JP2002136288A - A method for estimating the function of eukaryotic genes using yeast - Google Patents

Abstract

(57) [Summary] An object of the present invention is to provide a new method for efficiently estimating the function of a eukaryotic gene. After introducing a plasmid that expresses a eukaryotic gene into a wild-type yeast, a mutation is randomly introduced into the yeast genome, a mutant strain requiring the eukaryotic gene is identified, and the mutant strain is generated within the mutant strain. By identifying genetic mutations,
The function of the eukaryotic gene is estimated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for estimating the function of a eukaryotic gene using yeast.

[0002]

2. Description of the Related Art Gene function estimation includes methods of preparing antibodies and examining expression sites, intracellular localization, etc., and gene expression analysis using cDNA microarrays, GeneChip, etc. (Genome Function Research Protocol, Yodosha, 2000), a method of preparing a knockout mouse, a transgenic animal, etc., and observing the phenotype, etc. are known (protein nucleic acid enzyme transgenic animal, 40, 1995). There is also a method of estimating a function from information such as amino acid motifs and domain structures deduced from gene sequences, and the presence of similar proteins (Crosstalk between experimental medical domain structure and conventional transmission, 18, Yodosha, 2000). However, experiments using antibodies can provide information on intracellular localization, expression tissues, timing, etc., but these information rarely lead to elucidation of molecular functions.Similarly, expression profiling using microarrays, GeneChip, etc. There are few cases where it evolves from elucidation of molecular functions. In addition, the production of knockout mice and transgenic animals is costly and time-consuming, and there is often little information, such as embryonic lethality or a phenotype equivalent to that of a wild type even after the production. In addition, some phenotypes may not be directly linked to the molecular function of the knocked out gene. If there is a special motif or domain structure, or if there is a similar protein whose function is known, a method for detecting the activity in a test tube based on such information or a recombinant is prepared. By doing so, we can approach molecular function elucidation. However, it cannot be applied when information on motifs, domain structures, and similar proteins cannot be obtained.

Estimation of the function of eukaryotic genes using functional complementation of yeast has been performed for a long time (Nature,
327, 31-35, 1987), the conventional method was to introduce a human gene into a yeast established as a mutant strain and estimate the function of the introduced gene from its functional complementarity. This method has a problem that analysis is impossible unless a mutant strain has already been established.

[0004] As described above, in the prior art, there has been no known method for estimating the function of eukaryotic genes at low cost and efficiently.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and has as its object to provide a new method for efficiently estimating the function of eukaryotic genes. It is in.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, introduced a eukaryotic gene into a yeast mutant containing a certain functional defect and functionally complemented the yeast. The present inventors have conceived a reverse approach to the conventional method of estimating the function of the gene from gender (complementarity test).
In this method, first, a eukaryotic gene is introduced into yeast, a mutation is randomly introduced into the yeast genome, and a mutant requiring the gene is identified. Next, a gene mutation occurring in the mutant strain is identified, and the function of a eukaryotic gene is estimated from the function of the identified yeast gene.

The conventional complementation test requires that a mutant strain in which a gene in yeast which is considered to correspond to a eukaryotic gene has been mutated has already been established, and the complementation test is performed in a one-to-one correspondence. I was On the other hand, in the method conceived by the present inventors, mutants showing complementation are screened from a large number of mutant aggregates, selected, and further functional estimation is performed by gene identification. I thought it was a target.

Therefore, the present inventors examined whether or not the function of a eukaryotic gene can be estimated by the above method. As a test eukaryotic gene, an EWS / NOR-1 fusion gene derived from chromosomal translocation t (9; 22), which is a human gene, was used. First, an expression plasmid capable of controlling the expression of this human gene was constructed, and the plasmid was introduced into yeast. Next, using a mutagen, a DNA mutation was induced on the genome of the yeast into which the plasmid had been introduced. Thereafter, a mutant showing human gene auxotrophy was identified. This is because a strain that proliferates when the expression of the human gene is induced, and that is lethal or halts when the expression is suppressed is selected.In this process, it is considered that mutant strains depending on the expression induction conditions may be mixed. , A mutant strain exhibiting plasmid dependency was selected. It was confirmed that the human gene requirement of the mutant strain was derived from one gene mutation, and this gene mutation was identified. As a result, the gene having the mutation is small nu
It turned out to be a gene called SNU23 classified as clear ribonucleoprotein (snRNP). Since the EWS / NOR-1 fusion protein has an activity to complement the function of the SNU23 gene in yeast, the EWS / NOR-1 fusion gene
It was presumed to have a function as uclear ribonucleoprotein.

[0009] That is, the present inventors have found that the function of eukaryotic genes can be efficiently estimated by using a method based on a reverse approach to the conventional complementation test in yeast. The invention has been completed. The method of the present invention is greatly expected to be applied to basic research on human disease-related genes and the like, and development of therapeutic and diagnostic agents targeting disease-related genes.

The present invention relates to a new method for efficiently estimating the function of a eukaryotic gene, and more specifically, [1] a method for estimating the function of a eukaryotic gene, a) a step of introducing an expression plasmid containing a test eukaryotic gene into yeast, (b) a step of introducing a DNA mutation into the yeast genome obtained in step (a), and (c) a step obtained by step (b). Identifying a mutant strain exhibiting a test eukaryotic gene auxotrophy among the yeast mutant strains obtained, (d)
(E) estimating the function of a test eukaryotic gene from the gene identified in step (d), in the mutant strain identified in step (c), [2] identifying the gene in which the mutation has been introduced in step (c) by identifying a mutant strain that grows when the test eukaryotic gene is expressed and that is lethal or stops growth when the gene is not expressed. The method according to [1], which is performed by

[0011]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a new method for efficiently estimating the function of eukaryotic genes. In the method of the present invention, first, an expression plasmid containing a test eukaryotic gene is introduced into yeast (step (a)).

"Expression plasmid containing a test eukaryotic gene (also simply referred to as" test gene ")" means that a eukaryotic gene whose function is to be estimated is bound downstream of an expression control region; A plasmid having a structure capable of expressing the gene. The expression control region generally refers to a promoter and a DNA region that regulates transcription from the promoter. The expression control region in the present invention preferably contains a promoter capable of controlling transcription of a gene located downstream thereof. Examples of such a promoter include a GAL1 promoter, a PHO5 promoter, and a MET promoter. For the GAL1 promoter,
By culturing in a medium containing galactose, expression of a gene located downstream of the promoter can be induced.

[0013] The above-mentioned plasmid of the present invention preferably has a plurality of selectable markers in order to examine plasmid dependence in the introduced cells. Examples of the selection marker include URA3, ADE2, ADE1, TRP1, LEU2, HIS3 and the like. Expression of the selectable marker in cells is
It can be confirmed by determining the auxotrophy of cells by a replica method or the like. In particular, plasmid dependency can be examined by using the color of yeast colonies when using the ADE2 marker, and using 5FOA (5-Fluoroorotic Acid) sensitivity as an index when using the URA3 marker. In addition, the plasmid of the present invention is preferably a multicopy type which is easily removed in the absence of selection pressure by the above-mentioned selection marker.
For example, a plasmid having a 2 μm Ori as a replication origin, such as YEP13 and pYES2, can be suitably used.

[0014] Introduction of a eukaryotic gene expression plasmid into yeast is described in, for example, Methods in Yeast Genetics, Cold Spri.
ng Harbor Laboratory Press, 1997, using a lithium method, a spheroplast method, an electroporation method, or the like.

According to the present invention, the organism whose gene function can be estimated is not particularly limited as long as it is a eukaryote, but is preferably a higher eukaryote, for example, human, monkey, rat, mouse And the like.

In the present invention, the host cell into which the expression plasmid is introduced is Saccharomyces cerevis
iae) is preferably used, and fission yeast (Schizosaccharomyces
pombe) can also be used. In the present invention, in addition to yeast as a host cell, Drosophila (Dro
sophila megalogaster), nematode (Caenohabditis elegan)
s), zebrafish (Danio rerio) and the like may be used.

In the present invention, a DNA mutation is then introduced into the yeast genome obtained in step (a) (step (b)).

The introduction of a DNA mutation into the yeast genome can be carried out using, for example, ultraviolet light, a chemical mutation-introducing agent, or the like. Chemical mutagens include methanesulfonic acid ethyl ester (Methanesulfonic Acid Ethyl Ester), ENU (et
hylnitrosourea), TMP (4,5,8-trimethylpsoralen) and the like. Specifically, mutation can be introduced by treating yeast in the logarithmic growth phase with a chemical mutation-inducing agent such as methanesulfonic acid ethyl ester for a certain period of time.

Next, in the method of the present invention, a mutant showing the requirement for a test eukaryotic gene is identified from the yeast mutants obtained in the step (b) (step (c)).

"Test eukaryotic gene auxotrophy" refers to a state in which a yeast requires a specific eukaryotic gene in order for the gene mutation on the yeast genome to be functionally complemented.
Preferably, it refers to a state in which a yeast mutant requires expression of a test gene for growth or survival. In particular,
For example, when the test gene is expressed, the yeast mutant grows, but when the test gene is not expressed, the yeast is lethal or stops growing.

Isolation of a yeast strain exhibiting a test eukaryotic gene auxotrophy can be performed, for example, by a method of selecting an auxotrophic strain from non-auxotrophic strains. In this method,
First, a plasmid arranged under the control of a test eukaryotic gene, such as a GAL1 promoter capable of controlling the expression, a PHO5 promoter, or a MET promoter, is introduced into a yeast strain. Select strains that are lethal or stop growing. In this process, since there is a possibility that mutant strains depending on the expression induction conditions may coexist, next, a mutant strain exhibiting plasmid dependency is selected. In the absence of selective pressure, the test non-auxotrophic strain readily sheds the plasmid, so that only the test gene auxotroph shows plasmid dependence. This selection involves multiple selection markers on the plasmid, such as URA3, ADE2, ADE1, TR
By arranging IP1, LEU2, HIS3, etc., a clone that does not lose the plasmid even in the absence of selection pressure can be obtained by utilizing the phenotype of the marker. Expression of the marker in the yeast strain can be confirmed by determining the auxotrophy of the yeast strain by a replica method or the like. ADE2 or
When URA3 is used, there is a possibility that a gene involved in color development as a false positive or a mutant of a gene affecting 5FOA sensitivity may be included.
It is desirable to confirm the phenotype of +, URA +.

The mutants selected in this way are:
By mating with a diploid yeast wild strain and producing a diploid, it is possible to confirm that the gene mutation in the mutant strain is recessive and that the plasmid is not inserted (integrated) into the yeast genome. it can. A strain in which the gene mutation is recessive and has no plasmid inserted into the genome can be confirmed by growing even under promoter-suppressed conditions and eliminating the plasmid dependency.

[0023] If necessary, it is confirmed whether or not the eukaryotic gene requirement generated in the mutant strain is derived from one gene mutation. This can be performed using, for example, 4-spore analysis, backcross, and the like.

In the method of the present invention, the gene into which the mutation has been introduced in the mutant strain identified in step (c) is then identified (step (d)).

In the identification of a gene into which a mutation has been introduced, first, a mutation site generated on the genome of a yeast mutant is specified. When the mutation is found in a certain gene in yeast, the gene is identified as a gene into which the mutation has been introduced. It is also conceivable that the mutation is located on an expression control region such as a promoter and not necessarily on the gene ORF. In this case, the gene controlled by the expression control region is identified as a gene into which a mutation has been introduced. The mutation site on the genome can be specified by a method generally performed by those skilled in the art. For example, a gene mapping method (Methods in Yeast Genetics, Cold Spr
ing Harbor Laboratory Press, 1997), or a mutation identification method using a yeast genomic library.

Next, in the method of the present invention, the function of the test eukaryotic gene is estimated from the gene identified in step (d) (step (e)).

The function of the test eukaryotic gene can be estimated based on the function of the yeast gene identified in the above step (d). For example, the identified yeast gene is A
Since the test gene has an activity in yeast that complements the function of the yeast gene, the test gene is presumed to have the function A. Those skilled in the art can easily obtain information on the function of yeast genes. For example, Saccharomyce
s genome database (http://genome-www.stanford.edu/S
accharomyces /), Yeast protein database (http: // ww
w.proteome.com/databases/YPD/YPDsearch-quick.htm
l), Medline (http://www4.ncbi.nlm.nih.gov/PubMed/)
By using the above, information on the function of an arbitrary gene can be obtained.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[0029]

[Example 1] Introduction of human gene expression plasmid into wild-type yeast Chromosome translocation t (9; 2) frequently observed in mucinous chondrosarcoma
Gene product EWS / NOR-1 fusion gene (also known as EWS /
CHN, EWS / TEC; Human Mol. Genet., 4, 2219-2226, 199
We examined using 5). YEP-type plasmid pYES2ADE2 (U
RA3, ADE2, 2μm Ori,; pYES2 vector, Invitrogen
ADE2 inserted plasmid) downstream of the GAL1 promoter
Insert the WS / NOR-1 fusion gene cDNA into an expression vector (pYES2AD
E2-EWS / NOR-1) was constructed (FIG. 1). Germinating yeast W303 (MAT
a, leu2, ura3, trp1, ade2, his3) in YPD liquid medium (20g
/ l Difco peptone, 10g / l Yeast extract, 20g / l glucose) with shaking culture for 16 hours, wash twice with distilled water, then lithium solution (0.1M lithium acetate, 10mM Tris-HCl)
After suspending in 1 ml of pH 7.5, 1 mM EDTA, shake at 1.5 ° C. for 1.5 hours at 30 ° C., plasmid DNA 10 μg, heat-denatured carrier DNA (Shea
red herring testes DNA 10mg / ml) After mixing 50μl,
The mixture was further shaken at 30 ° C. for 30 minutes. Polyethylene glycol solution (40% polyethylene glycol average molecular weight 3350, 1
(0mM Tris-HCl, 1mM EDTA) 2.3ml was added and left for 1 hour,
DimethylSulfoxide (DMSO) 36
0 μl was added and heat shocked at 42 ° C. for 15 minutes. The yeast after the heat shock is washed once with distilled water, and is then washed on an SDA / -ura plate (6.7 g / l Difco yeast nitrogen base without a
mino acids, 20g / l glucose, 20g / l agarose, 30m
g / l L-isoleucine, 150mg / l L-valine, 20mg / l L-adenine hemisulfate, 20mg / l L-arginine HCl, 20mg / l
L-histidine HCl monohydrate, 100mg / l L-leucine, 30m
g / l L-lysine HCl, 20mg / l L-methionine, 50mg / l L-phenylalanine, 200mg / l L-threonine, 20mg / l L-tryptophan, 30mg / l L-tyrosine)
C. and the plasmid was introduced (lithium method).

Example 2 Mutagenesis Introduced into Yeast The budding yeast w303a into which pYES2ADE2-EWS / NOR-1 has been introduced was transformed into SDA /
Galactose medium (6.7g / l Difco yeast nitrogen base
without amino acids, 20g / l galactose, 10g / l raffinose pentahydrate, 30mg / l L-isoleucine, 150mg / l
L-valine, 20mg / l L-adenine hemisulfate, 20mg / l L-
Arginine HCl, 20mg / l L-Histidine HCl monohydrate, 1
00mg / l L-leucine, 30mg / l L-lysine HCl, 20mg / l L-
Methionine, 50mg / l L-Phenylalanine, 200mg / l L-
(Threonine, 20mg / l L-tryptophan, 30mg / l L-tyrosine, 20mg / l L-uracil) After liquid culture for 16 hours, wash twice with distilled water, and wash with phosphate buffer (pH 8.0)
Suspended in 10 ml. Methanesulfonic acid ethyl ester (Me
thanesulfonic Acid Ethyl Ester (Sigma))
Genetic mutation was introduced by holding at 0 ° C. for 40 minutes. The budding yeast into which the gene mutation has been introduced is neutralized with 10 ml of 10% Na-thiosulfate, washed with distilled water, shake-cultured in SDA / galactose liquid medium for 8 hours, the gene mutation is fixed, and the human gene-dependent strain is obtained. Was used for screening.

Example 3 Isolation of Human Gene-Dependent Mutant Yeast Strain into which the mutation was introduced was SDA / galactose / ADE plate (6.7 g / l Difco yeast nitrogen base without aminoa
cids, 20g / l galactose, 10g / l raffinose pentahydrate, 30mg / l L-isoleucine, 150mg / l L-valine, 15mg /
l L-adenine hemisulfate, 20mg / l L-arginine HCl,
20mg / l L-histidine HCl monohydrate, 100mg / l L-leucine, 30mg / l L-lysine HCl, 20mg / l L-methionine, 50mg
/ l L-phenylalanine, 200mg / l L-threonine, 20mg /
l L-tryptophan, 30 mg / l L-tyrosine, 20 mg / l L-uracil) at low density (300 cfu
/ 90mm dish) and culture for 36 hours, then SDA / ADE plate (6.7g / l Difco yeast nitrogen base without amino a
cids, 20g / l glucose, 20g / l agarose, 30mg / lL-
Isoleucine, 150mg / l L-valine, 15mg / l L-adenine
Hemi-sulfate, 20mg / l L-arginine HCl, 20mg / l L-histidine HCl monohydrate, 100mg / l L-leucine, 30mg / l L-
Lysine HCl, 20mg / l L-methionine, 50mg / l L-phenylalanine, 200mg / l L-threonine, 20mg / l L-tryptophan, 30mg / l L-tyrosine, 20mg / l L-uracil) . After the replica, both plates were cultured at 30 ° C. for 3 days. The phenotype is ADE2 + (white clone) and URA3 + (5FOA sensitive) because the human gene auxotroph always requires a plasmid even in the absence of selection pressure, but the non-auxotroph is ade2- (red clone). Yeasts showing ura3- (5FOA insensitive) appear with growth. In addition, since human gene expression is under the control of the GAL1 promoter, a human gene-requiring strain can grow on a medium containing galactose (GAL1 promoter-inducing conditions) and stop growing on a medium containing glucose (GAL1 promoter-suppressed conditions). I do. Therefore, from the aggregate of the yeast mutants, a clone that grew white on SDA / galactose / ADE and inhibited growth on SDA / glucose / ADE was selected. The obtained clones were then cultured in SDA / galactose liquid medium for 16 hours or more, and S containing 0.1% 5FOA.
It was applied to a DA / galactose plate and screened for 5FOA sensitivity. 5 Strain showing FOA sensitivity was seeded at low density on SDA / galactose plate, and SDA / -U
ra, -Ade / galactose plate (6.7g / l Difco yeast
nitrogen base without amino acids, 20g / l galactose, 10g / l raffinose pentahydrate, 20g / l agarose, 30mg / l L-isoleucine, 150mg / l L-valine, 20mg /
l L-arginine HCl, 20 mg / l L-histidine HCl monohydrate, 100 mg / l L-leucine, 30 mg / l L-lysine HCl, 20 mg /
l L-methionine, 50mg / l L-phenylalanine, 200mg / l
L-threonine, 20mg / l L-tryptophan, 30mg / l L-tyrosine) and all colonies are URA +, ADE +
Phenotype.

5 The clone showing FOA sensitivity, white clone, galactose-dependent growth, URA +, ADE + was wild-type w303α.
Methods in Yeast Genetics, Cold Spr
ingHarbor Laboratory Press, 1997) to produce a diploid yeast, and in the diploid, clones that lost all of the plasmid dependence, 5FOA sensitivity, and white trait were further selected.
The selected diploid strain was meiotically spread on a sporulation plate (10 g / l potassium acetate, 20 g / l agarose) and kept at 30 ° C for 48 hours, and was a genetically recessive mutation by tetraspore analysis And confirmed that the phenotype was based on 1 gene mutation (Methods in Yeast Genetics, Cold S
pring Harbor Laboratory Press, 1997), which was determined to be a yeast mutant requiring EWS / NOR-1.

[Example 4] Determination of mutant gene A budding yeast genomic library (YEP13 vector, LEU2,
The budding yeast genomic fragment 10-20 kb was inserted into the BamHI site and distributed by the Institute of Plant Science, The University of Tokyo) into the yeast mutant using the lithium method described above, and the SDA / -Leu plate (6.7 g / l Dif
co yeast nitrogen base without amino acids, 20g / l
Glucose, 20 g / l agarose, 30 mg / l L-isoleucine, 150 mg / l L-valine, 20 mg / l L-adenine hemisulphate, 20 mg / l L-arginine HCl, 20 mg / l L-histidine H
Cl monohydrate, 30mg / l L-lysine HCl, 20mg / l L-methionine, 50mg / l L-phenylalanine, 200mg / l L-threonine, 20mg / l L-tryptophan, 30mg / l L-tyrosine,
20 mg / l L-uracil), and the cells were cultured at 30 ° C. for 48 hours, and clones whose growth on a glucose medium was restored by plasmid introduction were selected. The selected yeast clones are purified by low-density reseeding and then purified in SDA / -Leu liquid medium (6.7 g / l D
ifco yeast nitrogen base without amino acids, 20g /
l glucose, 30mg / l L-isoleucine, 150mg / l L-valine, 20mg / l L-adenine hemisulfate, 20mg / l L-arginine HCl, 20mg / l L-histidine HCl monohydrate, 30mg
/ l L-lysine HCl, 20mg / l L-methionine, 50mg / l L-phenylalanine, 200mg / l L-threonine, 20mg / l L-tryptophan, 30mg / l L-tyrosine, 20mg / l L-uracil) After culturing for a time, the introduced plasmid was recovered.

Example 5 Plasmid Recovery Yeast was recovered from 3 ml of yeast culture by centrifugation, and a cell lysate (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM)
Tris-HCl, pH 8.0, 1 mM EDTA). A small amount of glass beads (425-600 nm in diameter), phenol
/ Chloroform / isoamyl alcohol (25: 24: 1) 200
After adding μl and vortexing for 5 minutes, the supernatant was recovered by centrifugation. The supernatant was precipitated with ethanol and then 100 μl T
E (10 mM Tris-HCl pH 8.0, 1 mM EDTA)
E. coli DH5α competent cells (Toyobo) were transformed according to a standard method using μl (Molecular Cloning, Cold Sp
ringHarbor Laboratory Press, 1989). The obtained E. coli colonies were cultured in LB / Amp + medium (bacto-tryptone 10 g / l, bact
o-yeast extract 5g / l, NaCl 10g / l, ampicillin 50m
g / l), and the plasmid containing the yeast genomic fragment was recovered. Sequence on YEP13 plasmid (Forward: TGG
AGC CAC TAT CGA CTA CG / SEQ ID NO: 1, Reverse: GAT
Using the CTT CCC CAT CGG TGA TG / SEQ ID NO: 2) as a primer, the sequences at the 5 ′ end and 3 ′ end of the yeast genomic fragment DNA were subjected to DNA sequencing (Current protocols in molecula).
r biology, John Wiley & Sons, Inc., 1998) and the Saccharomyces genome database (http: // genome
-www.stanford.edu/Saccharomyces/) to obtain the isolated yeast genomic fragment DNA sequence. Since the recovered DNA fragment corresponded to yeast chromosome 4 chromosome 283531-293684 bp and contained 7 genes, a deletion region was prepared using a restriction enzyme and the minimum region showing complementary activity was determined.

As a result, the minimum region retaining the activity was 2835
It was 31-286087 bp, and the further deleted 283531-285055 bp showed no complementary activity (FIG. 2). Since the SNU23 gene exists only in the minimum region where the complementation activity is lost,
The mutant gene in the mutant is small nuclear ribonucleoprot
It was strongly suggested that SNU23 might be classified as ein (snRNP).

[Example 6] Determination of gene mutation in mutant strain The mutant gene in the mutant strain is likely to be SNU23. Therefore, the DNA direct sequence method (Current protocols in mo
The SNU23 gene sequence in the mutant yeast strain was analyzed using lecular biology, John Wiley & Sons, Inc., 1998). SNU
As a result of determining the base sequence of all of the promoter region, protein coding region, and terminator region of 23 genes, a single base substitution was recognized in the protein coding region. This mutation occurs on codon 82 of the SNU23 gene,
One amino acid substitution from GC (Cys) to TAC (Tyr) occurred (FIG. 3). Since this mutation site corresponds to the first Cys constituting the zinc finger structure of the SNU23 protein,
This mutation was thought to be accompanied by the disruption of the zinc finger structure.

From the above experiments, it was shown that the EWS / NOR-1 fusion protein can have the activity to complement the function of the SNU23 gene in yeast. As a result, the yeast gene in which the mutation was found was the SNU23 gene, and the SNU23 gene was small nucl
Reported function as ear ribonucleoprotein (snRNP) (EM
BO J., 16, 4092-4106, 1997)
It can be estimated that the / NOR-1 fusion gene has a function as snRNP.

[0038]

According to the present invention, it has become possible to efficiently estimate gene functions of eukaryotes. According to the method of the present invention, a new therapeutic method, drug discovery, clinical diagnosis, and the like can be approached by estimating the biological function of a human disease-related gene. The method of the present invention is also expected to be used for basic research for elucidating gene functions of eukaryotes including humans.

[0039]

[Sequence List] SEQUENCE LISTING <110> National Cancer Center Research Institute The Organization for Pharmaceutical Safety and Research Genox Research, Inc. <120> Method for predicting function of an eukaryotic gene using yeast. <130> OPS-A0004 <140> < 141> <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 1 tggagccact atcgactacg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 2 gatcttcccc atcggtgatg 20

[Brief description of the drawings]

FIG. 1 shows the structure of a plasmid that expresses the human EWS / NOR-1 gene.

FIG. 2 is a diagram showing a minimum region where complementary activity disappears.
The position in parentheses indicates the position on the chromosome 4 of the budding yeast genome database.

FIG. 3 is a view showing a gene mutation observed in an EWS / NOR-1 auxotroph. Upper: wild-type SNU23 gene sequence, lower: E
Fig. 4 shows the sequence of the WS / NOR-1 auxotroph mutant SNU23 gene. Codon 8
The change of 2 TGC to TAC resulted in 1 amino acid substitution. Boxes indicate the four amino acids that make up the zinc finger structure.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Nagaya Okura 5-1-1 Tsukiji, Chuo-ku, Tokyo National Cancer Center Research Institute Cell Growth Factor Research Division (72) Inventor Toshihiko Tsukada 5, Tsukiji, Chuo-ku, Tokyo -1-1 F-term in Cell Growth Factors Research Division, National Cancer Center Research Institute 4B024 AA11 AA20 CA04 CA07 DA12 EA04 FA10 GA11 GA19 GA25 HA01 HA11 HA19 4B063 QA05 QA18 QA19 QQ07 QQ12 QQ13 QQ43 QR33 QR60 QR69 QR80 QS12 QS24 QS38

Claims (2)

[Claims] 1. A method for estimating the function of a eukaryotic gene, comprising the steps of: (a) introducing an expression plasmid containing a test eukaryotic gene into yeast; (b) obtaining the yeast obtained in step (a) Introducing a DNA mutation into the genome of (c),
Identifying a mutant strain exhibiting a test eukaryotic gene auxotrophy among the yeast mutant strains obtained in the step (b);
(D) in the mutant strain identified in step (c),
A method comprising: identifying a gene into which a mutation has been introduced; and (e) estimating the function of a test eukaryotic gene from the gene identified in step (d). 2. The step of identifying a gene into which a mutation has been introduced in step (c) by identifying a mutant strain that grows when the test eukaryotic gene is expressed, and that is lethal or stops growing when the gene is not expressed. The method of claim 1, wherein