JPH0998782A - Mutation gene inducing defect in mitochondria homologous recombination - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene coding for a polypeptide containing a sequence varied at a single amino acid of a specific position among amino acid sequences of a wild-type MHR1 protein and useful for the diagnosis, treatment, etc., of diseases relating to mitochondria. SOLUTION: This new variant mhr1 gene codes for a polypeptide containing an amino acid sequence varied at least the 99th amino acid in the amino acid sequence of a wild-type MHR1 protein expressed by the formula. It acts for the homologous recombination of mitochondria and is usable for the diagnosis and treatment of diseases caused by the genetic variation of mitochondria and the prevention of aging, etc., by the detection and correction of the varied gene of mitochondria. The gene can be produced by extracting a genom from a wild-type yeast to construct a genom library, selecting a strain containing wild-type MHR1 gene from the library, determining the base sequence, comparing with a base sequence determined after the PCR amplification of a genom extracted from an mhr1 mutant strain and determining the varied site.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to mutants on nuclear chromosomes which cause a defect in homologous recombination of mitochondria.
For the mhr1 gene.

[0002]

2. Description of the Related Art Mitochondria are organelles present in eukaryotic cells, and play a role in synthesizing ATP, which is energy by oxidative phosphorylation of substances, through an electron transport system. Mitochondria produce high levels of reactive oxygen species as a byproduct of oxygen respiration, which constantly damage mitochondrial DNA. In order to maintain mitochondrial DNA and maintain organ function, mitochondria must have a mechanism for repairing such DNA damage and suppressing DNA abnormalities so as not to cause mitochondrial DNA abnormalities. One of the functions considered to be important as the mechanism is homologous recombination, which is considered to be essential for nuclear DNA of eukaryotic cells and DNA repair of bacteria and viruses.

[0003] Homologous recombination means that, when a base sequence in a pair of DNA molecules has almost the same portion over several hundred base pairs or more, the DNA is located at a position exactly corresponding to each other in the portion.
A phenomenon commonly found in the biological world where DNA molecules are reconnected through strand breaks and reassociation. Then, as a result of the homologous recombination, the other DNA compensates for the damage in one DNA, thereby serving to maintain the normal function of the DNA. When the function of a homologous recombination gene decreases, not only DNA abnormalities increase due to the inability to repair DNA damage, but also when the homologous recombination gene functions abnormally, non-allelic Abnormal unequal crossovers occur between a pair of repeats at the site, leading to DNA abnormalities such as deletions. Mitochondria D
It has been pointed out that this type of deletion in NA may be associated with mitochondrial diseases (eg, mitochondrial encephalomyopathy caused by deficiency of mitochondrial energy-producing enzymes), aging, etc. (Holt, IJ) et al., Na
ture (London), 331, 717-719 (1988)).

[0004] Therefore, by isolating the mutant gene involved in the above homologous recombination abnormality and knowing its role, the detection and modification of the mutant gene can be used for diagnosis and treatment of diseases related to mitochondria, prevention of aging, etc. It is possible to do. By the way, most mitochondrial proteins are encoded on nuclear chromosomes, and it is predicted that homologous recombination of mitochondria also depends mostly on nuclear genetic information.

[0005] Therefore, as a step for understanding the role and mechanism of homologous recombination of mitochondria, isolation of a gene having a mutation causing a defect of homologous recombination of mitochondria, ie, a mutant gene in the nucleus, is required. Is mentioned.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a nuclear chromosome gene which causes homologous recombination of mitochondria and a mutant gene thereof.

[0007]

Means for Solving the Problems As a result of intensive studies based on the above-mentioned problems, the present inventors have found that by fusing nucleated cells and anucleated cells, homologous recombination of mitochondria in the cell nucleus is achieved. A working mutant gene was detected and successfully cloned, thereby completing the present invention.

That is, the present invention provides a mutant mhr1 encoding a polypeptide comprising an amino acid sequence in which at least one amino acid at position 99 of the amino acid sequence of the wild type MHR1 protein represented by SEQ ID NO: 1 is mutated. Is a gene. The mutated amino acid sequence includes that represented by SEQ ID NO: 2. Here, “MHR1 protein” refers to a protein obtained by expression of a wild-type MHR1 gene (a wild-type gene that works for mitochondrial homologous recombination in the cell nucleus), and “mutant mhr1 gene” refers to the MHR1 gene. Mutant, ie, a mutant gene that causes a loss of homologous mitochondrial recombination in the cell nucleus.

The "mutated amino acid sequence" includes an amino acid located at position 99 in the amino acid sequence represented by SEQ ID NO: 1 which is encoded by a gene having a function of causing loss of mitochondrial homologous recombination. It means that at least one amino acid is a sequence substituted, inserted or deleted by another amino acid. Therefore,
Substitution, insertion, deletion, etc., at the 99th amino acid, and as long as it has the function of causing the loss of mitochondrial homologous recombination, the amino acid other than the 99th amino acid,
It means that an amino acid different from the amino acid sequence may be substituted, the amino acid may be deleted, or an amino acid may be inserted.

[0010] Further, the present invention is a recombinant plasmid containing the gene. Hereinafter, the present invention will be described in detail. In cloning the gene of the present invention, first, the presence or absence of a mutant gene showing homologous recombination deficiency of mitochondrial DNA is detected. Next, a yeast genomic library containing a wild-type gene is introduced into cells that are found to have a mutant gene as a result of the detection, and the desired wild-type gene is cloned. The cloning is performed by picking up a gene that complements the mutation of a gene involved in a specific property mutated in the mutant cell, for example, a DNA damage repair defect, a recombination defect or a temperature-sensitive mutation, that is, a wild-type gene. Next, in order to clone the mutant gene of the present invention, primers are designed according to the nucleotide sequence of the wild type gene. Then, PCR is performed using the entire genomic DNA of the mutant cell as a template. Lastly, the gene of the present invention can be cloned by determining the nucleotide sequence of the obtained mutant gene and comparing it with the wild type nucleotide sequence to determine the mutation site. 1. Detection of Mutant Gene The detection of the presence or absence of the mutant gene is performed by detecting a nucleated cell containing a mitochondria having a specific marker gene (for example, an antibiotic resistance gene or the like) and a nucleated cell containing a mitochondria having a marker gene different from the marker gene. Cell fusion with cells, by selecting from the resulting fused cells with each antibiotic resistance corresponding to the marker gene, to measure the mitochondrial homologous recombination frequency,
It is performed by detecting the presence or absence of a mutation in a gene involved in mitochondrial homologous recombination from the decrease. Here, the presence or absence of the mutated gene can be detected regardless of whether it is dominant or recessive.

The method is performed as follows, and FIG. 1 shows an outline thereof. In FIG. 1, first, two types of cells to be subjected to cell fusion are prepared. One cell is the recipient cell 1
(Recipient cell) and the other cell as donor cell 2 (donor cell). The recipient cell 1 is a cell to be detected, and is a nucleated cell containing a nucleus 5 and a mitochondria 3 in the cell. Then, in the nucleus 5, either the mitochondrial homologous recombination normal gene or the recessive or dominant mutant gene is present. Examples of the type of the recipient cell 1 include a yeast cell or a cultured cell of an animal or a plant.

On the other hand, the donor cell 2 is a cell that plays a role in providing the mitochondria 4 in the cell to the recipient cell 1, and the nucleus 6 has been removed in advance by treatment with a drug (for example, nocodazole or the like). Nuclear cells containing 4 but not nucleus 6. Therefore, whether mitochondria in donor cell 2 undergo homologous recombination is determined by nuclear 6
Has nothing to do with the genetic information of The donor cell 2 includes cells of the same species as the recipient cell.

The preparation of the cells, the passage of the cells (culture), and the like can be performed using a conventionally generally known technique. Different marker genes are present in the DNAs of mitochondria 3 in the recipient cell 1 and mitochondria 4 in the donor cell 2, respectively. A resistance gene in mitochondria 3 is defined as a marker gene (I),
The resistance gene in mitochondria 4 is replaced with a marker gene (I
I). The marker gene is a gene required when selecting a fusion cell described below. For example, if the marker gene (I) is a chloramphenicol resistance gene,
Such a gene is necessary when the antibiotic used for selection is chloramphenicol, and the marker gene (II)
Is an oligomycin resistance gene, such a gene is necessary when the antibiotic used for selection is oligomycin.

However, the marker gene (I) or (II)
Are not the same gene, and their types are not particularly limited. For example, an oligomycin resistance gene,
A chloramphenicol resistance gene or an antimycin resistance gene can be used in appropriate combination. In the present invention, as an example, a gene resistant to chloramphenicol (hereinafter referred to as “Chl ^R ”) is used as a marker gene (I) for mitochondria 3 of recipient cell 1;
The mitochondria 4 of the donor cell 2 will be described with reference to a mitochondrial gene having a gene resistant to oligomycin (hereinafter referred to as “Oli ₁ ^R ”) as a marker gene (II).

Next, the recipient cell 1 and the donor cell 2 are subjected to cell fusion by a cell fusion method. The cell fusion method may be performed by a generally known cell fusion method using polyethylene glycol or the like after removing the cell walls of both cells with an appropriate drug (for example, litase or the like). In addition, a general method may be used for cell wall regeneration. When cell fusion is performed normally, mitochondria 3 and mitochondria 4 are mixed and contained in new recipient cells (fused cells 7). After cell fusion, homologous recombination of mitochondrial DNA depends (is governed) only on genetic information in the nucleus 5 derived from the recipient cell 1.
However, at this stage, it is unclear whether the homologous recombination gene in the nucleus 5 is normal or a recessive or dominant mutation.

Therefore, first, canavanine (2-amino-4-
By culturing in a medium containing (guanidinooxy) butyric acid), only cells having nucleus 5 are selected. For this purpose, the chromosome of the nucleus 5 is provided with a canavanine-resistant recessive mutant gene, for example, "can1" in advance. As a result, cells having a nucleus 6 which cannot be avoided in the donor cells, and fusion with cells having the nucleus 6 can be obtained 2
Diploid cells are completely eliminated. Also, during this culture, the individual cells will have either mitochondria 3, mitochondria 4 or both recombinant mitochondria 8 only.

Next, from the above-mentioned fused cells, a fused cell having mitochondria inherited from Oli ₁ ^R (a marker gene derived from recipient cell 1) by culturing in an oligomycin-containing medium is selected. Next, cells having recombinant mitochondria inherited from Chl ^R (marker gene derived from donor cell 2) are selected by culturing in a medium supplemented with chloramphenicol.

If the homologous recombination gene in the nucleus 5 is a mutant gene, the mutant gene cannot work for mitochondrial homologous recombination, and as a result of cell fusion and subsequent culture, 7, mitochondria 3 or mitochondria 4 exist independently. Therefore, cells having mitochondria 3 and cells having mitochondria 4 are removed by oligomycin and chloramphenicol, respectively, and finally cells containing recombinant mitochondria cannot be obtained.

On the other hand, when the homologous recombination gene in the nucleus 5 is a normal gene, the gene is the DN of mitochondria 3
A acts on homologous recombination between A and the DNA of mitochondria 4 and mitochondria 8 in which mitochondria from both recipient cell 1 and donor cell 2 are recombined (both Chl ^R and Oli ₁ ^R on the same mitochondrial DNA) Is obtained. Therefore, only cells with mitochondria 8 are obtained by oligomycin and chloramphenicol selection without killing.

When a cell containing recombinant mitochondria is finally obtained, the nucleus 5 in the recipient cell 1 is determined to have a normal gene for mitochondrial homologous recombination, and the recombinant mitochondria are determined. In the case where a cell containing the gene was not obtained, it can be determined that the nucleus 5 in the recipient cell 1 has a recessive or dominant mutant gene.

Whether the mutant gene is a dominant mutant gene or a recessive mutant gene is determined by the frequency of appearance of the recombinant obtained by the present invention and the conventional method (for cells that do not remove the nucleus of the donor cell). A method of cell fusion with a recipient cell).
That is, as a result of crossing haploid cells having a mutant gene and haploid cells having a normal gene by a conventional method,
When diploid cells having recombinant mitochondria appear at the same frequency as when haploid cells having normal genes are crossed with each other, the mutant gene is determined to be a recessive mutant gene. On the other hand, if no diploid cell having the recombinant mitochondria appears, the mutant gene is determined to be a dominant mutant gene. 2. Cloning of Gene The causative gene of the dominant or recessive mutation detected by the above detection method is cloned by a molecular biological technique.

In the present invention, in order to obtain the causative gene of the nuclear chromosome recessive mutant mhr1 which impairs homologous recombination of mitochondrial DNA, first, wild-type yeast DNA, MHR1
Clone. Next, using the nucleotide sequence of the peripheral region of the open reading frame of the obtained wild-type MHR1 gene as a primer, the total DNA
By performing PCR (Polymerase Chain Reaction) using as a template, a mutant gene can be cloned. (1) Preparation of wild-type yeast genomic DNA library As a method for extracting chromosomal DNA from wild-type cells detected by the above detection method, any known method can be used.

Next, the obtained chromosomal DNA was ligated with the restriction enzyme Sau3A.
Complete digestion with I yields the Sau3AI fragment of chromosomal DNA. Next, the Sau3AI fragment is subjected to ordinary agarose gel electrophoresis to cut out a gel containing a fragment of a desired chain length. The DNA fragment in the excised gel is purified by phenol / chloroform or the like, and further concentrated by ethanol precipitation or the like to obtain a purified fragment.

[0024] The purified Sau3AI fragment is converted into an appropriate vector.
A genomic DNA library can be prepared by inserting a recombinant DNA into a BamHI cleavage site of a DNA to prepare a recombinant DNA, and transforming or transducing a host cell using the recombinant DNA. Examples of the host cell used here include DH5α of Escherichia coli and JM109. (2) Extraction of plasmid DNA The introduced genomic library is introduced into a mutant strain.
The yeast transformation method is as follows (H. Ito et al.,
J. Bacteriol. 153 (1983), 163.).

At 30 ° C., a YPD liquid medium (1% yeast extract, 2%
(% Polypeptone and 2% glucose) are treated with 1 mM lithium chloride in the presence of 10 mM Tris-HCl buffer (pH 7.5). Thereafter, the genomic library is treated in the presence of about 75 mg / ml of salmon sperm and 40% polyethylene glycol 4000, and then transformed into a mutant cell.

The mutant strain thus introduced is spread on an SD agar medium (a medium containing 2% glucose, 0.67% amino acid-free yeast extract and 2% agar) containing histidine and tryptophan, and the growing colonies are separated. obtain. Next, the obtained colony is replicated on an agar medium containing glycerol to obtain a colony growing at 37 ° C. The obtained colony is cultured in an SD liquid medium (2% glucose, 0.67% amino acid-free yeast extract) containing histidine and tryptophan to obtain cultured cells. The yeast cells are disrupted with glass beads, the resulting cell-free extract is introduced into E. coli (eg, DH5α), and LB agar medium containing ampicillin (1% bactotryptone, 0.5% yeast extract, 0.5% Na
Cl, 2% agar, pH 7.2). Plasmid DNA can be extracted by further culturing the grown colonies in an LB liquid medium containing ampicillin. (3) DNA subcloning and nucleotide sequence determination The obtained plasmid is digested with restriction enzymes such as ClaI, BamHI, and MluI to cut out a DNA fragment. Among them, it shows complementation to recombination deficiency of mhr1 mutant, temperature-sensitive mutation or DNA damage repair deficiency caused by ultraviolet irradiation
Subclone the DNA fragment. For subcloning, a DNA fragment that has been cut short is inserted into a YCp50 plasmid, and the yeast is transformed into the yeast (H. Ito et al., J. Bacte.
riol. 153 (1983), 163.) to introduce these DNA fragments into mutant cells. The shortest DNA fragment obtained is further inserted into plasmid pUC118. Then, its base sequence is determined by a known method (eg, dideoxy method, maxam-Gilbert method, etc.).

The location of this gene in the yeast chromosome is determined by performing Southern hybridization on the 16 yeast chromosomes separated by pulse field electrophoresis using the fragment as a probe. You can find out. (4) Cloning of mhr1 gene and determination of mutation site The mhr1 gene of the mutant strain is cloned, and the mutation site of the gene is searched.

First, whole genomic DNA is prepared from the mhr1 mutant. That is, grown in a YPD liquid medium at 30 ° C. for 48 hours.
hr1 mutant cells were transformed with zymolyase 100T (Zymolyase-100T; S
After removing the cell wall with eikagaku Corporation, Tokyo, Japan, 1% SDS (sodium lauryl sulfate; sodium)
The cells are disrupted in the presence of Lauryl Sulfate). Then, a known DNA separation and purification procedure (C. Gut
hrie and GRFink; Molecular Biology Academic Pres
s Inc. San Diego, California, 1991).
Prepare the whole genomic DNA of the mutant strain.

PCR is performed on the whole genomic DNA obtained from the mhr1 mutant. PCR was performed using the cloned MHR1
The base sequence in the region around the open reading frame of the gene is used as a primer (the primer can be obtained by chemical synthesis). And 10 mM Tris-HCl,
1.5 mM MgCl ₂ , 50 mM KCl, 0.125 mM each dNTP, 1 pmol primer, 1.25 U taq DNA polymerase (Boehringe
r Mannheim).

Next, the obtained mhr1 gene was transformed into Hinc of pUC118.
Insert at position II and determine its nucleotide sequence. The method for determining the nucleotide sequence is the same as the above method.

[0031]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. Example 1 Detection of Mutant Gene (1) Preparation of Donor Cells Donor cells showing a mitochondrial genotype of ω ⁻ -Oli ₁ ^R (“ω ⁻ ” means that there is no ω intron in the mitochondrial gene, To prepare "ω ⁺ " means that there is an ω intron in the mitochondrial gene), OP11c-55R5 cells (derived from yeast Saccharomyces cerevisiae) were prepared by adding 15 μg / ml nocodazole containing 1% DMSO. (Inhibits nuclear translocation when forming new daughter cells (Jacobs, CW et al., J. Cell
Biol., 107, 1409-1426 (1988); Sigma)).
50 ml Falcon at 1.5 x 10 ⁶ cells / ml in 10 ml YPGly medium
Resuspended in tube. After incubating at 18 ° C. for 20 hours, the four tubes (about 2.2 × 10 ⁶ cells / ml each) were centrifuged (1,400 × g, 5 minutes at 4 ° C.), and 20 ml of K
The cells were washed once with a PS buffer (1.2 M sorbitol, 50 mM potassium phosphate buffer, pH 7.5), and the cells were collected in one 50 ml tube (Falcon). In order to separate the non-nucleated cells from the mother cells, the cells were resuspended in 10 ml of KPS buffer and sonicated at 0 ° C. for 3 minutes using an ultrasonic disruptor (UR-200P, manufactured by Tommy Seiko Co., Ltd.). (Intensity 4/11).

The present inventors have confirmed that complete non-nucleated cells were formed in the suspension by a fluorescence microscope and a phase contrast microscope after DAPI-staining. The treated suspension was centrifuged (1,400 × g, 4 ° C., 5 minutes), and the precipitate was resuspended in 200 μl of KPS buffer. This suspension was placed in a glass tube (12.5 (D) x 105 (L) mm), 1 ml each of 10% and 15 ml.
%, 20%, 25% and 30% Ficoll 400 (Pharmacia Biopro
cess Technology AB). Fifteen
After gradient centrifugation at 0 × g for 30 minutes at 30 ° C., 15% Ficoll
The fraction contained about 70% of anucleate cells on microscopic observation after DAPI staining. Therefore, collecting the 15% Ficoll fraction,
Dilute 2 fold with KPS buffer, and add 1400 xg for 5 min.
The cells were collected by centrifugation at ℃. 3 ml of K
After washing once with PS buffer, the mixture of mother cells and non-nucleated cells was centrifuged at 1,400 × g for 5 minutes at 30 ° C., and 1 ml of KP
Resuspended in S buffer.

The above OP11c-55R5 cells were obtained from Saccharomyce
It is called s cerevisiae OP11-55R5 and is deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-14988. (2) Preparation of Recipient Cells IL166-187 cells, a mutant of this cell line, and UV11 cells were prepared as recipient cells. IL166-187 cells are derived from the yeast Saccharomyces cerevi
siae) is 1 share of, have a normal gene "MHR1" in the nucleus, but wild-type mitochondrial gene type is "ω ⁺ Chl ^R". FL67, a variant of the IL166-187 cell line
The cell is a cell obtained by introducing a mutation into the IL166-187 cell by treatment with ethyl methanesulfonic acid, and the mutant gene “mhr
1 '' in the nucleus and mitochondrial genotype is `` ω ⁺ Chl
^R 's. In addition, UV11 cells also contain the IL166-187.
Cells are cells prepared by the same process as that used to prepare FL67 cells, and have a gene in the nucleus that is unknown whether it is a normal gene or a recessive or dominant mutant gene, and has a mitochondrial genotype of ω ⁺ Chl ^R ".

[0034] The above IL166-187 cells were Saccharomyce
s cerevisiae IL166-187, and FL67 cells were Saccha
romyces cerevisiae FL67, FERM P-14989, FERM P-149
Deposited as 87. (3) Cell fusion A suspension of anucleate cells and mother cells (donor cells prepared in the above (1)) and the recipient cells prepared in the above (2) were each 3: 1 (3 × 10 ⁶ cells : 1 × 10 ⁶ cells) at 100 μl
Mix in KPS buffer. 50 units of litase (Boe
hirnger Mannheim GmbH, 10,000 units / ml) was added to the mixture, and the mixture was incubated at 30 ° C for 30 minutes to form spheroplasts (cells from which cell walls had been removed). Spheroplast is 1,00
Collect by centrifugation at 0 × g for 5 minutes at 30 ° C., wash twice with 1 ml of KPS buffer, 1,000 × g
For 5 minutes at 30 ° C. To perform cell fusion on spheroplasts, pellet 250 μl of 35
% Polyethylene glycol 4,000 (dissolved in KPS buffer), incubated at 30 ° C. for 15 minutes, and 1,000 × g
And centrifuged at 30 ° C. for 5 minutes.
Wash the spheroplasts once with 1 ml of KPS buffer,
The treated spheroplasts were suspended in 2 ml of SD medium supplemented with the required amino acids and canavanine (1.5 μg / ml) (glass tube 12.5 (D) × 105 (L) mm), and were shaken by rotary shaking. C. (incubated at 93 rpm for 3 days). The culture was then diluted 100-fold and incubated under the same conditions for a further 3 days. Then, a fused cell having a nucleus derived from the recipient cell was obtained. (4) Treatment with antibiotics (selection of cells) Next, the fused cells were spread on a YPGly plate containing oligomycin (3 μg / ml), and cells that received the Oli ₁ ^R -mitochondrial marker from the donor cells were selected. . Colonies showing resistance to oligomycin (Ol
i ₁ ^R) were cultured with chloramphenicol (4 mg / ml) and YPG plate containing oligomycin. The ratio (%) of chloramphenicol-resistant (Chl ^R ) -colonies in Oli ₁ ^R colonies was calculated as the frequency of gene conversion at the ω-position.
Gene conversion is one type of genetic recombination. (5) Results The results are shown in Table 1. In Table 1, capital letters (for example, “MHR1”) in parentheses below each cell name indicate that the gene in the nucleus is a normal gene.
Those described in lower case (for example, “mhr1”) mean that the gene in the nucleus is a mutant gene. Also"[ ]"
What is described in the above means the mitochondrial genotype. ω ^+, ω ^-, the meaning of Chl ^R and Oli ₁ ^R are as defined above (hereinafter the same).

[0035]

[Table 1] From Table 1, it can be seen that IL166-187 cells, which are recipient cells, do not have a mutated gene because the gene in the nucleus caused homologous recombination of mitochondria (98.2% of recombinants were obtained). Turned out to be a cell. In contrast, FL67
The cells were found to have mutated genes because the gene in their nucleus did not cause mitochondrial homologous recombination (only 4% of the recombinants were obtained). . Furthermore, it was found that UV11 cells are cells that do not have a mutant gene involved in homologous recombination.

[Comparative Example 1] The mutant gene of FL67 is recessive by the conventional method without removing the nucleus of the donor cells, that is, the method of joining haploid cells having different mating types (α and a). A test was performed to determine whether there was or was dominant. Cells used for both recipient and donor cells were IL16
6-187 and its derivatives, FL67 derivatives and UV11 derivatives. All of these cells were generated by the inventor of IL166-18.
7 Prepared by treating cells with ethyl methanesulfonate.

Table 2 shows the results.

[0038]

[Table 2] From Table 2, it can be seen that a normal gene (for example,
Those that cause mitochondrial recombination, regardless of whether the gene in the nucleus of the recipient cell has a mutation. This result is in contrast to the result of No. 2 in Table 1 above.

Therefore, the mutant gene (mh
It can be seen that r1) is a recessive mutant gene that is hidden by the normal gene on the donor cell side and eventually causes mitochondrial recombination. As described above, the recessive mutant gene can be detected by the detection method of the present invention. [Example 2] Cloning of MHR1 gene In order to obtain a gene that controls the phenotype (DNA damage repair defect, recombination defect, and temperature-sensitive mutation caused by ultraviolet irradiation) of mutant FL67 (mhr1), the temperature of mhr1 mutant was determined. Cloning of the MHR1 gene was performed aiming at the gene that complements the sensitivity.

(1) Construction of genomic library Wild-type yeast IL166-187 cells described in Example 1 (FERM P-14
989), a genomic library of the MHR1 gene was constructed by a known method. For the construction of a genomic library, a single copy vector YCp50 was used. Next, mhr1 mutant FL67 (FERM P-14987; MET α, ura3, h
is1, trp1), the yeast genomic library constructed with YCp50 was introduced as follows.

At 30 ° C., a YPD liquid medium (1% yeast extract, 2%
% Polypeptone and 2% glucose) were treated with 1 mM lithium chloride in the presence of 10 mM Tris-HCl buffer (pH 7.5). Thereafter, the genomic library was treated in the presence of about 75 mg / ml of salmon sperm and 45% polyethylene glycol 4000, and then transformed into mutant cells.

The thus introduced mutant strain is spread on an SD agar medium (a medium containing 2% glucose, 0.67% amino acid-free yeast extract and 2% agar) containing histidine and tryptophan, and the growing colonies are separated. Obtained. next,
The obtained colony was replicated on an agar medium containing glycerol to obtain a colony growing at 37 ° C. The obtained colony is cultured in an SD liquid medium (2% glucose, 0.67% amino acid-free yeast extract) containing histidine and tryptophan to obtain cultured cells. The yeast cells are destroyed with glass beads, the obtained cell-free extract is introduced into E. coli DH5α, and LB agar medium containing ampicillin (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 2% Agar,
pH 7.2). The grown colonies were further cultured in an LB liquid medium containing ampicillin to extract plasmid DNA.

Next, histidine (8 mg / 20 ml plate)
Agar was cultured on SD agar medium (2% glucose, 0.67% amino acid-free yeast extract, 2% agar) containing tryptophan (8 mg / 20 ml plate) at 30 ° C for 5 days, and the growing colonies were replicated on glycerol agar medium. did. From these, colonies capable of growing on a glycerol agar medium at 37 ° C. were selected, and one candidate was obtained.

The candidate (colony) was mixed with 2 ml of histidine (8 mg / 20 ml plate) and tryptophan (8 mg / 20 ml).
Plate) containing SD liquid medium (2% glucose, 0.67%
, And cultured overnight at 30 ° C., and the yeast cells were destroyed with 0.5 mm glass beads. The obtained cell-free extract was subjected to D. Hanahan (J. Mol. Biol., 166 (19)
83), according to the method of 557-580), and introduced into E. coli DH5α,
It was spread on an LB agar medium (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar, pH 7.2) containing ampicillin (50 μg / ml).

Cultured at 37 ° C. for 1 day, one of the grown colonies was selected, and LB containing ampicillin (50 μg / ml) was added.
The cells were cultured in a liquid medium (1 day at 37 ° C). From this, plasmid was prepared using the alkaline method (Nucleic Acid Res., 7, 1513).
DNA was extracted. The BamHI site of the resulting plasmid
A 9.6 kbp DNA fragment was included.

The 9.6 kbp DNA fragment was digested with ClaI, BamHI, MluI
Cleavage with restriction enzymes such as recombination of the mhr1 mutant, and about 0.7 kbp of complementarity to temperature sensitivity
It was subcloned up to the DNA fragment. Add this to pUC
It was inserted into 118 HincII sites. And ALFDNA Seque
The nucleotide sequence was determined using ncer (Falmasha).

The result is shown in SEQ ID NO: 3. The nucleotide sequence represented by SEQ ID NO: 3 was obtained by cloning a gene complementary to the nature of the mutation, and is the nucleotide sequence of a wild-type gene. In SEQ ID NO: 3 there was a 459 bp open reading frame encoding a predicted 153 amino acids. The amino acid sequence in the open reading frame is shown in SEQ ID NO: 1.

Using this gene fragment as a probe, Southern hybridization was performed on 16 chromosomes of yeast separated by pulse field electrophoresis. As a result, it was found that this gene is on the XII-th chromosome. did. Given that all the phenotypes of the mutant strain were complemented by the wild-type MHR1 gene, it was concluded that this gene was definitely the target.

Example 3 Cloning of mhr1 Gene and Determination of Mutation Site The mhr1 gene of the mutant strain was cloned, and the mutation site of the gene was searched. mhr1 mutant (FL67; FERM P-149
87), whole genomic DNA was obtained by the following method.

Mhr1 grown in YPD liquid medium at 30 ° C. for 48 hours
Mutant cells were transformed with zymolyase 100T (Zymolyase-100T; Seik
agaku Corporation, Tokyo, Japan), 1% SDS (Sodium Lauryl Sulfate; Sodium Lauryl)
Cells were disrupted in the presence of Sulfate). Then, a known DNA separation / purification procedure (C. Guthrie and
GRFink; Molecular Biology Academic Press Inc. S
an Diego, California, 1991), the whole genomic DNA of the mhr1 mutant was prepared.

The PCR of 100 ng of the total genomic DNA obtained was
MTris-HCl, 1.5 mM MgCl ₂ , 50 mM KCl, 0.125 mM dNT
P, a reaction mixture containing 1 pmol of primer and 1.25 U of taq DNA polymerase (Boehringer Mannheim) was used. Incubation at 94 ° C. for 25 seconds, followed by annealing at 55 ° C. for 30 seconds, and further one minute at 68 ° C. for one minute of the polymerase reaction were repeated for 35 times. Thereafter, the polymerase reaction at 68 ° C. was extended to 7 minutes.
In addition, a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6) were used as primers.

The obtained mhr1 gene was inserted at the Hinc II position of pUC118, and its nucleotide sequence was determined. The result is shown in SEQ ID NO: 4. SEQ ID NO: 4 has a mutation (substitution from G to A) at position 296 on the open reading frame represented by SEQ ID NO: 3 (position 421 in the nucleotide sequence of SEQ ID NO: 3). )was there. This suggests that the amino acid at position 99 has been changed from glycine to aspartic acid at the protein level. The amino acid sequence is shown in SEQ ID NO: 2.

[0053]

Industrial Applicability According to the present invention, there are provided mutated genes which cause deficient homologous recombination of mitochondria and recombinant plasmids containing these genes. The gene of the present invention is essential for maintaining stable mitochondrial DNA, which is essential for maintaining the cell respiratory function of eukaryotes. Useful for breeding yeast strains.

[0054]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 153 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Met Cys Val Val Asn Tyr Lys Gln Ser Val His Leu Tyr 1 5 10 15 Gln Asn Leu Cys Arg Leu Arg Tyr Leu Arg Asp Val Ala Gln Arg Lys 20 25 30 Glu Ser Asp Lys Leu Arg Lys Lys Asp Ser Asn Gly His Val Trp Tyr 35 40 45 Ser Gly Gln Tyr Arg Pro Thr Tyr Cys Gln Glu Ala Val Ala Asp Leu 50 55 60 Arg Glu Ser Leu Leu Lys Val Phe Glu Asn Ala Thr Pro Ala Glu Lys 65 70 75 80 Gln Thr Val Pro Ala Lys Lys Pro Ser Ile Tyr Trp Glu Asp Pro Trp 85 90 95 Arg Met Gly Asp Lys Asp Lys His Trp Asn Tyr Asp Val Phe Asn Ala 100 105 110 Leu Gly Leu Glu His Lys Leu Ile Gln Arg Val Gly Asn Ile Ala Arg 115 120 125 Glu Glu Ser Val Ile Leu Lys Glu Leu Ala Lys Leu Glu Ser His Pro 130 135 140 Thr Glu Gln Thr Glu Val Ser Ser Gln 145 150 153

SEQ ID NO: 2 Sequence length: 153 Sequence type: amino acid Topology: linear Sequence type: protein Sequence: Met Cys Val Val Asn Leu Gln Asn Tyr Lys Gln Ser Val His Leu Tyr 1 5 10 15 Gln Asn Leu Cys Arg Leu Arg Tyr Leu Arg Asp Val Ala Gln Arg Lys 20 25 30 Glu Ser Asp Lys Leu Arg Lys Lys Asp Ser Asn Gly His Val Trp Tyr 35 40 45 Ser Gly Gln Tyr Arg Pro Thr Tyr Cys Gln Glu Ala Val Ala Asp Leu 50 55 60 Arg Glu Ser Leu Leu Lys Val Phe Glu Asn Ala Thr Pro Ala Glu Lys 65 70 75 80 Gln Thr Val Pro Ala Lys Lys Pro Ser Ile Tyr Trp Glu Asp Pro Trp 85 90 95 Arg Met Asp Asp Lys Asp Lys His Trp Asn Tyr Asp Val Phe Asn Ala 100 105 110 Leu Gly Leu Glu His Lys Leu Ile Gln Arg Val Gly Asn Ile Ala Arg 115 120 125 Glu Glu Ser Val Ile Leu Lys Glu Leu Ala Lys Leu Glu Ser His Pro 130 135 140 Thr Glu Gln Thr Glu Val Ser Ser Gln 145 150 153

SEQ ID NO: 3 Sequence length: 680 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Saccharomyces
cerevisiae) Strain name: IL166-187 Sequence: AGAGTATGGA CAAGTACTAT ATTCTCAATT TCCCAACTTT TCGCAAACAC AGGTGGATAA 60 GCTGTTTGTG AGACCAAACT GGAGCAACAG AAAGCCATCA TTGAGAAGGG ACATCTGGAA 120 AATGT ATG TGT GTA GTG AAG TAT GAG AAC TTG GAG AAC TTG GAG AAC TTG GAG AAC TTG GAG AAC TTG GAC ATG Gln Ser Val His 1 5 10 CTA TAC CAG AAC CTC TGC CGG TTG AGA TAC CTC CGT GAT GTG GCA CAG 215 Leu Tyr Gln Asn Leu Cys Arg Leu Arg Tyr Leu Arg Asp Val Ala Gln 15 20 25 30 CGT AAG GAG AGT GAC AAG CTA AGA AAA AAG GAC TCT AAC GGG CAC GTC 263 Arg Lys Glu Ser Asp Lys Leu Arg Lys Lys Asp Ser Asn Gly His Val 35 40 45 TGG TAT AGC GGA CAG TAT AGA CCT ACA TAT TGT CAA GAG GCA GTG GCA 311 Trp Tyr Ser Gly Gln Tyr Arg Pro Thr Tyr Cys Gln Glu Ala Val Ala 50 55 60 GAC TTG CGG GAG TCC TTG TTG AAG GTG TTT GAG AAT GCC ACA CCA GCA 359 Asp Leu Arg Glu Ser Leu Leu Lys Val Phe Glu Asn Ala Thr Pro Ala 65 70 75 GAA AAG CAG ACA GTA CCC GCC AAA AAA CCG TCC ATA TAC TGG GAG GAC 407 Glu Lys Gln Thr Val Pro Ala Lys Lys Pro Ser Ile Tyr Trp Glu Asp 80 85 90 CCA TGG AGG ATG GGT GAC AAG GAC AAA CAT TGG AAT TAC GAT GTG TTC 455 Pro Trp Arg Met Gly Asp Lys Asp Lys His Trp Asn Tyr Asp Val Phe 95 100 105 110 AAT GCT CTG GGG CTG GAA CAC AAG CTT ATT CAG CGT GTG GGG AAC ATT 503 Asn Ala Leu Gly Leu Glu His Lys Leu Ile Gln Arg Val Gly Asn Ile 115 120 125 GCG AGG GAA GAA AGC GTT ATT CTG AAG GAA CTA GCT AAG CTC GAA TCA 551 Ala Arg Glu Glu Ser Val Ile Leu Lys Glu Leu Ala Lys Leu Glu Ser 130 135 140 CAT CCT ACA GAG CAG ACG GAA GTG TCT TCC CAG TAG AACGCTTCGT 597 His Pro Thr Glu Gln Thr Glu Val Ser Ser Gln 145 150 CATTAGATAT ACAAATGCAG GTAGAAAAAA AAACATATAGCTAGATCATAGATCATAGAAGG TCC 680

SEQ ID NO: 4 Sequence length: 680 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Saccharomyces (Saccharomyces)
cerevisiae) Strain name: FL67 Sequence: AGAGTATGGA CAAGTACTAT ATTCTCAATT TCCCAACTTT TCGCAAACAC AGGTGGATAA 60 GCTGTTTGTG AGACCAAACT GGAGCAACAG AAAGCCATCA TTGAGAAGGG ACATCTGGAA 120 AATGT ATG TGT GTA GTG AAC TAG CAA GAT AAG TAG CAAG AAT GAT AGA TAG CAA GAT AGT GAT AGA GAT AGA TAG CAA GAG Ast GAG Ast G AAG TAG CAA GAG AAT GAT Ast GAG AAT GAT AAG GAT Asg Val His 1 5 10 CTA TAC CAG AAC CTC TGC CGG TTG AGA TAC CTC CGT GAT GTG GCA CAG 215 Leu Tyr Gln Asn Leu Cys Arg Leu Arg Tyr Leu Arg Asp Val Ala Gln 15 20 25 30 CGT AAG GAG AGT GAC AAG CTA AGA AAA AAG GAC TCT AAC GGG CAC GTC 263 Arg Lys Glu Ser Asp Lys Leu Arg Lys Lys Asp Ser Asn Gly His Val 35 40 45 TGG TAT AGC GGA CAG TAT AGA CCT ACA TAT TGT CAA GAG GCA GTG GCA 311 Trp Tyr Ser Gly Gln Tyr Arg Pro Thr Tyr Cys Gln Glu Ala Val Ala 50 55 60 GAC TTG CGG GAG TCC TTG TTG AAG GTG TTT GAG AAT GCC ACA CCA GCA 359 Asp Leu Arg Glu Ser Leu Leu Lys Val Phe Glu Asn Ala Thr Pro Ala 65 70 75 GAA AAG CAG ACA GTA CCC GCC AAA AAA CCG TCC ATA TAC TGG GAG GAC 407 Glu Lys Gln Thr Val Pro Ala Lys Lys Pro Ser Ile Tyr Trp Glu A sp 80 85 90 CCA TGG AGG ATG GAT GAC AAG GAC AAA CAT TGG AAT TAC GAT GTG TTC 455 Pro Trp Arg Met Asp Asp Lys Asp Lys His Trp Asn Tyr Asp Val Phe 95 100 105 110 AAT GCT CTG GGG CTG GAA CAC AAG CTT ATT CAG CGT GTG GGG AAC ATT 503 Asn Ala Leu Gly Leu Glu His Lys Leu Ile Gln Arg Val Gly Asn Ile 115 120 125 GCG AGG GAA GAA AGC GTT ATT CTG AAG GAA CTA GCT AAG CTC GAA TCA 551 Ala Arg Glu Glu Ser Val Ile Leu Lys Glu Leu Ala Lys Leu Glu Ser 130 135 140 CAT CCT ACA GAG CAG ACG GAA GTG TCT TCC CAG TAG AACGCTTCGT 597 His Pro Thr Glu Gln Thr Glu Val Ser Ser Gln 145 150 CATTAGATAT ACAAATGCAG GTAGAAAAAA AAACATATACT AGCTGCATCTAGA TTAGAGCATCAGAAAA 680

SEQ ID NO: 5 Sequence length: 20 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: GGACATCTGG AAAATGTATG

SEQ ID NO: 6 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TCTAATGACG AAGCGTTCTA

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a method for detecting a mutant gene involved in mitochondrial homologous recombination.

[Explanation of symbols]

1 ... recipient cell, 2 ... donor cell, 3 ... mitochondria, 4
... mitochondria, 5 ... nucleus, 6 ... nucleus, 7 ... fusion cells, 8
... mitochondria with recombinant DNA

Claims (3)

[Claims] 1. A mutant mhr1 gene encoding a polypeptide comprising an amino acid sequence in which at least one amino acid at position 99 in the amino acid sequence of the wild type MHR1 protein represented by SEQ ID NO: 1 is mutated. 2. The mutant mhr1 gene according to claim 1, wherein the mutated amino acid sequence is represented by SEQ ID NO: 2. 3. A recombinant plasmid containing the gene according to claim 1 or 2.