attempt is effective for Saccharomyces cerevisiae, which is baker's yeast and top-fermenting yeast for beer.

However, Saccharomyces c.
arlsbergensais) and other yeasts of the genus Saccharomyces, the presence of other HO genes in addition to the above-mentioned HO gene (hereinafter referred to as HO-SC gene) is observed.
The inactivation of the O-SC gene could not sufficiently inhibit the transfer of yeast to the above homothalism.

[0011]

Therefore, the problem to be solved by the present invention is to clarify the location of the HO gene other than the already known HO-SC gene and its nucleotide sequence, and to identify the above-mentioned HO-SC gene. It is intended to provide a means for sufficiently inhibiting the transition to yeast homothalism, by using the HO gene other than the HO-SC gene alone.

[0012]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and as a result, Saccharomyces curlsbergensis (Sac
charomyces carlsbergensais) and Saccharomyces
New HO in Ubarum (Saccharomyces uvarum)
It was clarified that the existence of the gene could be identified and the nucleotide sequence of the new HO gene could be clarified to solve the above problems.

That is, the gist of the present invention is as follows. (1) A yeast endonuclease gene having the amino acid sequence shown in SEQ ID NO: 1 and containing a nucleotide sequence encoding an endonuclease capable of cleaving the mating type locus. (2) A yeast endonuclease gene having the amino acid sequence shown in SEQ ID NO: 4 and containing a nucleotide sequence encoding an endonuclease capable of cleaving the mating type locus.

(3) The endonuclease gene according to (1) above, wherein the nucleotide sequence encoding the endonuclease is represented by SEQ ID NO: 3. (4) The endonuclease gene according to (2) above, wherein the base sequence encoding the endonuclease is represented by SEQ ID NO: 6. (5) A yeast homotalism gene containing the endonuclease gene according to (1) or (3) above.

(6) A yeast homothalism gene containing the endonuclease gene according to (2) or (4) above. (7) The homotalism gene according to (5) above, which is represented by SEQ ID NO: 2. (8) The homotalism gene according to (6) above, which is represented by SEQ ID NO: 5.

(9) A yeast homotalism gene fragment inactivated by suppressing the expression of at least a part of the endonuclease gene according to any one of (1) to (4) above. (10) Suppression of expression by restriction enzyme cleavage, insertion, deletion,
By substitution or addition, by restriction enzyme cleavage, insertion, deletion, substitution or addition to the promoter or terminator region of the endonuclease gene, by insertion or substitution of a marker gene for the promoter or terminator region, or the endonuclease Antisense R of gene
The homotalism gene fragment according to (9) above, which is due to the introduction of NA.

(11) A YIp-type plasmid for homotalism gene disruption, which comprises a part of the endonuclease gene and the marker gene according to any one of (1) to (4) above. (12) An inactivated YIp type plasmid for homotalism gene disruption, which incorporates the endonuclease gene and the marker gene according to any one of (1) to (4) above. (13) A transformed yeast transformed with the homotalism gene fragment according to (9) or (10) above.

(14) A transformed yeast transformed with the YIp type plasmid for disrupting the homotalism gene according to the above (11). (15) A transformed yeast transformed with the YIp type plasmid for disrupting the homotalism gene according to (12). (16) A method for breeding yeast, which comprises culturing the transformed yeast according to any one of (13) to (15) to breed heterozygous mating yeast.

The present invention will be described in detail below. A. A yeast homothalism gene (hereinafter referred to as HO gene or HO-B of the present invention) encoding the nucleotide sequence shown in SEQ ID NO: 2.
The cloning method of R gene or HO-UV gene) will be described.

The HO gene of the present invention, HO-BR gene (SEQ ID NO: 2) and HO-UV gene (SEQ ID NO: 5)
The endonucleases (SEQ ID NO: 1 and SEQ ID NO: 4, respectively) that cleave the yeast mating type locus (MAT) encoded therein identify the locus (MAT) that controls the mating type of Saccharomyces yeast. It is an enzyme that controls zygotic conversion by cleaving at the site and initiating recombination with silenced zygotic genes (HML, HMR) present at other loci.

The source of the HO-BR gene which is the HO gene of the present invention is not particularly limited as long as it is a yeast having a gene substantially containing the HO-BR gene. For example,
Saccharomyces, a bottom-fermenting yeast of beer. Saccharomyces carlsbergensis, Saccharomyces bayanus
(IFO 1127), Saccharomyces. Globosus (Saccha
romyces globosus) (IFO 10557), Saccharomyces monacensis (ATCC)
0220, IFO 279), Saccharomyces. Inucitatus (S
It can be cloned from accharomyces inusitatus) (IFO 1343) and the like. H which is one of the HO genes of the present invention
The source of the O-UV gene is Saccharomyces bayanus (IFO
1127), Saccharomyces inusitatus (IFO 1343), Sacch
aromyces uvarum (IFO 0615), Saccharomyces heteroge
It can be cloned from nicus (IFO 1620), Pilsen No. 1 (IFO 2031) or the like.

It should be noted that even if the yeast is classified as an incomplete bacterium because sporulation is not observed, any yeast having the HO-BR or HO-UV gene can be used as a DNA source. Is. In the present invention, the cloning step is usually performed by a known method (Molecular Cloning (198
9): It can be performed according to Methods in Enzymology 194 (1991)).

That is, the above-mentioned yeast gene library is prepared by extracting all the above-mentioned yeast DNA and introducing a gene introduction vector into which a DNA fragment derived from the above DNA is incorporated into a host. Then, by selecting a desired clone from such a gene library and amplifying the clone, the above cloning step can be carried out.

Preparation of the above-mentioned yeast gene library For the extraction of the above-mentioned yeast total DNA, for example, yeast protoplasts are prepared, and from these protoplasts, commonly known DNA extraction methods such as phenol extraction method and chloroform extraction method are used. It can be performed using a conventional method. In addition to the above-described method of preparing protoplasts in advance, DNA can be extracted by a cell disruption method using glass beads or the like, but it is easy to prepare a high molecular weight DNA. It is preferable to carry out the protoplast method.

A PCR method (PCR Technology Henry) using the above-mentioned total DNA as a template and a single-stranded DNA fragment considered to be common to various HO genes as a primer is used.
A. Erlich, Stockton press (1989)) can be used to specifically amplify the desired HO gene to make the cloning process more efficient.

Any gene transfer vector that is obtained by a conventional method such as restriction enzyme treatment and that incorporates the DNA fragment derived from the above DNA may be used, so long as it is known as a transfer vector for yeast. It is possible to For example, a YRp-type plasmid that is a yeast multicopy vector having an ARS sequence of a yeast chromosome as an origin of replication; a YEp-type plasmid that is a yeast multicopy vector having a yeast 2 μm DNA origin of replication; a yeast chromosome ARS sequence that replicates. YCp-type plasmid, which is a single copy vector for yeast having a centromere DNA sequence of the yeast chromosome as an origin, and YIP-type plasmid, which is a yeast chromosome-integrating vector having no origin of replication of yeast, and the like. . The above-described yeast transfer vector is publicly known (published by Asakura Shoten, ABC Series "Genetic Engineering for Substances", p68), and can be easily prepared. In addition, pBR strains, pUC strains, and Blue Scripts (Blue Scri
Commercially available commercially available transfer plasmids of the pt) strain can be used. Also, Charon system and EMBR
A wide variety of phages, cosmids, etc. for introducing strains can be used.

The host to be transformed or transduced with the prepared vector for introduction may be selected according to the kind of the above vector.

Selection of Clone The method of selecting a desired clone from the gene library and amplifying the clone can also be a generally known method.

That is, from the above gene library,
A clone having a desired HO-BR gene or HO-UV gene is selected by a colony hybridization method, a plaque hybridization method or the like using a labeled probe containing a sequence unique to the HO gene, and the desired HO-BR gene or HO- Amplification of the UV gene in E. coli cells or PC of the desired HO-BR gene or HO-UV gene using a primer containing a sequence unique to the HO gene
It can be obtained by directly amplifying by the R method.

As the probe used in the colony hybridization method or the plaque hybridization method, it is possible to use the entire base sequence of the HO gene, and it is also possible to use a partial synthetic oligonucleotide. In addition, when the hybridization and the probe are washed off, it is possible to carry out at a lower temperature as compared with the case of a perfectly matched gene.

The nucleotide sequence of the desired gene obtained by the above method can be determined and confirmed by, for example, Maxam-Gilbert, Meth. Enzym., 65 , 499-560.
(1980)) and dideoxynucleotide chain termination method using M13 phage (Messing, J. and Vieire, J., Gene, 19 , 269-276.
(1982)) and so on. Further, the homothalism gene of the present invention determined by the above method is chemically synthesized according to a commonly known chemical synthesis method, for example, a conventional method such as the phosphite-triester method (Nature, 310 , 105 (1984)). be able to.

Further, H which is the HO gene of the present invention can also be obtained by semisynthesis using a part of the above-mentioned HO gene derived from natural products
The O-BR gene or the HO-UV gene can be obtained. The HO gene of the present invention obtained by the above method includes the endonuclease gene (structural gene) encoding an endonuclease that cleaves a yeast homotalism gene having at least the amino acid sequence shown in SEQ ID NO: 1 or 4. Genes with other sequences upstream or downstream of, for example, so-called promoter region or terminator region of the structural gene are also included in the HO gene of the present invention.

B. HO-BR gene or H by transformation
Inactivation of O-UV Gene The inactivation of the HO-BR gene or the HO-UV gene by transformation is necessary to transfer the yeast of the homotalism type life cycle to the heterotalism type life cycle. here,
The “inactivation” means inhibiting the expression of the HO gene by suppressing the expression of the endonuclease gene contained in the HO gene.

The inactivation of the HO-BR gene or HO-UV gene in vitro can be carried out by suppressing the expression of the endonuclease gene by adopting a method usually used as a gene recombination technique. .

The means for suppressing the expression of the endonuclease gene is not limited to the case where at least a part of the endonuclease gene itself is cleaved with a restriction enzyme, and insertion, deletion, substitution, addition, etc. may be performed. Cleavage, insertion, deletion, substitution, addition, etc. by a restriction enzyme may be carried out within the promoter region or terminator region that regulates the expression of the nuclease gene. Also,
A marker gene can be inserted or replaced in the promoter region or terminator region. Furthermore, the expression of the endonuclease gene may be suppressed by introducing antisense RNA of the endonuclease gene.

In the present invention, the inactivated HO-BR gene or HO-UV gene is introduced into yeast, and
-In order to easily confirm that the BR gene or HO-UV gene has been inactivated, the marker gene was inserted into the endonuclease structural gene portion, promoter region or terminator region of the HO-BR gene or HO-UV gene. Inactivation is preferred and advantageous.

The marker used in the present invention is not particularly limited as long as it is a marker gene that can be used in yeast. For example, drug resistance genes against chemical substances such as G418, blasticidin S, cycloheximide, sulfometuron methyl, cerulenin, and CUP1
It is possible to further exemplify such heavy metal resistance genes as the above, and enzyme genes whose activity can be easily discriminated such as β-galactosidase and α-acetolactate decarboxylase (ALDC). These are dominant markers and can be mentioned as preferable markers in that they can be used for high-order polyploid yeast.

If a sequence specific to the sequence used for disruption is used, it can be easily detected by the PCR method, colony hybridization method or the like. The inactivation of the yeast HO-BR gene or HO-UV gene is carried out by inactivating the HO-BR gene fragment or HO-UV gene fragment by inserting the above marker; a part of the yeast homotalism gene endonuclease gene, And a YIp-type plasmid for disrupting a homothalism gene incorporating a marker gene; or a YIp-type plasmid for disrupting a homothalism gene disrupting a yeast homothalism gene endonuclease gene and a marker gene.
It is carried out by inducing recombination with the O-BR gene or the HO-UV gene.

The inactivated fragment is prepared as follows. That is, an inactivating gene in which a desired marker gene is incorporated into the HO gene is incorporated into a mild plasmid that can be amplified in a large amount in a host, the plasmid is amplified in a large amount in the host, and the amplified mild type is amplified. A desired inactivated fragment can be obtained by separating and purifying a plasmid, subjecting the plasmid to a specific restriction enzyme treatment, and separating the molecular weight by agarose electrophoresis or the like. The inactive fragments thus obtained are usually electroporated, lithium, or spheroplasted (Meth
ods in Enzymology, vol. 194 (1991)) and the like, and directly introduced into yeast intended for transformation.

Details of the above steps will be described in Examples described later. The HO-BR gene fragment or HO-UV gene fragment inactivated by insertion, deletion, substitution or addition should be incorporated into a yeast vector, for example, YIp type plasmid, and then introduced into yeast cells by a conventional method. Thus, yeast cells can be transformed with the inactivated fragment incorporated in the vector.

That is, the integrated yeast is transformed into H
Internal f that disrupts the O-BR or HO-UV gene
The ragment disruption method can also be adopted. Furthermore, mutations were added to the inside of the HO-BR gene or HO-UV gene, the gene was integrated into a YIp type plasmid, yeast was transformed, and then clones in which the normal HO-BR gene or HO-UV gene and the marker were excised were screened. It is possible to use the gene replacement (Popin / Popout) method (Metho
ds in Enzymology, Vol. 194, p281-301).

The above internal fragment disruption
The YIp-type plasmid for disrupting the homothalism gene, in which a part of the yeast homothalism gene endonuclease gene and the marker gene are used in the method, can be prepared by the HO endonuclease of HO-SC gene, HO-BR gene or HO-UV gene. The internal region of the structural gene portion encoding the amino acid sequence can be prepared by fragmentation by restriction enzyme cleavage or partial deletion by DNA degrading enzyme, and incorporation into the YIp type plasmid.

As the marker gene used for the YIp type plasmid, it is preferable to use the above dominant marker gene. Internal fragm obtained in this way
As a yeast transformation method using the YIp type plasmid for homotalism gene disruption used in the ent disruption method, a generally known method can be used.

Further, the YIp-type plasmid for disrupting the homothalism gene, which is used in the Popin / Popout method and has the yeast homothalism gene endonuclease gene and the marker gene, is prepared by the HO-SC or HO-BR or HO-UV gene. Can be created by deletion, substitution, insertion, or addition.
As the yeast transformation method using the YIp type plasmid for homotalism gene disruption used in the Popin / Popout method thus obtained, a generally known method can be used.

In the Popin / Popout method, yeast strains lacking a dominant marker were screened from the obtained transformants, and chromosomal DNA of the obtained candidate yeast and Southern analysis or PCR analysis was performed. Normal HO
By confirming that the gene is replaced with the mutant HO gene, the desired HO gene can be inactivated.

The type of inactivating fragment to be introduced into cells differs depending on the type of yeast cell intended to inactivate the HO gene. That is, in order to prevent the transfer of the yeast having both the HO-SC gene and the HO-BR gene to homothalism, it is necessary to inactivate both the HO-SC gene and the HO-BR gene of the yeast. Therefore, the type of inactivated fragment that needs to be introduced, in such a case, HO-SC
2 of inactivated fragment of gene and inactivated fragment of HO-BR gene
Need to introduce types. In addition, the HO-BR gene and H
In the case of yeast having both the O-UV gene, it is necessary to introduce two types, an inactivated fragment of the HO-BR gene and an inactivated fragment of the HO-UV gene.

For the method of isolating the HO-SC gene and the nucleotide sequence of the gene, see "David W. Russell et al., MO.
L. AND CELL BIOL., Vol. 6, No12, pp4281-4294 (1986) ", and an inactivated fragment can be prepared based on the HO-SC gene in the same manner as above.

Saccharomyces. Carlsbergensis (Sacchargensis) is a yeast that requires introduction of two types of inactivating fragments of the HO-BR gene and the HO-SC gene.
omycescarlsbergensis), Saccharomyces. Saccharomyces pastrianus (IFO 1167), Saccharomyces. Monacensis (Saccharomyces monacensi
s) (IFO 0220, IFO 279) and the like. Also,
As a yeast that is sufficient if only the HO-BR gene is inactivated,
Saccharomyces. Globosus (Saccharomyces globosu
s).

Saccharomyces bayanus is a yeast for which it is necessary to introduce two types of inactivating fragments of HO-UV gene and HO-BR gene.
(IFO 1127), Saccharomyces inucitatus (Sacch
aromyces inusitatus) (IFO 1343) and the like. In addition, as a yeast which is sufficient to inactivate only the HO-UV gene, Saccharomyces ubalm (Saccharomyces
ces ubarum) (IFO 0615), Saccharomyces heterogenicus (IFO 162)
0) and Pilsen No. 1 (IFO 2031).

However, since the taxonomic names mentioned above are based on sugar assimilation and fermentability, they are directly related to the existence of the HO-SC gene and HO-BR and HO-UV genes. is not. Therefore, it is necessary to obtain information in advance what kind of HO gene is present by Southern analysis, and to judge the use and combination of HO genes according to the information. In addition to the above method, inactivation of the HO gene
It can also be achieved by expressing the antisense RNA in yeast cells.

C. Separation of Transformant Retaining Mating Ability In order to obtain a transformant retaining conjugating ability, it is possible to destroy all the HO genes existing on the chromosome. From some of the resulting spore clones, some were inactivated to sporulate,
It is also possible and an effective method to isolate a clone showing a stable mating type in which all the HO genes are destroyed. Spore formation can be performed by a usual method, and a spore clone can be obtained by a micromanipulator or a random spor method. Junction type test is a type or α
This can easily be done by observing the mating of the mold with the laboratory strain.

[0052]

EXAMPLES The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited to the examples.

[Example 1] Cloning of HO-BR gene from brewer's yeast a) Preparation of library for limiting brewer's yeast molecular weight Extraction of DNA chain from brewer's yeast 1 liter of YPD medium (yeast extract 10 g / l, peptone 20 g) /
Saccharomyces, which is a bottom-fermenting brewer's yeast with l, glucose 20 g / l). Karlsbergensis (Saccharomyces c
arlsbergensis) KBY003 was cultured to an OD of around 10 and the cells were collected and washed with autoclaved water. This is mycelia
2ml per 1g SCE (sorbitol (1M), EDTA (0.125M), trisodium citrate (pH 7) (0.1M), "Zymoly Ace"
After suspending in 100T "(Seikagaku Corporation) (0.01%),
The yeast was completely protoplastized by gentle shaking at 37 ° C for about 2 hours. To this, 3.5 ml of Ly per 1 g of bacterial cells
sis Buffer (Tris-HCl buffer (pH9) (0.5M), EDTA (0.2M)
M) and sodium dodecyl sulfate (SDS) (3%)) were added and gently stirred. This was kept warm at 65 ° C for 15 minutes to completely lyse it. After lysing, cool it to room temperature and prepare 23.5 ml 10-40 of Hitachi ultracentrifuge tube 40PA.
% Sucrose density gradient solution (sodium chloride (0.8M),
10 ml of Tris-HCl buffer (pH8) (0.02M), EDTA (0.01M) and sucrose (10-40%) were gently placed. this,
Using a Hitachi ultracentrifuge SCP85H, centrifugation was performed at 4 ° C / 26000rpm / 3hr. After centrifugation, a Komagome pipette was used to collect about 5 ml of the solution from the place as close as possible to the bottom of the tube.
1 l of TE buffer (Tris-HCl (pH8))
(10 mM), EDTA (1 ml)) was dialyzed overnight.

Cleavage of DNA Strand by Restriction Enzyme Next, about 20 μg of the chromosomal DNA thus obtained was placed in an Eppendorf tube and added to H Buffer (Tris-HCl buffer (pH 7.5) (50 mM), MgCl ₂ (10 mM), Dithiothreitol. (DT
T) (1 mM), NaCl (100 mM)), 200 units of restriction enzyme EcoRI (Boehringer) was added, and the mixture was kept at 37 ° C. for 3 to 4 hours.

Limitation of preparative molecular weight by Southern analysis and preparation of inserted DNA A part of the chromosomal DNA completely digested with EcoRI was applied to 1% agarose gel (acetate buffer, EDTA) and electrophoresed at 100 V for 2 hours. The agarose gel was immersed in an ethidium bromide solution, stained with a DNA molecular weight marker and DNA, and the electrophoretic state was recorded by a photograph or the like. Southern analysis using this agarose gel (Molecular Cloninng, Manniatis et a
, Cold Spring Harbor). As a probe, a HO gene fragment derived from Saccharomyces cerevisiae labeled by the method described below was used. From the obtained autoradiogram, a strong signal band at about 7 Kb and about 4 Kb
A weak signal band was obtained in the vicinity.

20 μg of the chromosomal DNA completely cleaved with EcoRI described in the previous section was electrophoresed on a 1% low-melting point agarose gel, and from about 3.5 Kb to about 4.
The gel part corresponding to a molecular weight of 5 Kb was collected in three parts. DNA fragments were obtained from each low melting agarose gel by heat melting and phenol treatment followed by ethanol precipitation. A part of these DNAs was again subjected to Southern analysis, and the DNA of the fraction giving a positive signal was subjected to the following experiment.

Ligation to vector 1 μg of pUC119 (Takara Shuzo) plasmid DNA was ligated to Hbu
Cut with 30 units EcoRI in ffer. Precipitate and recover the plasmid DNA with twice the amount of ethanol, and after drying,
It was suspended in 0 μl of 500 mM Tris-HCl buffer (pH 8). One unit of Bacterial Alkaline Phosphatase (Takara Shuzo) was added, and the mixture was reacted at 65 ° C. for 1 hour. An equal amount of phenol was added, and the mixture was vigorously stirred with a vortex mixer, and separated into two layers by centrifugation at 12000 rpm / 5 minutes.
The aqueous layer was collected and placed in another tube. After repeating this phenol treatment three times, add an equal amount of phenol / chloroform (1: 1) to the aqueous layer, stir vigorously with a vortex mixer, separate into two layers by centrifugation 12000 rpm / 3 minutes, and put it in another tube. Two times the amount of ethanol was added to the transferred aqueous layer to recover the plasmid DNA by precipitation. Dissolve the precipitate in an appropriate amount of TE buffer.

The thus treated pUC119 (50 ng) and the above-mentioned yeast chromosomal DNA fragment (50 ng) were ligated with Ligation Kit (Takara Shuzo). A part of this Ligation Mixture was used to transform Escherichia coli DH5 strain (Toyobo). Each method followed the experimental method of the Kit.

Preparation of probe Known HO gene (HO-SC gene) sequence (David W, Ru
ssell et al., Mol. Cell. Biol., 6 , 4281-4294 (1986)).
It is used as a probe for cloning the BR gene, and the above-mentioned nucleotide sequence is known. The DNA chain encoding the HO-BR gene synthesizes a part of the known sequence of the yeast gene library and uses it as a probe. It can be easily obtained. In addition, it can be easily obtained by synthesizing a primer based on the known sequence information and performing a PCR reaction using the primer.

Therefore, the chromosomal DNA of the yeast Saccharomyces cerevisiae was extracted by a commonly known method, and this was completely digested with HindIII,
From a gene library prepared by separating a DNA fragment around 2.5 Kb, an oligonucleotide synthesizing a sequence near the N-terminal of the HO gene was labeled with ³² P and used as a probe to obtain the HO-SC gene, and pH03 Was used in subsequent experiments. The HindIII fragment of pH03 (HO-S used as a probe for cloning the HO-BR gene)
The C gene) can be labeled with a radioisotope as long as it can be labeled,
We used a multiprime DNA labeling system (Amersham), and the method followed the attached protocol.

Cloning by colony hybridization 200 transformants per LB plate medium containing 50 μg / ml of ampicillin (bactotryptone 10 g / l, yeast extract 5 g / l, sodium chloride 5 g / l, agar 15 g / l) -300 colonies were formed. A nitrocellulose filter was placed on the same plate medium, and the transformant that appeared was transferred to the nitrocellulose filter by the replica method. As the filter, any filter can be used as long as it can be used for various colony hybridizations on the market.

The method of colony hybridization is based on Molecular Cloning (Cold Spring ha
rbor Laboratory, 1982) (p315, p326-328). The hybridization and washing of the filter were performed at 60 ° C. Colonies on the master plate at positions giving a positive signal on the autoradiograph were separated, and pHOBR-5, which had an insertion direction opposite to that of pHOBR-4, was subjected to the following steps.

B) Determination of the nucleotide sequence of the cloned HO-BR gene The HO-BR gene cloned at the EcoRI site of pUC119 was made into a series of deletions by EXOIII, and the nucleotide sequence was determined by the dideoxy method using a fluorescent primer. Was decided.

That is, a stepwise deletion plasmid was prepared from the above-mentioned pHOBR-4 and pHOBR-5 and used for the determination of the nucleotide sequence. First, 10 μg of plasmid was digested with restriction enzyme PstI,
Subsequent cut with BamHI and an equal volume of water saturated phenol /
Treat with chloroform (50:50) and add DN with ethanol.
A was collected as a precipitate. The precipitate was washed with 70% ethanol and dried under reduced pressure. The deletion from this DNA was prepared using the Takara Shuzo Kiroderation Kit. The method followed the attached protocol. The nucleotide sequence of the obtained partially deleted plasmid was determined by using 373A DNA sequencer manufactured by Applied Biosystems, and the reaction was performed by using a dye primer cycle sequence kit (manufactured by Applied Biosystems). The procedure of nucleotide sequence determination followed the attached protocol.

As a result, the HO gene of the present invention, HO-B
It was revealed that the R gene has the base sequence shown in SEQ ID NO: 2. In addition, the nucleotide sequence portion deduced to be the structural gene portion encoding the homothalism gene endonuclease of the present invention deduced from the nucleotide sequence,
It is as shown in SEQ ID NO: 3, and the amino acid sequence of the homotalism gene endonuclease of the present invention deduced from such a nucleotide sequence is as shown in SEQ ID NO: 1.

The pHOBR-4 used in the above steps was incorporated into Escherichia coli DH5α. Such a transformant was deposited as EKB102 on June 7, 1993, at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, as FERM BP-4323.

[Example 2] Inactivation of HO gene by transformation a) Construction of vector Construction of G-418 resistance marker A 2.9 Kb HindIII fragment containing the PGK gene of yeast (JP-A-2-265488) was pUC18. (Takara Shuzo). From this fragment, a plasmid pUCPGK21 having a PGK promoter and a terminator was prepared by the procedure shown in FIG. The G-418 resistance gene was prepared from the plasmid pNEO (Pharmacia) by the procedure shown in FIG. 1, and pUCPGK21
To prepare pPGKneo2.

Preparation of blasticidin resistance marker of plasmid pSY114P (JP-A-2-265488) having a yeast GPD promoter and a yeast GPD terminator.
Hind of pSY114H with HindIII linker linked to SmaI site
The HindIII fragment of the plasmid pSVSbsr (manufactured by Funakoshi) having the blasticidin resistance gene at the III site was ligated to prepare pGPDBSR by the procedure shown in FIG.

Construction of vector for HO-SC disruption pHO containing 2.5 Kb HindIII fragment containing HO-SC gene
A HindIII-SacII fragment containing no ARS sequence (origin of replication) near the C-terminal of the HO gene was prepared from 3 and blunted with Klenow enzyme, ligated with EcoRI linker, and ligated to EcoRI site of pUC18. PHOK22 was prepared. From pHOK22, Klenow was used to remove BamHI existing inside the HO endonuclease coding region of the HO gene by the procedure shown in FIG.
The SalI fragment containing the G-418 resistance gene of pPGKNEO2 was blunted with Klenow enzyme and ligated to the enzyme-blunted site to prepare pHONEO.

Construction of HO-BR Disruption Vector As shown in FIG. 4, the region corresponding to the ARS sequence of the HO-SC gene was deleted from the pHOBR 4 plasmid containing the HO-BR gene. Using the partially deleted plasmid pHOBR Δ6 prepared, HO-B on this plasmid was used.
After cutting the BglII site of R gene, XhoI linker blunted with Klenow enzyme was ligated to prepare pHOBRX. pHOB
The SalI fragment containing the blasticidin resistance gene of pGPDBRS was ligated to the XhoI site of RX to prepare pHOBRBSR.

Transformation The bottom brewer's yeast BFY268 was transformed as follows. Add yeast to 100 ml of YPD medium until OD = 16
The cells were cultured with shaking at ℃. After collecting the cells, the cells were washed once with sterile water. Then, the cells were washed once with 135 mM Tris-HCl buffer (pH 8.0) and suspended in the same buffer at a bacterial concentration of 2 × 10 ⁹ cells / ml. 300 μl of this bacterial solution was added to 10 μg of pHOBRBSR SslI.
Fragment, 10 μg pHONEO EcoRI fragment and carrier DNA
20 μg of Calf thymus DNA (Sigma) was added. To this, 1200 μl of 35% PEG4000 (sterilized with a filter) was added and stirred well. This stirred liquid 75
0 μl was transferred to a cuvette dedicated to Gene Pulser (Bio-Rad), and an electric pulse treatment was performed once using the Gene Pulser under the condition of 1 μF / 1000V. The bacterial solution was transferred from the cuvette to a 15 ml tube and left at 30 ° C. for 1 hour. Fungus
The cells were collected by centrifugation at 3000 rpm for 5 minutes, suspended in 1 ml of YPD medium, and shaken at 30 ° C. for 4 hours. After collecting the cells, suspend the cells in 600 μl of sterilized water, and add 150 μl each of 50 μg / ml G-418 and 100 μl.
It was applied to YPD agar medium containing g / ml of blasticidin S. This agar medium was kept warm at 30 ° C., and the colonies that appeared were obtained as transformants.

B) Separation of transformants retaining zygosity Separation of meiotic variants retaining zygosity by meiosis Transformants BFY268-6 showing resistance to both G-418 and blasticidin S were The platinum loop was inoculated into 50 ml of YPD medium and cultured at 20 ° C overnight. Wash the cells thoroughly with sterile water, suspend in sporulation medium (potassium acetate 0.5%), and
Shaking culture was performed at ° C. After confirming sporulation under a microscope, all yeast cells are collected by centrifugation at 3000 rpm for 5 minutes.

The suspension was suspended in 5 ml of sterilized water, 0.5 ml of a 1 mg / ml zymolyase aqueous solution was added, and the mixture was shaken at 30 ° C. overnight. Add 5 ml of a 1.5% (W / V) solution of the surfactant NP-40 and add it.
After ice-cooling for 15 minutes, an ultrasonic generator (Tomy Seiko Co., Ltd., Powersonic UR-20P type) was used to output for 30 seconds, and spores were dispersed at 50-75%. The spore dispersion was centrifuged to collect the spores, which was then suspended in 5 ml of sterile water. About one plate medium
Dilute appropriately so that 300-400 colonies appear, and add 50 μ
YP with g / ml G-418 and 100 μg / ml blasticidin C
It was spread on a D plate medium and stored at 30 ° C. for several days. Thus, the colonies that appeared were picked up, and the ability to mate with the conjugative-type assay yeast was examined.

That is, a small amount of yeast was collected from the obtained colonies, and 1 platinum loop of a-type tester strain YP was collected on YPD medium.
H499 (MATa, ura3-52, lys2-801, ade2-101, trp1-Δ62, his
3-Δ200, leu2- Δ1, CUP ^r ) was mixed and cultured at 30 ° C. overnight. A part of the culture solution was observed under a microscope to observe the presence of zygotes. Furthermore, a part of this mixed bacterial cell was collected and assay medium (M
_{gSO 4 · 7H 2 O (0.2} %), (NH 4) 2 SO 4 (0.2%), KH 2 PO 4 (0.3%),
_{CaCl 2. 2H 2 O (0.0025%} ), Yeast Nitrogen Base W / O AA
(DIFCO) (0.3%), casamino acid (0.5%), glucose (4%),
It was applied on CuSO ₄ (1 mM), agar (2%)). It was cultured overnight at 30 ° C., and when growth was observed, it was designated as α type. Similarly, the same test was performed using the α-type tester strain YPH500 (MAT α, the gene markers other than the zygote type are the same as those of YPP499), and when proliferation was observed in such a test, it was designated as a-type.

The results are shown in Table 1. HO-SC and HO
-BR and inactivated brewer's yeast BFY268 include a type and α
A yeast strain having a mating type of type was isolated. In Table 1, when the HO gene was not inactivated, confirmation by a microscope was performed.

[0076]

[Table 1]

C) Breeding using conjugatable transformants Yeast having inactivation of the HO gene and having conjugation ability can be obtained by the usual a
Type or α-type conjugation ability. Therefore, the joining method used conventionally can be used as it is.

Separation of mating type capable strains from Sake Yeast Society No. 7 Yeast Sake Yeast Society No. 7 yeast was appropriately diluted so that 100-200 colonies appeared in one plate medium, and plated on YDP plate medium. Well separated colonies were picked up, grown on YDP plate medium and stored as master plates. A small amount of each strain was taken from each clone stored on the master plate and placed in a 96-well microtiter plate containing YPD liquid medium, one for each clone.
The wells were inoculated. Yeast strain TESTa strain (a strain in which YPH499 was transformed with EcoRI digestion product of pHONEO and imparted with G-418 resistance) preliminarily cultured in YPD liquid medium was added little by little to each well. After statically culturing this at 30 ° C. overnight, it was centrifuged (3000 rpm, 5 minutes), and the separated product was washed once with sterilized water.
Resuspend this in sterile water and add a small amount of the suspension in each well.
SD + G-418 Plate Medium (Yeast, Nitrogen, Base (Difco) 6.7g / l, Glucose 20g / l, Agar 20g / l, G-
418 50 mg / l). Incubate at 30 ℃ for 2-3 days,
In the wells in which the growth was observed, the added two types of yeast were conjugated, so it was judged that the mating type of the clone of the corresponding master plate was a type.

In this way, K7-a3 exhibiting a-type mating type was separated from the Sake Yeast Society No. 7 yeast and used for the Examples described later.

Breeding example by conjugation of sake yeast and brewer's yeast Yeast showing a-type conjugation ability isolated from brewer's yeast BFY268
268-a5 (MEL +, gal-ho-sc :: G-418 ^R , ho-br :: BS ^R ),
Yeast K7-α3 (m with α-type conjugation ability isolated from sake yeast
el-, GAL +, ho) for each one platinum loop and 1 ml of YPD
The liquid medium was inoculated. This was statically cultured at 30 ° C. overnight. The culture solution is appropriately diluted and plated on YPGal + G-418 plate medium (yeast extract 10 g / l, bactopeptone 20 g / l, galactose 20 g / l, G-418 50 mg / l, agar 20 g / l). This was cultured at 30 ° C. Five colonies that appeared were picked up and assayed for growth on a YPD + BS 100 mg / l plate medium, and all showed BS resistance. These colonies are YPGa
l + 5-Bromo-4chloro-3-indolyl-α-D-galactopyranoside
(Wako Pure Chemical Industries, Ltd.) Colonies were stained blue with a 50 mg / l plate medium, and were positive for melibiase.

That is, these colonies were 268-a5 and K7.
The characteristics of -α3 conjugating strain (MEL +, GAL +, G-418 ^R , BS ^R ) were shown, and it was shown to be a hybrid strain of brewer's yeast and sake yeast.

[Example 3] Cloning of HO-UV gene a) Saccharomyces uvarum
of molecular weight-limited library from um)

D from Saccharomyces ovarum
Extraction of NA chain Saccharomyces ovarum (Sacc) was added to 5 ml of YPD medium.
haromyces uvarum IFO0615) was cultured at 30 ° C. for 1 to 2 days until saturation was reached, and the cells were collected by centrifugation and washed with sterile water. The cells were suspended in 0.5 ml of sorbitol solution (0.9 M sorbitol, 0.1 M EDTA, Tris buffer pH 8.0), 50 μL of Zymolyase 100T, 0.3 mg / mL) and 1 μL of β-mercaptoethanol were added, and the temperature was 37 ° C. Keep it warm for 1 hour. Collect protoplasts by centrifugation and add 0.5 mL of Tr.
is ・ EDTA solution (50mM Tris pH8.0, 20mM EDTA)
Suspended in. Add 50 μL of 10% SDS solution and incubate at 65 ° C for 20 minutes. Add 200 μl of 5M potassium acetate and add 30 μl on ice.
Leave for more than a minute. Centrifuge at 12,000 rpm for 5 minutes, and collect the supernatant in another centrifuge tube. Ethanol is added to collect DNA as a precipitate, which is transferred to another centrifuge tube and dissolved in 300 μL of TE buffer. Add 50 μL of RNase A solution (1 mg / mL),
After incubating at 37 ° C for 1 hour, isopropyl alcohol is added and the mixture is stirred to collect the generated DNA precipitate. About 20 mL of the yeast culture solution was treated by this method to extract the DNA chain.

Cleavage of DNA Strand by Restriction Enzyme About 10 μg of the obtained chromosomal DNA was placed in an Eppendorf tube, and M buffer (Tris buffer pH 7.5, 10 mM,
MgCl ₂ , 10 mM, Dithiothreitol (DTT) 1 mM, NaCl 50 m
M), and the restriction enzyme HindIII (Boehringer)
100 units were added, and the mixture was kept at 37 ° C for 4 to 5 hours.

Determination of preparative molecular weight by Southern analysis and preparation of insert DNA 1 of chromosomal DNA completely digested with restriction enzyme HindIII
1% agarose gel (Tris acetate buffer, EDT
It was applied to A) and electrophoresed at 100 V for 2 hours. The agarose gel is immersed in an ethidium bromide solution, the DNA molecular weight marker and the DNA are stained, and the migration state is recorded by a photograph or the like. Southern analysis using this agarose gel (Molecula
r Cloning, Manniatis et al., Cold Spring Harbor). As the probe, the labeled HO gene fragment derived from Saccharomyces cerevisiae obtained by the method described in Example 1 was used. A band was observed in the vicinity of 4 to 5 Kb from the obtained autoradiograph. 20 μg of chromosomal DNA completely cleaved with HindIII as described in the previous section
Was electrophoresed on a 1% low melting point agarose gel, and a gel portion corresponding to a molecular weight of about 4 Kb to about 5 Kb was recovered using the molecular weight marker as an index. DNA fragments were obtained from a low melting agarose gel by heat melting and phenol treatment followed by ethanol precipitation. A part of these DNAs was again subjected to Southern analysis, and it was confirmed that it gave a positive signal.
The following experiments were carried out.

Ligation into vector pUC118 was cut with HindIII to give Bacterial Alkaline P
A library was prepared using hosphatase (BAP) treated vector commercially available from Takara Shuzo. That is, this vector DNA (50 ng) and the above-mentioned 4-5 Kb
Ligation Kit (Takara Shuzo) was ligated to the chromosomal DNA fragment (50 ng). Escherichia coli DH5 strain (Toyobo) was transformed with a part of this Ligation Mix. Each method followed the method attached to the said Kit.

Cloning by colony hybridization Ampicillin 50 μg / prepared in a large Petri dish (90 mm)
About 1000 colonies were formed per LB plate medium containing 10 ml of bactotryptone (5 g / l of yeast extract, 5 g / l of sodium chloride, 15 g / l of agar) containing ml. A nitrocellulose filter was placed on this plate medium, and the transformant that appeared was transferred to the nitrocellulose filter by the replica method. As the filter, any filter can be used as long as it can be used for various colony hybridizations on the market. This time, Hibond N +, which is commercially available from Amersham, was used. The colony hybridization was carried out at 60 ° C. according to the method attached to the filter. The washing of the filter also followed the procedure attached to the filter, but the washing process was stopped at the step of 1 × SSPE, 0.1% SDS, and the washing temperature was 65 ° C. A colony on the master plate at a position giving a positive signal on the autoradiograph was separated, and the plasmid contained in the clone was named pHO-UV1 and used for the subsequent experiments.

B) Determination of the nucleotide sequence of the cloned HO-UV gene About 4.5 k cloned into the HindIII site of pUC118
The insert fragment of b was recovered from the low melting point agarose by a conventional method, and was inserted in the HindIII site of pUC118 in the reverse direction, and
O-UV2 was prepared. The base sequence was determined using the above pHO-UV1 and pHO-UV2. Nucleotide sequence determination was performed using Applied Biosystems' 373A DNA sequencer, and the reaction was performed using the Di Primer Cycle Sequencing Kit and Di Deoxy Terminator.
A cycle sequencing kit (manufactured by Applied Biosystems) was used. The procedure followed the attached protocol.

As a result, the HO gene of the present invention, HO-U
It was revealed that the V gene has the base sequence shown in SEQ ID NO: 5. In addition, the base sequence portion presumed to be a structural gene encoding the homothalism gene endonuclease of the present invention deduced from the base sequence is 1758 from the 2057th position to the 3814th position in the base sequence shown in SEQ ID NO: 5. (SEQ ID NO: 6). The amino acid sequence of the homotalism gene endonuclease of the present invention deduced from such a nucleotide sequence is as shown in SEQ ID NO: 4. The pHO-UV1 used in the above steps was incorporated into Escherichia coli DH5α. Such a transformant is EKB1
FERM BP-4697 on June 15, 1994, at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, on June 15, 1994.
Has been deposited as.

Example 4 Construction of HO-UV Gene Disruption Vector A construction diagram of the gene disruption vector is shown in FIG. That is,
The pHO-UV1 was cleaved with BglII, the 5 ′ overhang was filled in with Klenow enzyme, the Sal1 linker was ligated, and recyclized,
It was named O-UV-12.

On the other hand, the marker gene is Nakazawa et al. (J. Ferm
ent. Bioeng., Vol. 76, No. 1, p60-63, 1993), which used the PDR4 gene conferring cerulenin resistance.
The base sequence of the PDR4 gene is Hussain et al. (Gene, Vol. 101,
p149-152, 1991) and is already known. This gene was obtained by amplifying a KpnI fragment of about 2 kb, which was reported to be able to express cerulenin resistance by Nakazawa et al., By the PCR method. The resulting PCR fragment was inserted into the KpnI site of pUC119 and named pYT63. A 40 bp synthetic oligonucleotide was ligated between the EcoRI and HindIII sites of pUC18 to prepare pKN1. About 2 Kb from pYT63 containing PDR4 at the KpnI site of pKN1
KpnI fragment was inserted and named pKN2.

The XhoI fragment of the obtained plasmid pKN2 was ligated to the SalI site of pHO-UV-12, and the HO-UV gene was inactivated with a marker gene.
Got 31

Example 5 Separation of Transformant Retaining Mating Ability from Brewer's Yeast 10 μg of plasmid pHO-UV-31 was cleaved with PstI and StuI, and DNA was recovered from the reaction solution by ethanol precipitation. The precipitate was dissolved in 10 μg of sterilized water. This DNA solution 5
μl was used for the following transformations. pHOBRG PDBSR 10
μg was digested with SalI, DNA was recovered from the reaction solution by ethanol precipitation, and the precipitate was dissolved in 10 μg of sterilized water. 5 μl of this DNA solution was used for the following transformation. Yeast Sa
Transformation of ccharomyces bayanus (IFO 1127) was performed by the electroporation method. The method is Becker's method (Methods in Enzymology, Vol.194, p182, Academic
Press Inc.), but 0.4 ml of YPD liquid medium was added to 1 ml of the yeast suspension recovered with the sorbitol solution after the electric pulse, and the mixture was cultured at 28 ° C. for 4 hours with shaking. An appropriate amount of cerulenin (manufactured by Sigma) was plated on a YPD plate medium containing a final concentration of 5 μg / ml and blasticidin C of a final concentration of 100 μg / ml, and incubated at 28 ° C. for several days until colonies appeared. The colonies that appeared were streaked and a large number of single clones were isolated. This clone was cultured on a YPD plate medium at 30 ° C., and the obtained cells were sporulated medium (0.
It was applied to 5% potassium acetate, 2% agar) and kept at 20 ° C. Confirm sporulation, put an appropriate amount of yeast cells at 3000 rpm,
Collect by centrifugation for 5 minutes. Suspend in 5 ml of sterile water, 1
Add 0.5 ml of mg / ml zymolyase aqueous solution and 10 μl of β-mercaptoethanol, and shake at 30 ° C. overnight. After adding 5 ml of 1.5% (W / V) solution of surfactant NP-40 and cooling with ice for 15 minutes, 30 seconds output with an ultrasonic generator (Tomy Seiko, Powersonic UR-20P type) 50- Disperses spores at 75%. Centrifuge the spore dispersion and collect the spores in the precipitate,
Suspended in 5 ml of sterile water. About 300 to about 4 per plate
YP containing 5μg / ml cerulenin and 100μg / ml blasticidin C diluted appropriately so that 00 colonies appear
Spread on D plate medium and incubate at 30 ℃ for several days. The colonies that appeared were picked up and examined for their mating ability with the mating-type assay yeast.

That is, a small amount of yeast was collected from the obtained colonies, and 1 platinum loop of a-type tester Y was collected on YPD medium.
PH499 (MATa, ura3-52, lys2-801, ade2-101, tr
p1-Δ62, his3-Δ200, leu2-Δ1, CUP ^r ), and the mixture was incubated at 30 ° C. overnight. The mixed portion was collected from the cells on the assay medium _{[MgSO 4 .7H 2 O, 0.2%} . (NH 4) 2 SO 4, 0.2%. KH 2
PO ₄ , 0.3%. CaCl ₂ .2H ₂ O, 0.0025%. Yeast Nitrogen Bas
e W / O AA (DIFCO), 0.3%. Camino acid, 0.5%. Glucose,
4%. CuSO ₄ , 1 mM. Agar, 2%]. It was cultured overnight at 30 ° C., and when growth was observed, it was designated as α type. Similarly, the same test was performed using α-type tester strain YPH500 (MATα, the gene markers other than the zygote type are the same as YPH499), and when proliferation was observed, it was set to a-type.

Furthermore, laboratory yeasts of known mating type and YPD
The cells were mixed and cultured on an E plate medium, cultured overnight at 30 ° C., and examined the presence or absence of zygotes by microscopic examination on the next day. Sacc in which the HO-UV and HO-BR genes were inactivated by these two confirmation methods
A yeast strain having a-type and α-type mating type was isolated from haromyces byanus.

Example 6 Southern Analysis of HO Gene Group In order to investigate the distribution of the HO gene in various brewer's yeasts,
Southern analysis was performed. The test yeasts are X-2180 (laboratory yeast), TFY069 (top-fermenting yeast), KBY003 (bottom-fermenting yeast), KBY011 (bottom-fermenting yeast), KBY268.
(Bottom fermenting yeast), Saccharomyces byanus (IFO 112
7) ， Saccharomyces uvarum (IFO 0615), Pilsen No.1
(IFO 2031) was used.

One platinum loop of the above yeast was cultivated in 5 mL of YPD medium and shake-cultured at 30 ° C. for 1 to 2 days to give a) of Example 1.
Chromosomal DNA was extracted by the same method as described in 1. 5
Completely cut μg of chromosomal DNA with EcoRI to obtain 0.8%
Was subjected to agarose gel electrophoresis. After electrophoresis, the DNA is denatured and neutralized, transferred from the gel to a membrane, and subjected to Southern analysis (Molecular Cloning, Manniatis et.al. Cold Spring.
Harbor). As the probe, a labeled HO gene fragment derived from Saccharomyces cerevisiae whose method was described in Example 1 a) was used.

The results are shown in FIG. Top fermenting yeast (TFY
069; lane 2) has a homologous gene (HO-SC gene) of the HO gene at the same molecular weight position (about 7 kbp) as the laboratory yeast (X-2180; lane 1), but the bottom fermentation yeast [K
BY003 (lane 3), KBY011 (lane 4), KBY
268 (lane 5)] is another type of homologous gene (HO-BR gene; about 4 kb with a different molecular weight) together with the HO-SC gene.
It became clear that they had p). All bottom-fermenting yeasts investigated this time had the HO-SC and HO-BR genes. Saccharomyces byanus (lane 6), Sa
ccharomyces uvarum (lane 7) and Pilsen No. 1 (lane 8) are HO homologous genes (HO-U) with different molecular weights.
V gene; about 2.5 kbp and 1.5 kbp) were retained.

[0099]

According to the present invention, the already known HO-S
The location of the HO gene other than the C gene and its nucleotide sequence were clarified, and the transition to yeast homothalism was achieved by combining the HO gene with the HO-SC gene and by using the HO gene other than the HO-SC gene alone. The means for sufficiently inhibiting the above are provided.

[0100]

[Sequence list]

SEQ ID NO: 1 Sequence length: 586 Sequence type: Amino acid Topology: Unknown Sequence type: Peptide Sequence characteristics Origin Biological name: Saccharomyces. Karlsbergensis (Sac
charomyces carlsbergensis) Strain name: KBY003 Sequence:

[0101]

[0102]

SEQ ID NO: 2 Sequence length: 3899 Sequence type: Nucleic acid Number of strands: Double strand Topology: Unknown Sequence type: Genomic DNA Sequence features Origin Biological name: Saccharomyces. Karlsbergensis (Sac
charomyces carlsbergensis) Strain name: KBY003 Sequence:

[0104]

[0105]

[0106]

SEQ ID NO: 3 Sequence length: 1758 Sequence type: Nucleic acid Number of strands: Double strand Topology: Unknown Sequence type: Genomic DNA Sequence features Origin Biological name: Saccharomyces. Karlsbergensis (Sac
charomyces carlsbergensis) Strain name: KBY003 Sequence:

[0108]

[0109]

SEQ ID NO: 4 Sequence length: 586 Sequence type: Amino acid Topology: Unknown Sequence type: Peptide Sequence characteristics Origin: Organism name: Saccharomyces
uvarum) Strain name: EKB103 Sequence: Met Leu Ser Glu Asn Thr Thr Ile Leu Met Ala Asn Gly Lys Ile Glu 1 5 10 15 Asp Ile Ala Asn Val Thr Ala Asn Ser Tyr Val Met Cys Glu Asp Gly 20 25 30 Ser Ala Ala Arg Val Ile Ser Val Thr Gln Gly Cys Gln Lys Ile Tyr 35 40 45 Asn Ile Gln Gln Lys Thr Lys His Arg Ala Phe Glu Gly Glu Pro Gly 50 55 60 Arg Leu Asp Pro Arg Arg Arg Thr Ile Tyr Gln Arg Leu Ala Leu Gln 65 70 75 80 Cys Thr Ala Gly His Lys Leu Ser Val Arg Val Pro Thr Lys Pro Leu 85 90 95 Leu Glu Lys Ser Gly Arg Ser Ala Thr Lys Tyr Lys Val Arg Trp Arg 100 105 110 Asn Leu Gln Gln Cys Gln Thr Leu Asp Gly Arg Ile Ile Thr Ile Pro 115 120 125 Lys Asn His His Lys Thr Phe Pro Met Thr Val Glu Gly Glu Phe Ala 130 135 140 Ala Lys Arg Phe Ile Glu Glu Met Glu Arg Leu Lys Gly Glu Tyr Phe 145 150 155 160 Asn Phe Asp Ile Glu Val Arg Asp Leu Asp Tyr Leu Asp Ala Gln Leu 165 170 175 Arg Ile Ser Ser Cys Ile Arg Phe Ser Pro Val Ile Thr Gly Asn Gly 180 185 190 Val Leu Ser Lys Phe Leu Thr Gly Arg Asn Asp Leu Val Thr Pro Ala 195 200 205 Val Lys Ser Met Ala Trp Met Leu Gly Leu Trp Leu Gly Asp Gly Thr 210 215 220 Thr Lys Glu Pro Glu Ile Ser Val Asp Ser Leu Asp Pro Lys Leu Met 225 230 235 240 Glu Ser Leu Arg Lys Gln Ala Lys Ile Trp Gly Leu Tyr Leu Thr Val 245 250 255 Cys Asp Asp His Val Pro Leu Arg Ala Lys His Val Arg Leu His Tyr 260 265 270 Gly Asp Gly Pro Asp Glu Asn Arg Lys Thr Arg Asn Leu Arg Lys Asn 275 280 285 Asn Pro Phe Trp Asn Ala Val Thr Lys Leu Lys Phe Lys Arg Glu Leu 290 295 300 Asp Gly Glu Lys Gln Ile Pro Glu Phe Met Tyr Ser Glu His Val Glu 305 310 315 320 Val Arg Glu Ala Phe Leu Ala Gly Leu Ile Asp Ser Asp Gly Tyr Val 325 330 335 Val Lys Lys Gly Glu Gly Pro Glu Ser Tyr Lys Ile Ala Ile Gln Thr 340 345 350 Val Tyr Ser Ser Ile Met Asp Gly Val Ile His Ile Ser Arg Ser Leu 355 360 365 Gly Met Ser Ala Thr Val Thr Thr Arg Ser Ala Arg Glu Glu Ile Ile 370 375 380 Glu Gly Arg Lys Val Gln Cys Gln Phe Thr Tyr Asp Cys Asn Val Ala 385 390 395 400 Gly Gly Thr Thr Leu Gln Asn Val Leu Ser Tyr Cys Arg Ser Gly His 405 410 415 Lys Thr Arg Glu Ile Pro Pro Ile Val Lys Arg Glu Pro Val Tyr Phe 420 425 430 Gly Phe Thr Asp Asp Phe Gln Gly Glu Ser Thr Val Tyr Gly Leu His 435 440 445 Ile Glu Gly His Lys Asn Phe Leu Leu Gly Asn Lys Ile Glu Val Lys 450 455 460 Ser Cys Gly Gly Tyr Cys Glu Gly Glu Gln Pro Lys Leu Ser Gln Arg 465 470 475 480 Lys Asn Leu Lys His Cys Ile Ala Cys Pro Arg Lys Gly Ile Lys Tyr 485 490 495 Phe Tyr Lys Asp Trp Ser Gly Lys Asn Arg Val Cys Ala Arg Cys Tyr 500 505 510 Gly Arg Tyr Lys Phe Ser Gly His His Cys Ile Asn Cys Lys Tyr Val 515 520 525 Pro Glu Ala Arg Glu Val Lys Lys Ala Lys Asp Lys Gly Glu Lys Leu 530 535 540 Gly Ile Thr Pro Glu Gly Leu Pro Phe Lys Gly Pro Glu Cys Leu Arg 545 550 555 560 Cys Gly Gly Ile Leu Gln Phe Asp Ala Val Arg Gly Pro Asn Lys Ser 565 570 575 Cys Ala Thr Asn Ile Gly Val Arg Ile Cys 586 580 585

SEQ ID NO: 5 Sequence length: 4442 Sequence type: Nucleic acid Number of strands: Double strand Topology: Unknown Sequence type: Genomic DNA Sequence features Origin: Organism name: Saccharomyces saccharomyces
(uvarum) Strain name: EKB103 Sequence: 10 20 30 40 50 60 AAGCTTAGAG TTGCCCGCAG GCTTAACCAG GGCAAGCGAT CGATGCGTAC CAAAAAAAGT 70 80 90 100 110 120 AGTAAGGTGA GAATGATCCT TACCGTTTAA CTTCACTA GAATTCA 130ACT 150AAAATGCAATATAAAAACATTAT 130AGTAAAATGCAATATAAAATGCAATATATAAAATGCAATATATAAAATGCAATAT 130AAAAATGCAATATATAAAATGCAATATATAAAATGCAATATATAAAATGCAATATATATAAAATGCAATATATAAAATTCA TTTACCGCGT TAAAACCTAC ATCAAAAAAG GGCGGATCAA TACGATGAAA AAAAAGGTGT 250 260 270 280 290 300 GCAAGAAAAA GTAGCATTCC AACGCCGATT GTAGCCTCTA GGATAATAAA GAAACTTGAA 310 320 330 340 350 360 ATCGTACGTA GTATTCGCGT CTTTAAGCGG TTTTGTTTGA TCTTAGTTCT CACAATCCCG 370 380 390 400 410 420 TATTTCTATG GAACTATCAT CCTCAAAGCA GCGACAGATC ATACAATTAC TCTGTGAAGT 430 440 450 460 470 480 GCTACTATCA CCATTCTGTC AGGCCCTCCT TGATTTCTTG GCTCTTGGAT ACACGTATCT 490 500 510 520 530 540 GAATCACTTG TTGGACCTTA GAATTCCACG TGCCGCTGAA CGTAGCAGTC ATCACTGTAC 550 560 570 580GGCC 660 CG CG 610 CG 650 CG CG 610 CG CG 650 CG 650 650 CG GTGA TGAGATAAAT CAATTTAATG 670 680 690 700 710 720 AAAACCAGCA CACTATAACG CTGGAGCAAT TGTCCAAGCA ACAAGAAAAA AAACCTTGAC 730 740 750 760 770 780 AATAATTAGC TGAAAGGGAT GAAGTAACTG ATTTGTCTTT TCTCTCTGCG GATGATCTGT 790 800 810 820 830 840 GGGAAAATGA TTTGGGCTAA ATCACGTAAA AAGTTTGATT CCGCGGCTAA TATCTCTGAG 850 860 870 880 890 900 GGGGTGTCAA CGAGCTCGTA GAGTCTTCAT TTTGAGGCAA CCAATGTATA ATGTCCAAGG 910 920 930 940 950 960 AGTTGCTCAA AGAAACAGCC GCAGAATGTT AAGAACGAAT CGGTTGGCAT CAGAGTATGC 970 980 990 1000 1010 1020 ACGATTTGTC CTGATCATGG CAATTAAGTA ACTCCTAGAT CGATTTGGCG TCTTGAAATC 1030 1040 1050 1060 1070 1080 ATATTAACGC CAAACGAAAT ATCCCATTAC TTTTCCTATT TGAGATCATC TTAGTTACGG 1090 1100 1110 1120 1130 1140 CTACAGAGAA GAAAAAGGGT CACATGCCAT AATAATACAG ATTACGTTCA CGAAAATTGG 1150 1160 1170 1180 1190 1200 CCCTATGGCA CATATTTTCG ATTCACGAAA ACCCGATGAC TGTATAGACG TGAAATCCAC 1210 1220 1230 1240 1250 1260 GAAAACAAGT CTGATTGCAC ACGTAGGCGA TCCACGAAAA CAGATGCGAC CGTATACCTG 1270 1280 1290 1300 1310 1320 CAGG TTGAT GCAATTGTAT ACATGAATGC CTCACGAAAC TTGATGTAGC 1330 1340 1350 1360 1370 1380 TGTTCACATG ATTTCCGCGA AGGGTGATTT AGTGGAATAC ACGACTACCA CGAAAACAGA 1390 1400 1410 1420 1430 1440 TGTAATCGCA TACACGAATG CCACAAATGG CGATGTAATA TCACACACGA GTGTCACGAA 1450 1460 1470 1480 1490 1500 ACTGATGTAA ATTCACACAC GAGTGTCACG AAACTGATGT AAATTCATAC ACGAGTTTCA 1510 1520 1530 1540 1550 1560 CGAAATTGAT GTAATCGTAT ACACAAGTGC CACGAAACTG ATGTGATTGT CTGCAGGAGT 1570 1580 1590 1600 1610 1620 ACCACGAAAA ACTGATGTAA TGGACTACAC AATGTGTTGA AAAATGCTTC CGTTATGACA 1630 1640 1650 1660 1670 1680 CACGTCAGTG ATTCACGAAA ACGAAAGCTA TTGTATACAC CAGATATTCA CGAAAATGAT 1690 1700 1710 1720 1730 1740 GCTATTATTT ACATCAACCT GCCGCGAAAA CTGATGTAAT TGTTTACATC AATATCTTCG 1750 1760 1770 1780 1790 1800 CAGAAGAGCA ATCCATTTAC TTCGTAGGAG CTGCCGGTAC TACTGGCAAA TACACTAGCT 1810 1820 1830 1840 1850 1860 TCTGGGCGAA CTACAAGGTG GAAAACCACG AAAAGTTAGA ACTACGTTCA GGCAAAGCAG 1870 1880 1890 1900 1910 1920 TGGAAGAATC CAAGCGTTCT CTTGCTTCCG TCGAAGTTCG TTAAAGTGTT C TAAATGTGC 1930 1940 1950 1960 1970 1980 TGTTAATCAT CGAATATTTA AAGCTCATAG GATCGTAGCC AATTCATACT CTTAGCCCTT 1990 2000 2010 2020 2030 2040 TCTTCTTTCC ATTATAATCA ATTACTCTCA AGCCACATCT ATATAGACAG CAATCACTTC 2050 2060 2070 2080 2090 2100 CATCTAGCCT TTAAACATGC TTTCCGAAAA TACAACTATA TTGATGGCCA ATGGTAAAAT 2110 2120 2130 2140 2150 2160 CGAAGACATC GCTAATGTCA CCGCTAATTC CTACGTTATG TGCGAAGATG GTTCTGCCGC 2170 2180 2190 2200 2210 2220 CCGCGTTATT AGTGTCACAC AAGGCTGCCA AAAAATCTAC AATATTCAGC AAAAAACTAA 2230 2240 2250 2260 2270 2280 GCATAGAGCT TTTGAAGGCG AACCTGGTAG ATTGGACCCT AGACGTAGAA CTATTTACCA 2290 2300 2310 2320 2330 2340 ACGCCTGGCC TTGCAATGTA CTGCAGGTCA TAAACTGTCT GTAAGGGTTC CTACTAAACC 2350 2360 2370 2380 2390 2400 ATTGTTAGAG AAAAGCGGTA GAAGTGCCAC GAAATATAAA GTGAGATGGA GAAACTTACA 2410 2420 2430 2440 2450 2460 ACAATGTCAG ACACTTGACG GAAGAATAAT AACAATTCCA AAAAATCACC ACAAAACGTT 2470 2480 2490 2500 2510 2520 TCCAATGACT GTCGAAGGTG AATTTGCTGC AAAACGTTTC ATAGAAGAAA TGGAGCGTTT 2530 2540 2550 2560 2570 2580 GAAAGGTGAA TACTTTAATT TTGACATCGA AGTCAGAGAT TTGGATTATC TCGACGCTCA 2590 2600 2610 2620 2630 2640 GTTGAGAATT TCTAGCTGTA TAAGATTCAG TCCGGTTATT ACTGGGAACG GTGTTTTATC 2650 2660 2670 2680 2690 2700 TAAATTTCTC ACCGGACGTA ATGACCTAGT GACTCCCGCT GTGAAAAGTA TGGCTTGGAT 2710 2720 2730 2740 2750 2760 GCTTGGTTTA TGGTTAGGTG ACGGTACAAC AAAAGAGCCC GAGATCTCTG TAGATAGTTT 2770 2780 2790 2800 2810 2820 GGACCCTAAA TTAATGGAAA GTCTACGAAA ACAAGCCAAA ATTTGGGGGC TCTATCTTAC 2830 2840 2850 2860 2870 2880 AGTGTGTGAT GACCATGTTC CGCTGCGTGC CAAACATGTG AGGCTACATT ATGGGGATGG 2890 2900 2910 2920 2930 2940 GCCAGATGAA AATAGAAAGA CCAGGAATTT AAGAAAGAAC AATCCATTTT GGAACGCTGT 2950 2960 2970 2980 2990 3000 CACAAAGTTG AAATTCAAAA GGGAACTTGA CGGTGAGAAG CAGATCCCAG AATTCATGTA 3010 3020 3030 3040 3050 3060 TAGCGAGCAC GTAGAGGTCC GTGAAGCATT TTTGGCAGGT TTAATCGATT CAGATGGATA 3070 3080 3090 3100 3110 3120 TGTTGTGAAG AAGGGCGAAG GCCCAGAATC TTACAAAATA GCAATTCAAA CTGTGTATTC 3130 3140 3150 3160 3170 3180 ATCAATTATG GACGGAGTTA TTCATATTTC CAGATCT CTT GGAATGTCCG CTACTGTGAC 3190 3200 3210 3220 3230 3240 CACCAGATCC GCCAGAGAAG AAATTATTGA GGGAAGGAAA GTTCAATGCC AATTCACATA 3250 3260 3270 3280 3290 3300 TGACTGTAAT GTTGCTGGAG GAACAACATT ACAAAACGTT TTATCATACT GTCGAAGCGG 3310 3320 3330 3340 3350 3360 CCATAAAACT AGAGAAATTC CCCCAATTGT GAAAAGAGAA CCAGTGTATT TCGGGTTCAC 3370 3380 3390 3400 3410 3420 GGACGATTTT CAAGGTGAGA GTACAGTATA CGGACTTCAT ATAGAGGGGC ATAAAAATTT 3430 3440 3450 3460 3470 3480 CTTATTAGGC AATAAAATAG AAGTTAAGTC CTGTGGGGGC TATTGCGAAG GAGAACAACC 3490 3500 3510 3520 3530 3540 GAAATTATCC CAGAGGAAAA ACTTGAAGCA CTGTATTGCG TGTCCTAGAA AGGGCATTAA 3550 3560 3570 3580 3590 3600 ATATTTTTAT AAAGATTGGA GCGGTAAAAA CCGAGTGTGT GCAAGATGTT ACGGAAGATA 3610 3620 3630 3640 3650 3660 CAAGTTTAGT GGCCATCACT GTATAAACTG CAAATATGTA CCAGAAGCAC GTGAGGTTAA 3670 3680 3690 3700 3710 3720 AAAAGCGAAA GATAAAGGTG AAAAATTGGG CATAACGCCC GAAGGCCTGC CGTTCAAAGG 3730 3740 3750 3760 3770 3780 ACCAGAATGT TTACGATGTG GGGGGATTTT GCAATTTGAC GCTGTTCGTG GACCCAATAA 3790 3800 3810 3820 3830 3840 GAGTTGTGCG ACAAATATTG GGGTACGGAT TTGTTAAAAT ATGTAAATTA GTTTAAAAAA 3850 3860 3870 3880 3890 3900 AGTAGGTTGT ATGTATTATA TGTAAAAGTA AGTCGTATGT ATTTTATGTA AAAAGTAAGT 3910 3920 3930 3940 3950 3960 TGTATGTGTT ATATGTAATT TTTAATATTT TTTTCTGTCA GAACAGTATT TGCAGGATTT 3970 3980 3990 4000 4010 4020 TCTTTTATTT GAAATGATTA AATTAGAAAA TACATCTACC CACCAATTAC AAAAATTCTA 4030 4040 4050 4060 4070 4080 CTAAAACTGG TAACACGAAA AGTACCAATA CGGTCACTCC ATTGATCGAT ATAATTGAAT 4090 4100 4110 4120 4130 4140 GCAAACAAAA AGTTTTAATT GTGATACTTG TGAATTTTAT TTTATTAAGG ATACAAAGTT 4150 4160 4170 4180 4190 4200 AAGAGAAAAC AAAATTTATA TACAATAAGT AATATTCATA TATATGTGAT GGATGCAATC 4210 4220 4230 4240 4250 4260 TTAACGAGAA GACATGGCCT TGGTGACAAC TCTCTTCAAA CCAACTTCAG CCTTTCTCAA 4270 4280 4290 4300 4310 4320 TTCATCAGTA GATGCGTCTT CGATTTGCAA AGCGGCCATA GCATCAGATA AAGCGGCTTC 4330 4340 4350 4360 4370 4380 GATCTTGGAC TTGGAACCTC TCTTCAATTT AGAAGATAAG ATTGGGTCAG TGACATTTTG 4390 4400 4410 4420 4430 4440 TTCGATGGAA GCAACGTAGG AT TCCAATCT TTGTCTGGCT TCATGCTTCT TGGCGAAAGC TT

SEQ ID NO: 6 Sequence length: 1758 Sequence type: Nucleic acid Number of strands: Double strand Topology: Unknown Sequence type: Genomic DNA Sequence features Origin: Organism name: Saccharomyces
uvarum) strain name: EKB103 sequence: ATGCTTTCCG AAAATACAAC TATATTGATG GCCAATGGTA AAATCGAAGA CATCGCTAAT 60 GTCACCGCTA ATTCCTACGT TATGTGCGAA GATGGTTCTG CCGCCCGCGT TATTAGTGTC 120 ACACAAGGCT GCCAAAAAAT CTACAATATT CAGCAAAAAA CTAAGCATAG AGCTTTTGAA 180 GGCGAACCTG GTAGATTGGA CCCTAGACGT AGAACTATTT ACCAACGCCT GGCCTTGCAA 240 TGTACTGCAG GTCATAAACT GTCTGTAAGG GTTCCTACTA AACCATTGTT AGAGAAAAGC 300 GGTAGAAGTG CCACGAAATA TAAAGTGAGA TGGAGAAACT TACAACAATG TCAGACACTT 360 GACGGAAGAA TAATAACAAT TCCAAAAAAT CACCACAAAA CGTTTCCAAT GACTGTCGAA 420 GGTGAATTTG CTGCAAAACG TTTCATAGAA GAAATGGAGC GTTTGAAAGG TGAATACTTT 480 AATTTTGACA TCGAAGTCAG AGATTTGGAT TATCTCGACG CTCAGTTGAG AATTTCTAGC 540 TGTATAAGAT TCAGTCCGGT TATTACTGGG AACGGTGTTT TATCTAAATT TCTCACCGGA 600 CGTAATGACC TAGTGACTCC CGCTGTGAAA AGTATGGCTT GGATGCTTGG TTTATGGTTA 660 GGTGACGGTA CAACAAAAGA GCCCGAGATC TCTGTAGATA GTTTGGACCC TAAATTAATG 720 GAAAGTCTAC GAAAACAAGC CAAAATTTGG GGGCTCTATC TTACAGTGTG TGATGACCAT 780 GTTCCGCTGC GTGCCAAACA TGTGAGGCTA CATTATGGGG A TGGGCCAGA TGAAAATAGA 840 AAGACCAGGA ATTTAAGAAA GAACAATCCA TTTTGGAACG CTGTCACAAA GTTGAAATTC 900 AAAAGGGAAC TTGACGGTGA GAAGCAGATC CCAGAATTCA TGTATAGCGA GCACGTAGAG 960 GTCCGTGAAG CATTTTTGGC AGGTTTAATC GATTCAGATG GATATGTTGT GAAGAAGGGC 1020 GAAGGCCCAG AATCTTACAA AATAGCAATT CAAACTGTGT ATTCATCAAT TATGGACGGA 1080 GTTATTCATA TTTCCAGATC TCTTGGAATG TCCGCTACTG TGACCACCAG ATCCGCCAGA 1140 GAAGAAATTA TTGAGGGAAG GAAAGTTCAA TGCCAATTCA CATATGACTG TAATGTTGCT 1200 GGAGGAACAA CATTACAAAA CGTTTTATCA TACTGTCGAA GCGGCCATAA AACTAGAGAA 1260 ATTCCCCCAA TTGTGAAAAG AGAACCAGTG TATTTCGGGT TCACGGACGA TTTTCAAGGT 1320 GAGAGTACAG TATACGGACT TCATATAGAG GGGCATAAAA ATTTCTTATT AGGCAATAAA 1380 ATAGAAGTTA AGTCCTGTGG GGGCTATTGC GAAGGAGAAC AACCGAAATT ATCCCAGAGG 1440 AAAAACTTGA AGCACTGTAT TGCGTGTCCT AGAAAGGGCA TTAAATATTT TTATAAAGAT 1500 TGGAGCGGTA AAAACCGAGT GTGTGCAAGA TGTTACGGAA GATACAAGTT TAGTGGCCAT 1560 CACTGTATAA ACTGCAAATA TGTACCAGAA GCACGTGAGG TTAAAAAAGC GAAAGATAAA 1620 GGTGAAAAAT TGGGCATAAC GCCCGAAGGC CTGCCGTTCA AAGGACCAGA ATGTTTACGA 1680 TGTGGGGGGA TTTTGCAATT TGACGCTGTT CGTGGACCCA ATAAGAGTTG TGCGACAAAT 1740 ATTGGGGTAC GGATTTGT 1758

[Brief description of drawings]

FIG. 1 Plasmid pUCPGK21 and plasmid pPGKneo2
Manufacturing process drawing of.

FIG. 2 is a process drawing of the plasmid pGPDBSR.

FIG. 3 is a process drawing of plasmid pHONEO.

FIG. 4 is a process drawing of plasmid pHOBRBSR.

FIG. 5 is a diagram showing a construction diagram of a gene disruption vector.

FIG. 6 is a diagram showing the results of Southern analysis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 15/09 ZNA C12R 1:86) (C12N 15/09 ZNA C12R 1:85) (C12N 1 / 19 C12R 1:86) (C12N 1/19 C12R 1:85) C12R 1:86) (C12N 15/00 ZNA A C12R 1:85)

Claims (16)

[Claims] 1. An amino acid sequence represented by SEQ ID NO: 1,
And a yeast endonuclease gene, which comprises a nucleotide sequence encoding an endonuclease capable of cleaving the mating type locus. 2. Having the amino acid sequence shown in SEQ ID NO: 4,
And a yeast endonuclease gene, which comprises a nucleotide sequence encoding an endonuclease capable of cleaving the mating type locus. 3. The endonuclease gene according to claim 1, wherein the nucleotide sequence encoding the endonuclease is represented by SEQ ID NO: 3. 4. The endonuclease gene according to claim 2, wherein the nucleotide sequence encoding the endonuclease is represented by SEQ ID NO: 6. 5. A yeast homothalism gene comprising the endonuclease gene according to claim 1 or 3. 6. A yeast homothalism gene containing the endonuclease gene according to claim 2 or 4. 7. The homothalism gene according to claim 5, wherein the homothalism gene is represented by SEQ ID NO: 2. 8. The homothalism gene according to claim 6, wherein the homothalism gene is represented by SEQ ID NO: 5. 9. A yeast homotalism gene fragment inactivated by suppressing the expression of at least a part of the endonuclease gene according to any one of claims 1 to 4. 10. Suppression of expression by restriction enzyme cleavage, insertion, deletion, substitution or addition, restriction enzyme digestion, insertion, deletion, substitution or addition to the promoter or terminator region of the endonuclease gene, The homothalism gene fragment according to claim 9, which is obtained by inserting or substituting a marker gene for the promoter or terminator region, or by introducing an antisense RNA of the endonuclease gene. A YIp-type plasmid for disrupting a homotalism gene, which comprises a part of the endonuclease gene according to any one of claims 1 to 4 and a marker gene. 12. The inactivated one of claims 1 to 4.
A YIp type plasmid for disrupting a homotalism gene, which comprises the endonuclease gene and the marker gene according to the above item. 13. A transformed yeast transformed with the homotalism gene fragment according to claim 9. 14. A transformed yeast transformed with the YIp type plasmid for disrupting the homotalism gene according to claim 11. 15. A transformed yeast transformed with the YIp type plasmid for disrupting the homotalism gene according to claim 12. 16. A method for breeding yeast, which comprises culturing the transformed yeast according to any one of claims 13 to 15 to breed heterozygous mating type yeast.