JP3705768B2 - Mutant yeast cells and mutant histones H2A and H2B and uses thereof - Google Patents

Description

【００７０】
実施例４
実施例２で作成したヒストンH2B変異酵母細胞を用いて、実施例３と同様の方法によりSPT表現型の変化を調べた。生育状況の一例は図４に示したとおりである。この図４では、実施例５において良好なSPT表現型を示したH2A変異酵母（FA004#1[K4A]）を正のコントロールとして表示している。この図４に示した例では、117位Gly、118位Thr、121位ValをそれぞれAlaに置換した変異型ヒストンH2Bを発現する変異酵母細胞が、それぞれ異なった程度でSPT表現型を示している。
【００７１】
作製した112種類のH2B変異酵母細胞のうち、SPT表現型を示した変異酵母のアミノ酸置換部位と、SPT表現型の程度は表２に示したとおりである。作成した112種類のヒストンH2B変異酵母細胞のうち、表２に示した21種類の変異型ヒストンH2Bを発現する酵母細胞が、野生型酵母細胞に比べて、少なくとも３倍以上のSPT表現型の変化を示した。
【００７２】
これら21種類のヒストンH2B変異酵母細胞は、変異型ヒストンH2Bの発現によりヌクレオソームおよび染色体クロマチン構造を変化させることによって、遺伝子発現形態を改変していることが確認された。従って、これらのヒストンH2B変異酵母細胞を使用することによって、遺伝子発現形態に影響を及ぼす因子を探索することが可能であることが確認された。また、各々の変異型ヒストンH2Bまたはそれらを個々にコードするポリヌクレオチドを細胞に導入することによって、細胞の遺伝子発現形態を改変することが可能であることが確認された。
【００７３】
【表２】

【００７４】
実施例５
実施例１で作成したヒストンH2A変異酵母細胞を使用して、6AUに対する各細胞の感受性を調べた。
【００７５】
具体的には、実施例１で作成したヒストンH2A変異酵母細胞と野生型酵母細胞をそれぞれ培地上で生育させ、遠心分離操作および水による洗浄操作により培地成分を除去した株をそれぞれ1mg/mlの濃度の6AUを含有する培地および6AUを全く含有しない培地上に塗布した。塗布の方法は、あらかじめ顕微鏡観察により計数した細胞濃度に基づいて、１スポット当たり約10万細胞、約3万細胞、約1万細胞、約3000細胞、約1000細胞、約300細胞、約100細胞、約30細胞になるように段階的に希釈し、それぞれ5μlのスポットとして塗布した。このプレートを
30度で約２日〜４日間保温し、各培地上での生育状況を観察した。生育状況の一例を図５に示した。この図５に示した例では、65位Gluおよび66位LysをそれぞれAla残基に置換した変異型ヒストンH2Aを発現する変異酵母細胞が6AUに対して有意に高い感受性を示した。
【００７６】
作製した112種類のH2A変異酵母細胞のうち、6AUに対して感受性を増強した変異酵母のアミノ酸置換部位と、6AU感受性増強の程度は表３に示したとおりである。作成した112種類のヒストンH2A変異酵母細胞のうち、表３に示した10種類の変異型ヒストンH2Aを発現する酵母細胞が、野生型酵母細胞に比べ、少なくとも３倍以上の6AU感受性を示した。
【００７７】
これら10種類のヒストンH2A変異酵母細胞は、変異型ヒストンH2Aの発現によって、6AUに対する感受性を増強させていることが確認された。6AUは細胞のRNA転写およびDNA複製の阻害剤であることから、これらのヒストンH2A変異酵母細胞は、6AUと同様の作用を有する因子を高感度で探索するための系として有用であることが確認された。また、各々の変異型ヒストンH2Aまたはそれらを個々にコードするポリヌクレオチドを細胞に導入することによって、細胞の6AUまたは類似因子に対する感受性を増強することが可能であることが確認された。
【００７８】
【表３】

【００７９】
実施例６
実施例２で作成したヒストンH2B変異酵母細胞を使用し、実施例５と同様にして6AUに対する各細胞の感受性を調べた。酵母細胞の生育状況の一例を図６に示した。この図６に示した例では、123位LysがAlaに置換された変異型ヒストンH2Bを発現する変異酵母細胞が、6AUに対する感受性を増強させている。
【００８０】
この変異酵母細胞の6AUに対する感受性増強の程度は表４に示したとおりである。作成した112種類のヒストンH2B変異酵母細胞のうち、表４に示した1種類の変異型ヒストンH2Bを発現する酵母細胞が、野生型酵母細胞に比べ、300倍以上の6AU感受性を示した。
【００８１】
このヒストンH2B変異酵母細胞は、変異型ヒストンH2Bの発現によって、6AUに対する感受性を増強させていることが確認された。従って、このヒストンH2B変異酵母細胞は、6AUと同様の作用を有する因子を高感度で探索するための系として有用であることが確認された。また、変異型ヒストンH2Bまたはそれをコードするポリヌクレオチドを細胞に導入することによって、細胞の6AUまたは類似因子に対する感受性を増強することが可能であることが確認された。
【００８２】
【表４】

【００８３】
実施例７
実施例１で作成したヒストンH2A変異酵母細胞を使用して、TBZまたはBenomylに対する各細胞の感受性を調べた。
【００８４】
具体的には、実施例１で作成したヒストンH2A変異酵母細胞と野生型出芽酵母をそれぞれ培地上で生育させ、遠心分離操作および水による洗浄操作により培地成分を除去した株をそれぞれ25μg/mlの濃度のTBZを含有する培地、10μg/mlの濃度のBenomylを含有する培地、および薬剤を含有しない培地上に塗布した。なお、薬剤を含有しない培地においてもTBZおよびBenomylの水溶化のために用いた溶媒のDMSOを等量含有させた。塗布の方法は、あらかじめ顕微鏡観察により計数した細胞濃度に基づいて、１スポット当たり約10万細胞、約3万細胞、約1万細胞、約3000細胞、約1000細胞、約300細胞、約100細胞、約30細胞になるように段階的に希釈し、それぞれ5μlのスポットとして塗布した。このプレートを30度で約２日〜４日間保温し、各培地上での生育状況を観察した。図７はTBZ含有培地における生育状況の一例であり、図８はBenomyl含有培地における生育状況の一例である。これらの図に示した例では、65位Gluまたは93位のGluがアラニンに置換された変異型ヒストンH2Aを発現する変異酵母細胞が、TBZまたはBenomylに対する感受性を増強させている。
【００８５】
作製した112種類のH2A変異酵母細胞のうち、TBZまたはBenomylに対して感受性を増強した変異酵母のアミノ酸置換部位と、感受性増強の程度は表５（TBZ）および表６（Benomyl）に示したとおりである。作成した112種類のヒストンH2A変異酵母細胞のうち、表５および表６に示した７種類の変異型ヒストンH2Aを発現する酵母細胞が、野生型酵母細胞に比べ、TBZまたはBenomylに対して少なくとも10倍以上の感受性を示した。
【００８６】
これら７種類のヒストンH2A変異酵母細胞は、変異型ヒストンH2Aの発現によって、TBZまたはBenomylに対する感受性を増強させていることが確認された。TBZまたはBenomylは細胞分裂阻害剤であることから、これらのヒストンH2A変異酵母細胞は、TBZまたはBenomylと同様の作用を有する細胞分裂阻害因子を高感度で探索するための系として有用であることが確認された。また、各々の変異型ヒストンH2Aまたはそれらを個々にコードするポリヌクレオチドを細胞に導入することによって、細胞分裂阻害因子に対する感受性を増強することが可能であることが確認された。
【００８７】
【表５】

【００８８】
【表６】

【００８９】
実施例８
実施例１で作成したヒストンH2A変異酵母細胞を使用して、各細胞の生育の程度を調べた。
【００９０】
具体的には、実施例１で作成したヒストンH2A変異酵母細胞と野生型酵母細胞をそれぞれ培地上で生育させ、遠心分離操作および水による洗浄操作により培地成分を除去した株を通常酵母が生育可能な栄養豊富寒天培地上に塗布した。塗布の方法は、あらかじめ顕微鏡観察により計数した細胞濃度に基づいて、１スポット当たり約10万細胞、約3万細胞、約1万細胞、約3000細胞、約1000細胞、約300細胞、約100細胞、約30細胞になるように段階的に希釈し、それぞれ5μlのスポットとして塗布した。このプレートを30度で約３日〜５日間保温し、培地上での生育状況を観察した。
【００９１】
結果は表７に示したとおりである。作成した112種類のヒストンH2A変異酵母細胞のうち、表７に示した２種類の変異型ヒストンH2Aを発現する酵母細胞が、野生型酵母細胞に比べ、生育の程度を1/10以下に低下させた。
【００９２】
これら２種類のヒストンH2A変異酵母細胞は、変異型ヒストンH2Aの発現によって、生育の程度を有意に低下させていることが確認された。従って、これらのヒストンH2A変異酵母細胞は、細胞の生育に影響を及ぼす因子を高感度で探索するための系として有用であることが確認された。また、各々の変異型ヒストンH2Aまたはそれらを個々にコードするポリヌクレオチドを細胞に導入することによって、細胞の生育を抑制することが可能であることが確認された。
【００９３】
【表７】

【００９４】
【発明の効果】
以上詳しく説明したとおり、この発明によって、染色体の一方の構成要素であるヒストンに点変異を導入することによって、ゲノムDNAがコードする遺伝子やその転写産物を直接操作することなく、新たな形質を獲得した新しい酵母細胞株が提供される。
【００９５】
また、この出願の発明によって、酵母細胞または非酵母細胞に新たな形質を導入する方法、各種有用物質のスクリーニング方法、さらにはそれら有用物質のヒストンに対する作用点をスクリーニングする方法が提供される。
【００９６】
【配列表】

【図面の簡単な説明】
【図１】ヒストンH2Aの種間の配列相同性を示したアミノ酸配列図である。「.」は最上段のアミノ酸と同一であること、「*」は最上段に示したアミノ酸が全種で保存されていること、数字はアミノ酸配列のN端からの位置を示す。
【図２】ヒストンH2Bの種間の配列相同性を、図１と同様に示したアミノ酸配列図である。
【図３】実施例５におけるヒストンH2A変異酵母細胞の65時間培養後の生育状況である。左図はヒスチジンを含有しない培地、右図はヒスチジンとリジンを含有しない培地における酵母細胞の生育状況である。A〜Hは1μl中の細胞数（A:約20,000、B:約6,000、C:約2,000、D:約600、E:約200、F:約60、G:約20、H:約6）である。
【図４】実施例６におけるヒストンH2B変異酵母細胞の64時間培養後の生育状況である。左図はヒスチジンを含有しない培地、右図はヒスチジンとリジンを含有しない培地における酵母細胞の生育状況である。細胞数（A〜H）は図３と同一。
【図５】実施例７におけるヒストンH2A変異酵母細胞の65時間培養後の生育状況である。左図は6AUを含有しない培地、右図は6AU(1mg/ml)を含有する培地における酵母細胞の生育状況である。細胞数（A〜H）は図３と同一。
【図６】実施例８おけるヒストンH2B変異酵母細胞の65時間培養後の生育状況である。左図は6AUを含有しない培地、右図は6AU(1mg/ml)を含有する培地における酵母細胞の生育状況である。細胞数（A〜H）は図３と同一。
【図７】実施例９におけるヒストンH2A変異酵母細胞の46時間培養後の生育状況である。左から、薬剤非含有培地、DMSO含有培地、DMSO+TBZ(25μl/ml)含有培地、DMSO+TBZ(100μl/ml)含有培地における酵母細胞の生育状況である。細胞数（A〜H）は図３と同一。
【図８】実施例９におけるヒストンH2A変異酵母細胞の46時間培養後の生育状況である。左から、薬剤非含有培地、DMSO含有培地、DMSO+Benomyl(10μl/ml)含有培地、DMSO+Benomyl(500μl/ml)含有培地における酵母細胞の生育状況である。細胞数（A〜H）は図３と同一。[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to mutant yeast cells and uses thereof. More specifically, the invention of this application expresses mutant histone H2A or histone H2B in which one specific amino acid residue is substituted with an Ala residue, and these mutant proteins, and is used for screening various useful substances. It relates to useful mutant yeast cells.
[0002]
[Prior art]
Histone is a basic protein that exists widely in the nucleus of eukaryotic cells and ionically binds to DNA. It usually consists of 5 types of components (H1, H2A, H2B, H3 and H4), and the components other than H1 are highly similar across species. For example, in the case of histone H2A, 89% of budding yeast histone H2A.1 (76 amino acid sequence from the 45th to 120th position from the N end) and human histone H2A.1 (76 amino acid sequence from the 44th to 119th position from the N end) The amino acid sequences are identical and the difference is only 8 residues. Similarly, in the case of histone H2B, budding yeast histone H2B.1 (92 amino acid sequence from the 36th to 127th position from the N end) and human histone H2B.1 (92 amino acid sequence from the N end to the 33rd to 124th position) 82% of the amino acid sequences are identical and the difference is 17 residues. In the case of histone H3, 93% amino acid sequence in budding yeast histone H3 (119 amino acid sequence from the 1st to the 119th from the N end) and human histone H3.3 (119 amino acid sequence from the 1st to the 119th from the N end) Match and the difference is 8 residues. In the case of histone H4, the budding yeast histone H4 (full length 102 amino acid sequence from the 1st to the 102nd from the N end) and human histone H4 (full length 102 amino acid sequence from the 1st to the 102nd from the N end) are 92% amino acid sequences Match and the difference is 8 residues. No other highly eukaryotic protein is known to be so highly conserved among species. Budding yeast is a eukaryotic species that can be easily genetically and biochemically analyzed, and is also referred to as baker's yeast, and is a useful microorganism widely used for fermentation of bread and other foods and alcoholic beverages. Similarly to budding yeast, harmful microorganisms belonging to the fungus genus such as Candida, Aspergillus, and Ringworm have histones as well as budding yeast, and the amino acid sequence is very similar to the amino acid sequence of budding yeast. (FIGS. 1 and 2).
[0003]
DNA is folded by regular binding to this histone, and both complexes form a basic structural unit called a nucleosome. These nucleosomes aggregate to form a chromosomal chromatin structure. By maintaining or specifically transforming this chromatin structure, reactions that occur on chromosomal DNA such as gene expression, DNA replication, and DNA repair are controlled. ing.
[0004]
On the other hand, proteins constituting eukaryotic cells are expressed from genes encoded by chromosomal DNA sequences (genomes). Analysis of the expression form and gene function of each gene, or industrial use of individual genes, The focus was on directly manipulating DNA-encoded genes and their transcripts. For example, expression of an isolated gene in a host-vector system, introduction of a foreign gene into an individual organism (transgenic), deletion of a specific gene in an individual organism (knockout), or the like.
[0005]
However, as described above, the maintenance of the chromatin structure of the chromosome is indispensable for proper gene expression, DNA replication, DNA repair, and other reactions from the genome. To that end, the regularity of the nucleosomes and the structure of histones are required. It is essential that the function is maintained normally. Therefore, gene expression, DNA replication, DNA repair, etc. can be controlled by introducing mutations into histones and changing the normal chromatin structure of the chromosome. For example, Hischhorn JN et al. (Mol. Cell. Biol. 15: 1999-2009, 1995) reported a transcription defect in a specific gene by introducing a mutation into histone H2A of a yeast cell (Saccharomyces cerevisiae). Yes.
[0006]
[Problems to be solved by the invention]
The invention of this application introduces a new yeast cell that has acquired a new trait without directly manipulating the gene encoded by genomic DNA or its transcription product by introducing a point mutation into histone, which is one component of the chromosome. It is an issue to provide.
[0007]
The invention of this application also provides a mutant histone capable of imparting a new character to yeast or non-yeast cells, a polynucleotide encoding the same, and further to yeast cells or non-yeast cells using these as materials. It is an object to provide a method for introducing a new character.
[0008]
Furthermore, an object of the invention of this application is to provide a screening method for various useful substances using the mutant yeast cells, and a method for screening an action point of these useful substances for histones.
[0009]
[Means for Solving the Problems]
In this application, as a first invention for solving the above-mentioned problems, the wild-type histone genes H2A and H2B are deleted, and a Ty sequence or a δ sequence is inserted into the essential gene promoter region of the yeast cell genome. , A yeast cell having a polynucleotide construct that expresses mutant histone H2A and wild type histone H2B, wherein the mutant histone H2A represents the amino acid sequence of the N-terminal region of wild type histone H2A. 4th Lys, 5th Gly, 26th Phe, 29th Gly, 46th Ser, 47th Gly, 66th Leu, 90th Asn, 91st Asp, 92nd Asp, Histone H2A mutation, which is a mutant histone H2A in which any one amino acid residue at position 94 Leu, position 99 Gly, position 103 Ile, position 107 Gly or position 115 Asn is replaced with an Ala residue Yeast cells are provided.
[0010]
This application provides, as a second invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B and expresses the mutant-type histone H2A and the wild-type histone H2B, H2A is the 65th position Glu, the 66th position Leu, the 73rd position Asp, the 74th position Asn, the 86th position Leu, the 93rd position Glu in SEQ ID NO. Histone H2A which is a mutant histone H2A in which any one amino acid residue of 94th position Leu, 103rd position Ile, 112th position Ile, 113th position His or 116th position Leu is substituted with Ala residue Mutant yeast cells are provided.
[0011]
This application provides, as a third invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B and expresses the mutant-type histone H2A and the wild-type histone H2B, H2A is the 65th position Glu, the 93rd position Glu, the 112th position Ile, the 113th position His, the 115th position Asn, the 116th position Leu in SEQ ID NO: 1 showing the N-terminal region amino acid sequence of wild type histone H2A or Provided is a histone H2A mutant yeast cell which is a mutant histone H2A in which any one amino acid residue at position 117 Leu is substituted with an Ala residue.
[0012]
This application provides, as a fourth invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B and expresses the mutant-type histone H2A and the wild-type histone H2B, Mutant histone in which any one amino acid residue at position 93 Glu or position 94 Leu in SEQ ID NO: 1 in which H2A represents the N-terminal region amino acid sequence of wild-type histone H2A is substituted with an Ala residue A histone H2A mutant yeast cell that is H2A is provided.
[0013]
In this application, as a fifth invention, the wild type histone genes H2A and H2B are deleted, and the Ty sequence or δ sequence is inserted into the essential gene promoter region of the yeast cell genome. A yeast cell having a polynucleotide construct that expresses histone H2B, wherein the mutant histone H2B is the first position Ser and the third position Lys in SEQ ID NO: 2 showing the amino acid sequence of the N-terminal region of wild-type histone H2B. , 5th place Glu, 6th place Lys, 7th place Lys, 8th place Pro, 10th place Ser, 90th place Ser, 92nd place Ile, 93rd place Ser, 105th place Leu, 106th place Pro 107th Gly, 108th Glu, 111th Lys, 114th Val, 117th Gly, 118th Thr, 121st Val, 122th Thr or 125th Ser A histone H2B mutant yeast cell is provided which is a mutant histone H2B in which the amino acid residues are substituted with Ala residues.
[0014]
This application provides, as a sixth invention, a yeast cell having a polynucleotide construct that lacks wild-type histone genes H2A and H2B and expresses wild-type histone H2A and mutant-type histone H2B, Provided is a histone H2B mutant yeast cell in which H2B is a mutant histone H2B in which the 123rd position Lys in SEQ ID NO: 2 showing the N-terminal region amino acid sequence of wild-type histone H2B is substituted with an Ala residue.
[0015]
As the seventh invention, this application is directed to the fourth position Lys, the fifth position Gly, the 26th position Phe, the 29th position Gly, the 46th position in SEQ ID NO: 1 showing the amino acid sequence of the N-terminal region of wild type histone H2A. Ser, 47th Gly, 66th Leu, 90th Asn, 91st Asp, 92nd Asp, 94th Leu, 99th Gly, 103rd Ile, 107th Gly or 115th Provided is a mutant histone H2A in which any one amino acid residue of Asn is substituted with an Ala residue.
[0016]
As the eighth invention, this application is directed to the 65th position Glu, the 66th position Leu, the 73rd position Asp, the 74th position Asn, the 86th position in SEQ ID NO: 1 showing the amino acid sequence of the N-terminal region of wild type histone H2A. A mutation in which one of the amino acid residues of Leu, 93rd Glu, 94th Leu, 103rd Ile, 112th Ile, 113th His or 116th Leu is replaced with an Ala residue Provides type histone H2A.
[0017]
As the ninth invention, this application is directed to the 65th position Glu, the 93rd position Glu, the 112th position Ile, the 113th position His, the 115th position in SEQ ID NO: 1 showing the amino acid sequence of the N-terminal region of wild type histone H2A. Asn, a mutant histone H2A is provided in which any one amino acid residue at position 116 Leu or position 117 Leu is replaced with an Ala residue.
[0018]
In this application, as the 10th invention, any one amino acid residue at position 93 Glu or position 94 Leu in SEQ ID NO: 1 showing the amino acid sequence of the N-terminal region of wild-type histone H2A is an Ala residue. Provided are substituted mutant histone H2A.
[0019]
As the eleventh aspect of the present application, this application is directed to the 1st position Ser, 3rd position Lys, 5th position Glu, 6th position Lys and 7th position in SEQ ID NO: 2 showing the amino acid sequence of the N-terminal region of wild type histone H2B. Lys, 8th Pro, 10th Ser, 90th Ser, 92nd Ile, 93rd Ser, 105th Leu, 106th Pro, 107th Gly, 108th Glu, 111th Lys, mutation in which one amino acid residue at position 114 Val, position 117 Gly, position 118 Thr, position 121 Val, position 122 Thr or position 125 Ser is replaced with an Ala residue Provide type histone H2B.
[0020]
This application provides, as a twelfth invention, a mutant histone H2B in which position 123 Lys in SEQ ID NO: 2 showing the amino acid sequence of the N-terminal region of wild-type histone H2B is substituted with an Ala residue.
[0021]
This application provides, as a thirteenth invention, a polynucleotide encoding the mutant histone H2A of the seventh invention.
[0022]
This application provides, as a fourteenth invention, a polynucleotide encoding the mutant histone H2A of the eighth invention.
[0023]
This application provides, as a fifteenth invention, a polynucleotide encoding the mutant histone H2A of the ninth invention.
[0024]
This application provides, as a sixteenth invention, a polynucleotide encoding the mutant histone H2A of the tenth invention.
[0025]
This application provides, as the seventeenth invention, a polynucleotide encoding the mutant histone H2B of the eleventh invention.
[0026]
This application provides, as an eighteenth invention, a polynucleotide encoding the mutant histone H2B of the twelfth invention.
[0027]
This application is, as a nineteenth invention, a method for affecting the chromosomal chromatin structure or gene expression form of yeast cells or non-yeast cells, wherein the mutant histone H2A of the seventh invention or the polymorphism of the thirteenth invention is used. There is provided a method comprising the step of introducing a nucleotide into a cell.
[0028]
This application is, as a twentieth invention, a method for affecting the chromosomal chromatin structure or gene expression form of yeast cells or non-yeast cells, wherein the mutant histone H2B of the eleventh invention or the polymorphism of the seventeenth invention is used. There is provided a method comprising the step of introducing a nucleotide into a cell.
[0029]
This application is, as a twenty-first invention, a method for enhancing the sensitivity of yeast cells or non-yeast cells to an inhibitor of RNA transcription or DNA replication, the mutant histone H2A of the eighth invention, or the fourteenth invention. A method comprising introducing a polynucleotide of the above into a cell.
[0030]
This application is, as a twenty-second invention, a method for enhancing the sensitivity of yeast cells or non-yeast cells to an inhibitor of RNA transcription or DNA replication, the mutant histone H2B of the twelfth invention, or the eighteenth invention. A method comprising introducing a polynucleotide of the above into a cell.
[0031]
This application provides, as a twenty-third invention, a method for enhancing the sensitivity of a yeast cell or a non-yeast cell to a mitotic inhibitor, wherein the mutant histone H2A of the ninth invention or the polynucleotide of the fifteenth invention is used. There is provided a method comprising the step of introducing into a cell.
[0032]
This application is, as a twenty-fourth invention, a method for reducing the growth of yeast cells or non-yeast cells, wherein the mutant histone H2A of the tenth invention or the polynucleotide of the sixteenth invention is introduced into a cell. A method characterized by comprising:
[0033]
This application is, as a 25th invention, a method for screening factors affecting the chromosomal chromatin structure or gene expression form of a yeast cell or non-yeast cell, wherein the mutant yeast cell of the first invention or the fifth invention is used. A method comprising identifying a candidate factor that changes the SPT phenotype of a mutant yeast cell as a target factor, comprising introducing a candidate factor.
[0034]
This application is, as a twenty-sixth invention, a method for searching factors affecting RNA transcription or DNA replication in yeast cells or non-yeast cells, wherein candidate factors are added to the mutant yeast cells of the second invention or the sixth invention. A method comprising identifying a candidate factor that changes the mortality rate of a mutant yeast cell as a target factor, comprising the step of introducing the mutant yeast cell.
[0035]
This application is, as a twenty-seventh invention, a method for screening a factor that affects cell division of a yeast cell or a non-yeast cell, comprising the step of introducing a candidate factor into the mutant yeast cell of the third invention, Provided is a method characterized by identifying a candidate factor that changes the mortality rate of a mutant yeast cell as a target factor.
[0036]
This application is, as a twenty-eighth invention, a method for screening a factor affecting the growth of yeast cells or non-yeast cells, which comprises the step of introducing a candidate factor into the mutant yeast cell of the fourth invention, Provided is a method characterized by identifying a candidate factor that alters the growth of yeast cells as a target factor.
[0037]
This application is, as a twenty-ninth invention, an assembly of yeast cells 112 having a polynucleotide construct that lacks wild-type histone genes H2A and H2B and expresses mutant-type histone H2A and wild-type histone H2B, Mutant histone H2A of each yeast cell is replaced with an Ala residue other than the 1st to 131st Ala residues in SEQ ID NO: 1 showing the N-terminal amino acid sequence of wild type histone H2A Provided is a collection of histone H2A mutant yeast cells, which are different mutant histone H2A.
[0038]
This application, as a thirtieth invention, is a collection of yeast cells 112 having a polynucleotide construct that lacks wild-type histone genes H2A and H2B and expresses wild-type histone H2A and mutant histone H2B, Mutant histone H2B of each yeast cell is replaced with an Ala residue other than the 1st to 130th Ala residues in SEQ ID NO: 2 showing the N-terminal amino acid sequence of wild type histone H2B. Provided is a collection of histone H2B mutant yeast cells, which are different mutant histone H2Bs.
[0039]
This application provides, as a thirty-first invention, a method for screening an action point of a factor that acts on histone H2A on histone H2A, wherein each histone H2A mutant yeast cell constituting the yeast cell aggregate of the twenty-ninth invention is used. A method is provided in which a factor is introduced into each of these, and a substituted amino acid site of mutant histone H2A expressed in yeast cells in which the effect of the factor is changed is identified as the site of action of the factor.
[0040]
This application is, as a thirty-second invention, a method for screening an action point of a factor acting on histone H2B on histone H2B, wherein each histone H2B mutant yeast cell constituting the yeast cell aggregate of the thirtieth invention is used. A method is provided in which a factor is introduced into each of the above, and a substituted amino acid site of mutant histone H2B expressed in yeast cells in which the action of the factor is changed is identified as the site of action of the factor.
[0041]
Hereinafter, embodiments of the present invention will be described in detail.
[0042]
DETAILED DESCRIPTION OF THE INVENTION
The mutant yeast cells of the first to sixth inventions are deficient in the original wild type histone H2A gene and histone H2B gene, respectively, and one amino acid residue of either histone H2A or histone H2B is an alanine residue. It possesses a polynucleotide construct (expression vector) that expresses the mutant histone H2A or histone H2B substituted in the above.
[0043]
Yeast cells deficient in wild-type histone H2A gene and histone H2B gene (hereinafter referred to as “H2A / H2B deficient yeast cells”. In this description, “/” means “and”) are known target gene replacements. It can also be made by gene knockout using the method, but preferably, MATa his3Δ200 leu2Δ1 trp1Δ63 ura3-52 lys2-128δ Δ (hta1-htb1) :: LEU2 Δ (hta2-htb2) :: TRP1 + pSAB6
Saccharomyces cerevisiae strain FY406 (Hirschhorn JN et al., Mol. Cell. Biol. 15: 1999-2009, 1995) can be used. This FY406 strain lacks two wild-type histone H2A genes and two wild-type histone H2B genes in the genome, and possesses one histone H2A gene and one histone H2B gene, and pSAB6 plasmid carrying the URA3 gene. (Hereinafter referred to as “H2A / H2B wild-type plasmid”. In this description, “/” means “and”).
[0044]
A construct obtained by cloning a histone H2A protein containing alanine point mutation or a polynucleotide encoding a histone H2B protein into such an H2A / H2B-deficient yeast cell (specifically, a plasmid. Hereinafter, “H2A-H2B mutant plasmid”) In this description, “-” means “or”). The H2A-H2B mutant plasmid carries the HIS3 gene. Since the FY406 strain loses the function of the HIS3 gene, it cannot grow in a medium that does not contain histidine, but can grow by possessing this H2A-H2B mutant plasmid. The H2A / H2B-deficient yeast strain into which this H2A-H2B mutant plasmid has been introduced is referred to as A strain. Strain A is spread on agar plates containing 5-Fluorootic Acid (5-FOA) at a concentration of 1 mg / ml and uracil at a concentration of 0.05 mg / ml. If the URA3 gene functions in budding yeast, 5-FOA is metabolized to a lethal intermediate and cannot grow on the plate. On the other hand, Saccharomyces cerevisiae without the function of the URA3 gene cannot biosynthesize uracil intracellularly, but can grow on the plate if uracil is added exogenously as in the above plate. Although the A strain has lost the function of the URA3 gene in the genome, the H2A / H2B wild-type plasmid carries the URA3 gene, so as long as the H2A / H2B wild-type plasmid is retained in the cell, It cannot grow. However, it is known that plasmids are generally distributed to daughter cells along with the division of parent cells, but are not distributed to one daughter cell at a low frequency and fall off from host cells. In the case of the A strain, the same phenomenon is confirmed, and even when it is applied on the plate, some colonies grow. This strain is called B strain. The B strain is deficient in the histone H2A gene and histone H2B gene in the genome and carries the H2A-H2B mutant plasmid. That is, the B strain is established as a budding yeast strain in which only one specific amino acid of histone H2A or histone H2B is selectively substituted with alanine.
[0045]
A method of substituting one amino acid residue of histone H2A protein or H2B protein with an alanine residue is the Kunkel method (Kunkel, TA Proc. Natl. Acad. Sci. USA 82) based on the hybridization of oligonucleotide and single-stranded DNA. : 488, 1985 and Kunkel, TA, et al. Methods in Enzymology 154: 367, 1987). That is, Escherichia coli (BW313, CJ236, etc.) represented by dut and ung genotypes is deficient in dUTPase (Dut) and Uracil-DNA glycosylase (Ung). Therefore, DNA in which a part of thiamine (T) in the DNA is replaced with deoxyuracil (dU) is synthesized. Using this Escherichia coli as a host fungus, a single-stranded DNA of a plasmid into which a histone H2A or H2B gene targeted for mutagenesis is cloned is prepared. The histone H2A gene and the histone H2B gene can be obtained by using, for example, a yeast cDNA synthesized with an oligonucleotide probe synthesized based on known sequence information (H2A: GenBank Accession No. CAA88505.1; H2B: GenBank Accession No. CAA88504.1, etc.) It can be obtained by a screening method, a PCR amplification method using oligonucleotide primers, or the like. Moreover, the publicly available well-known clone can also be utilized.
[0046]
This single-stranded DNA is hybridized in vitro with an oligonucleotide designed to substitute the target residue with alanine, and a complementary DNA strand is synthesized by a DNA polymerase reaction and a DNA ligase reaction. Ung this DNA ⁺ When introduced into E. coli strains (DH5α, etc.), the original DNA strand containing dU is degraded by Ung, but the complementary DNA strand synthesized in a test tube is replicated without degradation. In this way, a mutation-introducing plasmid obtained by cloning a gene encoding a histone H2A protein or a histone H2B protein containing an alanine point mutation can be obtained by selectively amplifying the DNA strand into which the mutation has been introduced.
[0047]
In addition, the mutant yeast cells of the first and fifth inventions have a Ty sequence or δ sequence inserted in the essential gene promoter region of the yeast cell genome in addition to the configuration as described above. Such a budding yeast strain is represented by, for example, a genotype of lys2-128δ. The budding yeast strain FY406 also has this lys2-128δ genotype. This means that the δ sequence has been inserted into the promoter region of the LYS2 gene in the genome and expression of the LYS2 gene no longer occurs. Such a strain cannot grow unless it contains lysine on the medium. However, when a quantitative or qualitative change in the expression of a gene occurs, a strain having the lys2-128δ genotype can grow on a medium containing no lysine. Such a phenotype is called a suppressor of Ty (SPT) phenotype (Winston, F. and Carlson, M. Trends Genet. 8: 387-391, 1992). Since the mutant yeast cells of the first and fifth inventions grow on a medium not containing lysine, it was confirmed that they exhibit an SPT phenotype. There are many reports of genetic mutations that lead to the SPT phenotype, including histones and other genetic mutations that encode molecules involved in the maintenance or conversion process of the genomic chromatin structure. It is an important indicator for detecting structural changes and gene expression changes. Therefore, the mutant yeast cells of the first and fifth inventions are useful as a system for examining how specific amino acid point mutations of histones H2A and H2B affect chromosomal chromatin structure and gene expression form, respectively. . It is also useful as a system (25th invention) for searching for factors affecting chromosomal chromatin structure and gene expression form. Furthermore, mutant H2A protein and mutant H2B protein (7th invention, 11th invention) possessed by these mutant yeast cells, or polynucleotides encoding these mutant proteins (13th invention and 17th invention) Can be used to change the chromosomal chromatin enzyme or gene expression form of yeast or non-yeast cells (19th invention, 20th invention).
[0048]
In the invention of this application, “factor” includes unknown and known compounds, proteins, peptides, polynucleotides, oligonucleotides and the like. The screening method of the present invention targets “non-yeast cells” in addition to yeast cells. As described above, the histone structure (amino acid sequence) is highly conserved across species, This is because the knowledge of yeast can be reliably applied to species other than yeast. Furthermore, non-yeast cells are intended not only for cultured cells (in vitro) but also for living cells within an individual organism (in vivo).
[0049]
On the other hand, the mutant yeast cells of the 2-4th invention and the 6th invention are yeast cells exhibiting a phenotype different from that of the wild-type yeast cell under specific conditions.
[0050]
The mutant yeast cells of the second and sixth inventions were identified as cell lines having significantly higher sensitivity to 6-azauracil (6AU) than the wild type. 6AU is an agent that affects RNA transcription and DNA replication by changing intracellular nucleotide concentration (Nakanishi T. et al. J Biol. Chem. 270: 8991-8995, 1995, Hampsey M. Yeast 13: 1099-1133, 1997). Accordingly, since these mutant yeast cells react sensitively to factors that affect RNA transcription or DNA replication, they are useful as a system for searching for these factors (the twenty-sixth invention). Furthermore, mutant H2A protein and mutant H2B protein (8th invention, 12th invention) possessed by these mutant yeast cells, or polynucleotides encoding these mutant proteins (14th invention and 18th invention), It can be used to enhance the sensitivity of yeast or non-yeast cells to inhibitors of RNA transcription or DNA replication (21st invention, 22nd invention).
[0051]
The mutant yeast cell of the third invention was identified as a cell line having significantly higher sensitivity to TBZ (Thiabendazole) and Benomyl (Methyl 1- (butylcarbamoyl) -2-benzimidazolecarbamate) than the wild type. Both TBZ and Benomyl have a physiological activity that inhibits cell division by inhibiting intracellular microtubule polymerization (Hampsey M. Yeast 13: 1099-1133, 1997). Therefore, since these mutant yeast cells react sensitively to factors that affect cell division, they are useful as a system for searching for these factors (the 27th invention). Furthermore, the mutant H2A protein possessed by these mutant yeast cells (the ninth invention), or the polynucleotide encoding this mutant protein (the fifteenth invention) is sensitive to inhibitors of cell division in yeast or non-yeast cells. It can be used to enhance (23rd invention).
[0052]
The mutant yeast cell of the fourth invention has been identified as a cell line having a lower degree of growth than the wild type. Accordingly, since these mutant yeast cells react sensitively to factors that affect the degree of growth, they are useful as a system for searching for these factors (the 28th invention). Furthermore, the mutant H2A protein possessed by these mutant yeast cells (the tenth invention) or the polynucleotide encoding this mutant protein (the sixteenth invention) is used for reducing the growth of yeast or non-yeast cells. (24th invention).
[0053]
The seventh to twelfth inventions are a mutant histone H2A protein and a mutant histone H2B protein that are expressed by each of the mutant yeast cells. These mutant proteins can also be obtained by a method of synthesizing a peptide by a known chemical synthesis method based on the sequence information of SEQ ID NOS: 1 and 2 and each amino acid substitution. Yeast cells can be obtained by a method of culturing yeast cells by a known method, and isolating and purifying the target protein from the culture by a known means. Alternatively, a polynucleotide encoding each mutant protein (13th to 18th inventions; hereinafter referred to as “mutant polynucleotide”) is linked to an appropriate expression vector, whereby an in vitro transcription / translation system, Mutant proteins can be isolated and purified from cultures of yeast cells (microorganisms, insect cells, mammalian cells, plant cells, etc.).
[0054]
For example, when a mutant protein is expressed in an in vitro transcription / translation system, a recombinant vector is prepared by inserting the mutant polynucleotide into a vector having an RNA polymerase promoter. If this vector is added to an in vitro transcription / translation system such as a rabbit reticulocyte lysate or wheat germ extract containing RNA polymerase corresponding to the promoter, the target mutant protein can be produced in vitro. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, pT7 / 319, and pBluescript II.
[0055]
When the mutant protein is expressed in microorganisms such as E. coli, the mutant polynucleotide is recombined into an expression vector having an origin, promoter, ribosome binding site, DNA cloning site, terminator, etc. that can replicate in the microorganism. To create an expression vector. By transforming host cells with this expression vector, transformant cells that express the mutant protein can be obtained. When this transformant is cultured, the target mutant protein is mass-produced from the culture. be able to. Examples of the expression vector for E. coli include pUC system, pBluescript II, pET expression system, and pGEX expression system.
[0056]
Further, when the mutant protein is expressed in eukaryotic cells, the mutant polynucleotide is inserted into a expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, etc. Create a vector. If this vector is introduced into a eukaryotic cell and cultured, a transformed eukaryotic cell expressing the target mutant protein can be obtained. Examples of expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYES2. As eukaryotic cells, mammalian cultured cells such as human fetal kidney-derived cell HEK293T, monkey kidney cell COS7, Chinese hamster ovary cell CHO, or primary cultured cells isolated from human organs can be used. Budding yeast, fission yeast, silkworm cells, Xenopus egg cells and the like can also be used. In order to introduce the expression vector into cells, known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method can be used.
[0057]
In order to isolate and purify the mutant protein expressed in the mutant yeast cell or transformed cell of the present invention, it can be performed by combining known separation procedures. For example, treatment with denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
[0058]
A protein expressed in a cell may be subjected to various modifications in the cell after being translated. Therefore, modified proteins are also included in the scope of the mutant protein of the present invention. Such post-translational modifications include N-terminal methionine elimination, acetylation, glycosylation, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation, ubiquitination, ADP ribosylation, methylation Etc.
[0059]
Mutant histones H2A and H2B obtained as described above, and mutant polynucleotides encoding them can be used in the nineteenth to twenty-fourth inventions as described above. In these method inventions, in order to introduce a mutant protein into a test cell, the mutant protein is added to a pharmacologically acceptable carrier solution without changing the structure or function of the mutant protein. Are mixed and introduced into cells by, for example, a microinjection method. Alternatively, if a recently developed intracellular introduction method using lipids (BioPORTER (Gene Therapy Systems, USA), Chariot (Active Motif, USA), etc.) is used, a mutant protein is introduced into living cells within an organism. In addition, the action can be exerted in vivo.
[0060]
In addition, when a mutant polynucleotide is introduced into a cell, a construct (expression vector) having the mutant polynucleotide is prepared by known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method. The method introduced in can be adopted. In addition, by incorporating an expression vector into a hollow nanoparticle, a retrovirus, an adenovirus, an adeno-associated virus, or the like, a mutant polynucleotide can be introduced and expressed in vivo by a gene therapy technique.
[0061]
The 29th and 30th inventions are a collection of mutant yeast cells that express mutant histone H2A protein or mutant histone H2B. Since these yeast cell populations express mutant proteins having different amino acid point mutations, factors affecting histone H2A or histone H2B (known factors or those identified in the 25th to 28th inventions above) It is useful as a system (the 31st invention and the 32nd invention) for specifying to which amino acid residue of histone the novel factor) acts. Such elucidation of the action point is effective for selecting a drug (for example, a cytotoxic drug) that acts only on a specific animal species. That is, histones are highly conserved across species as described above. However, as shown in FIGS. 1 and 2, yeast (sce) histones are more potent than Aspergius (hum). asp), Candida albicans (cal) and other fungi histones are highly homologous. Therefore, for example, if yeast histones substituted with amino acid residues peculiar to human histones have reduced susceptibility to toxic drugs, this drug has no effect on human cells, and is only effective against yeast and fungi. Can be judged.
[0062]
Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.
[0063]
【Example】
Example 1
Has a polynucleotide construct that lacks the wild-type histone genes H2A and H2B, has a δ sequence inserted into the essential gene promoter region of the yeast cell genome, and expresses mutant histone H2A protein and wild-type histone H2B Histone H2A mutant yeast cells were prepared. The mutant histone H2A protein is a mutant histone H2A protein in which each amino acid residue of SEQ ID NO: 1 showing the N-terminal region amino acid sequence of the wild type histone H2A protein is substituted with an Ala residue.
[0064]
Specifically, according to the Kunkel method, a plasmid having a polynucleotide encoding a mutant histone H2A and a wild type histone H2B in which each amino acid residue other than the alanine residue of SEQ ID NO: 1 is substituted with an alanine residue is yeast. The cells were introduced into FY406 strain. This strain is called A strain. Strain A was spread on agar plates containing 5-Fluorootic Acid (5-FOA) at a concentration of 1 mg / ml and uracil at a concentration of 0.05 mg / ml. If the URA3 gene functions in budding yeast, 5-FOA is metabolized to a lethal intermediate and cannot grow on the plate. On the other hand, Saccharomyces cerevisiae without the function of the URA3 gene cannot biosynthesize uracil intracellularly, but can grow on the plate if uracil is added exogenously as in the above plate. Although the A strain has lost the function of the URA3 gene in the genome, the H2A / H2B wild-type plasmid carries the URA3 gene, so as long as the H2A / H2B wild-type plasmid is retained in the cell, It cannot grow. However, it is known that plasmids are generally distributed to daughter cells along with the division of parent cells, but are not distributed to one daughter cell at a low frequency and fall off from host cells. The same phenomenon was confirmed in the case of strain A, and some colonies grew even when applied on the plate. This strain is called B strain. The B strain is a strain that lacks the histone H2A gene and the histone H2B gene in the genome and possesses a plasmid obtained by cloning a gene encoding a histone H2A protein containing an alanine point mutation and a wild type histone H2B protein.
[0065]
As described above, a histone H2A mutant yeast cell 131 strain in which only one specific amino acid of histone H2A was selectively substituted with alanine was established.
Example 2
A histone having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B, has a δ sequence inserted into the essential gene promoter region of the yeast cell genome, and expresses wild-type histone H2A and mutant histone H2B H2B mutant yeast cells were created. Mutant histone H2B is the same method as in Example 1 except that each amino acid residue other than the alanine residue of SEQ ID NO: 2 showing the N-terminal region amino acid sequence of wild-type histone H2B is substituted with an alanine residue. Created with.
Example 3
Using the histone H2A mutant yeast cells prepared in Example 1, changes in the SPT phenotype were examined.
[0066]
Specifically, histone H2A mutant yeast cells and wild-type budding yeast prepared in Example 1 are grown on a medium, respectively, and strains from which medium components have been removed by centrifugation and washing with water each contain lysine. It was spread on medium and medium without lysine. The application method is based on the cell concentration previously counted by microscopic observation, about 100,000 cells, about 30,000 cells, about 10,000 cells, about 3000 cells, about 3000 cells, about 1000 cells, about 300, about 100 cells, Diluted stepwise to approximately 30 cells and spread as 5 μl spots each. This plate was incubated at 30 degrees for about 2 to 4 days, and the growth state on each medium was observed (see Hartzog GA et al. Mol Cell Biol. 16: 2848-2856, 1996). An example of the growth situation is as shown in FIG. In the example shown in FIG. 3, the H2A mutant yeast (FA004 # 1 [K4A]) in which the 4th-position Lys is substituted with Ala grows well even in a medium not containing lysine (right figure) and shows the SPT phenotype ing.
[0067]
Among the 112 types of H2A mutant yeast cells produced, the amino acid substitution sites of the mutant yeast that showed the SPT phenotype and the degree of the SPT phenotype are as shown in Table 1. Among the 112 types of histone H2A mutant yeast cells that were created, the yeast cells that express the 15 types of mutant histone H2A shown in Table 1 showed at least a three-fold change in SPT phenotype compared to wild-type yeast cells. showed that.
[0068]
These 15 types of histone H2A mutant yeast cells were confirmed to have altered gene expression by changing the nucleosome and chromosomal chromatin structure by the expression of mutant histone H2A. Therefore, it was confirmed that by using these histone H2A mutant yeast cells, it is possible to search for factors that affect the gene expression form. Moreover, it was confirmed that the gene expression form of cells can be modified by introducing each mutant histone H2A or a polynucleotide encoding them individually into the cells.
[0069]
[Table 1]

[0070]
Example 4
Using the histone H2B mutant yeast cells prepared in Example 2, changes in the SPT phenotype were examined by the same method as in Example 3. An example of the growth situation is as shown in FIG. In FIG. 4, the H2A mutant yeast (FA004 # 1 [K4A]) that showed a good SPT phenotype in Example 5 is displayed as a positive control. In the example shown in FIG. 4, mutant yeast cells expressing mutant histone H2B in which Gly at position 117, Thr at position 118, and Val at position 121 are each replaced with Ala show SPT phenotypes to different degrees. .
[0071]
Among the 112 types of H2B mutant yeast cells produced, the amino acid substitution sites of the mutant yeast that showed the SPT phenotype and the degree of the SPT phenotype are as shown in Table 2. Among the 112 types of histone H2B mutant yeast cells that were prepared, the yeast cells that express the 21 types of mutant histone H2B shown in Table 2 showed at least a three-fold change in SPT phenotype compared to wild-type yeast cells. showed that.
[0072]
These 21 types of histone H2B mutant yeast cells were confirmed to have altered gene expression by changing the nucleosome and chromosomal chromatin structure by the expression of mutant histone H2B. Therefore, it was confirmed that by using these histone H2B mutant yeast cells, it is possible to search for factors that affect the gene expression form. It was also confirmed that the gene expression form of the cells can be modified by introducing each mutant histone H2B or a polynucleotide encoding them individually into the cells.
[0073]
[Table 2]

[0074]
Example 5
Using the histone H2A mutant yeast cells prepared in Example 1, the sensitivity of each cell to 6AU was examined.
[0075]
Specifically, the histone H2A mutant yeast cells and wild-type yeast cells prepared in Example 1 were grown on a medium, respectively, and 1 mg / ml each of the strains from which medium components were removed by centrifugation and washing with water. It was spread on a medium containing a concentration of 6AU and a medium containing no 6AU. The application method is based on the cell concentration counted by microscopic observation in advance, and approximately 100,000 cells, approximately 30,000 cells, approximately 10,000 cells, approximately 3000 cells, approximately 1000 cells, approximately 300 cells, approximately 100 cells per spot. , Diluted stepwise to about 30 cells and spread as 5 μl spots each. This plate
The culture was kept at 30 degrees for about 2 to 4 days, and the growth state on each medium was observed. An example of the growth situation is shown in FIG. In the example shown in FIG. 5, mutant yeast cells expressing mutant histone H2A in which 65th-position Glu and 66th-position Lys were substituted with Ala residues, respectively, showed significantly higher sensitivity to 6AU.
[0076]
Among the 112 types of H2A mutant yeast cells produced, the amino acid substitution sites of the mutant yeast having enhanced sensitivity to 6AU and the degree of 6AU sensitivity enhancement are as shown in Table 3. Among the 112 types of histone H2A mutant yeast cells prepared, the yeast cells expressing the 10 types of mutant histone H2A shown in Table 3 showed at least 3 times more 6AU sensitivity than the wild type yeast cells.
[0077]
These ten types of histone H2A mutant yeast cells were confirmed to have enhanced sensitivity to 6AU by expression of mutant histone H2A. Since 6AU is an inhibitor of cellular RNA transcription and DNA replication, it was confirmed that these histone H2A mutant yeast cells are useful as a system for searching for factors having the same action as 6AU with high sensitivity. It was done. Moreover, it was confirmed that the sensitivity of cells to 6AU or similar factors can be enhanced by introducing each mutant histone H2A or a polynucleotide encoding them individually into the cells.
[0078]
[Table 3]

[0079]
Example 6
Using the histone H2B mutant yeast cells prepared in Example 2, the sensitivity of each cell to 6AU was examined in the same manner as in Example 5. An example of the growth status of yeast cells is shown in FIG. In the example shown in FIG. 6, mutant yeast cells that express mutant histone H2B in which Lys at position 123 is replaced with Ala have enhanced sensitivity to 6AU.
[0080]
The degree of enhancement of sensitivity of the mutant yeast cells to 6AU is as shown in Table 4. Among the 112 types of histone H2B mutant yeast cells prepared, the yeast cells expressing one type of mutant histone H2B shown in Table 4 showed 6 AU sensitivity more than 300 times that of wild-type yeast cells.
[0081]
This histone H2B mutant yeast cell was confirmed to have enhanced sensitivity to 6AU due to the expression of mutant histone H2B. Therefore, it was confirmed that this histone H2B mutant yeast cell is useful as a system for searching for a factor having the same action as 6AU with high sensitivity. In addition, it was confirmed that the sensitivity of cells to 6AU or similar factors can be enhanced by introducing mutant histone H2B or a polynucleotide encoding the same into the cells.
[0082]
[Table 4]

[0083]
Example 7
The histone H2A mutant yeast cells prepared in Example 1 were used to examine the sensitivity of each cell to TBZ or Benomyl.
[0084]
Specifically, the histone H2A mutant yeast cells and wild-type budding yeast prepared in Example 1 were grown on a medium, and the strains from which medium components were removed by centrifugation and washing with water were each 25 μg / ml. It was spread on medium containing TBZ at a concentration, medium containing Benogyl at a concentration of 10 μg / ml, and medium containing no drug. In addition, even in a medium not containing a drug, an equal amount of DMSO as a solvent used for water-solubilization of TBZ and Benomyl was contained. The application method is based on the cell concentration counted by microscopic observation in advance, and approximately 100,000 cells, approximately 30,000 cells, approximately 10,000 cells, approximately 3000 cells, approximately 1000 cells, approximately 300 cells, approximately 100 cells per spot. , Diluted stepwise to about 30 cells and spread as 5 μl spots each. The plate was incubated at 30 degrees for about 2 to 4 days, and the growth state on each medium was observed. FIG. 7 shows an example of the growth situation in the TBZ-containing medium, and FIG. 8 shows an example of the growth situation in the Benomyl-containing medium. In the examples shown in these figures, mutant yeast cells expressing mutant histone H2A in which Glu at position 65 or Glu at position 93 is substituted with alanine have enhanced sensitivity to TBZ or Benomyl.
[0085]
Among the 112 types of H2A mutant yeast cells produced, the amino acid substitution sites and the degree of sensitivity enhancement of the mutant yeast with enhanced sensitivity to TBZ or Benomyl are shown in Table 5 (TBZ) and Table 6 (Benomyl). It is. Among the 112 types of histone H2A mutant yeast cells prepared, the yeast cells expressing the seven types of mutant histone H2A shown in Tables 5 and 6 are at least 10 times more resistant to TBZ or Benomyl than the wild type yeast cells. It was more than twice as sensitive.
[0086]
These seven types of histone H2A mutant yeast cells were confirmed to have enhanced sensitivity to TBZ or Benomyl by the expression of mutant histone H2A. Since TBZ or Benomyl is a cell division inhibitor, these histone H2A mutant yeast cells may be useful as a system for highly sensitive search for cell division inhibitors having the same action as TBZ or Benomyl. confirmed. It was also confirmed that the sensitivity to mitotic inhibitors can be enhanced by introducing each mutant histone H2A or a polynucleotide encoding them individually into cells.
[0087]
[Table 5]

[0088]
[Table 6]

[0089]
Example 8
Using the histone H2A mutant yeast cells prepared in Example 1, the degree of growth of each cell was examined.
[0090]
Specifically, histone H2A mutant yeast cells and wild-type yeast cells prepared in Example 1 are grown on a medium, and normal yeast can grow in a strain from which medium components have been removed by centrifugation and washing with water. It was applied on a nutrient rich agar medium. The application method is based on the cell concentration counted by microscopic observation in advance, and approximately 100,000 cells, approximately 30,000 cells, approximately 10,000 cells, approximately 3000 cells, approximately 1000 cells, approximately 300 cells, approximately 100 cells per spot. , Diluted stepwise to about 30 cells and spread as 5 μl spots each. This plate was kept warm at 30 degrees for about 3 to 5 days, and the growth state on the medium was observed.
[0091]
The results are as shown in Table 7. Among the 112 types of histone H2A mutant yeast cells that were prepared, yeast cells that express the two types of mutant histone H2A shown in Table 7 reduced the degree of growth to 1/10 or less compared to wild-type yeast cells. It was.
[0092]
These two types of histone H2A mutant yeast cells were confirmed to significantly reduce the degree of growth due to the expression of mutant histone H2A. Therefore, it was confirmed that these histone H2A mutant yeast cells are useful as a system for searching for factors that affect cell growth with high sensitivity. It was also confirmed that cell growth could be suppressed by introducing each mutant histone H2A or a polynucleotide encoding them individually into cells.
[0093]
[Table 7]

[0094]
【The invention's effect】
As explained in detail above, by introducing point mutations in histones, one of the components of the chromosome, this invention acquires new traits without directly manipulating the genes encoded by genomic DNA and their transcripts. New yeast cell lines are provided.
[0095]
The invention of this application also provides a method for introducing a new character into yeast cells or non-yeast cells, a method for screening various useful substances, and a method for screening the action points of these useful substances on histones.
[0096]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an amino acid sequence diagram showing sequence homology between histone H2A species. “.” Is the same as the amino acid at the top, “*” indicates that the amino acid shown at the top is conserved in all species, and the number indicates the position from the N end of the amino acid sequence.
FIG. 2 is an amino acid sequence diagram showing the sequence homology between histone H2B species as in FIG.
FIG. 3 shows the growth of histone H2A mutant yeast cells in Example 5 after culturing for 65 hours. The left figure shows the growth of yeast cells in a medium containing no histidine, and the right figure shows a medium containing no histidine and lysine. A to H are the number of cells in 1 μl (A: about 20,000, B: about 6,000, C: about 2,000, D: about 600, E: about 200, F: about 60, G: about 20, H: about 6) It is.
FIG. 4 shows the growth status of the histone H2B mutant yeast cells in Example 6 after culturing for 64 hours. The left figure shows the growth of yeast cells in a medium containing no histidine, and the right figure shows a medium containing no histidine and lysine. The number of cells (A to H) is the same as FIG.
FIG. 5 shows the growth of the histone H2A mutant yeast cells in Example 7 after culturing for 65 hours. The left figure shows the growth of yeast cells in a medium not containing 6AU, and the right figure shows a medium containing 6AU (1 mg / ml). The number of cells (A to H) is the same as FIG.
FIG. 6 shows the growth status of histone H2B mutant yeast cells in Example 8 after culturing for 65 hours. The left figure shows the growth of yeast cells in a medium not containing 6AU, and the right figure shows a medium containing 6AU (1 mg / ml). The number of cells (A to H) is the same as FIG.
FIG. 7 shows the growth status of the histone H2A mutant yeast cells in Example 9 after culturing for 46 hours. From left to right, the growth of yeast cells in a drug-free medium, DMSO-containing medium, DMSO + TBZ (25 μl / ml) -containing medium, and DMSO + TBZ (100 μl / ml) -containing medium. The number of cells (A to H) is the same as FIG.
FIG. 8 shows the growth of histone H2A mutant yeast cells in Example 9 after culturing for 46 hours. From the left, the growth state of yeast cells in a drug-free medium, DMSO-containing medium, DMSO + Benomyl (10 μl / ml) -containing medium, and DMSO + Benomyl (500 μl / ml) -containing medium. The number of cells (A to H) is the same as FIG.

Claims (27)

  Polynucleotide construct that is a yeast cell that lacks the wild-type histone genes H2A and H2B, does not express the LYS2 gene by inserting a δ sequence into the LYS2 gene promoter region, and expresses the mutant-type histone H2A and wild-type histone H2B Wherein the mutant histone H2A is the 4th Lys, 5th Gly, 26th Phe, 29th position in SEQ ID NO: 1 showing the N-terminal region amino acid sequence of wild type histone H2A. Gly, 46th Ser, 47th Gly, 66th Leu, 90th Asn, 91st Asp, 92nd Asp, 94th Leu, 99th Gly, 103rd Ile, 107th A mutant histone H2A in which any one amino acid residue of Gly or Asn at position 115 is substituted with an Ala residue, and histone H2A has an SPT phenotypic change of 3 times or more compared to wild type yeast. Mutant yeast cells.   A yeast cell having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B and expresses the mutant-type histone H2A and the wild-type histone H2B, wherein the mutant-type histone H2A is the N-terminus of the wild-type histone H2A. 65th position Glu, 66th position Leu, 73rd position Asp, 74th position Asn, 86th position Leu, 93rd position Glu, 94th position Leu, 103rd position Ile in SEQ ID NO: 1 showing the region amino acid sequence A histone H2A mutant yeast cell which is a mutant histone H2A in which any one amino acid residue at position 112 Ile, position 113 His or position 116 Leu is substituted with an Ala residue.   A yeast cell having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B and expresses the mutant-type histone H2A and the wild-type histone H2B, wherein the mutant-type histone H2A is the N-terminus of the wild-type histone H2A. Any one of the 65th position Glu, the 93rd position Glu, the 112th position Ile, the 113th position His, the 115th position Asn, the 116th position Leu or the 117th position Leu in SEQ ID NO: 1 showing the region amino acid sequence A histone H2A mutant yeast cell, which is a mutant histone H2A in which an amino acid residue is replaced with an Ala residue.   A yeast cell having a polynucleotide construct that lacks the wild-type histone genes H2A and H2B and expresses the mutant-type histone H2A and the wild-type histone H2B, wherein the mutant-type histone H2A is the N-terminus of the wild-type histone H2A. A histone H2A mutant yeast cell which is a mutant histone H2A in which any one amino acid residue at position 93 Glu or position 94 Leu in SEQ ID NO: 1 showing the region amino acid sequence is substituted with an Ala residue.   A polynucleotide construct that is a yeast cell that lacks the wild-type histone genes H2A and H2B, does not express the LYS2 gene by inserting a δ sequence into the LYS2 gene promoter region, and expresses wild-type histone H2A and mutant histone H2B Wherein the mutant histone H2B is the first position Ser, the third position Lys, the fifth position Glu, and the sixth position in SEQ ID NO: 2, which shows the amino acid sequence of the N-terminal region of the wild-type histone H2B. Lys, 7th Lys, 8th Pro, 10th Ser, 90th Ser, 92nd Ile, 93rd Ser, 105th Leu, 106th Pro, 107th Gly, 108th Glu, 111th position Lys, 114th position Val, 117th position Gly, 118th position Thr, 121st position Val, 122th position Thr or 125th position Ser any one amino acid residue as Ala residue A histone H2B mutant yeast cell which is a substituted mutant histone H2B and has an SPT phenotypic change of 3 times or more compared to wild type yeast.   A yeast cell carrying a polynucleotide construct that lacks wild-type histone genes H2A and H2B and expresses wild-type histone H2A and mutant-type histone H2B, wherein the mutant-type histone H2B is the N-terminus of wild-type histone H2B. A histone H2B mutant yeast cell comprising a polynucleotide construct that expresses a mutant histone H2B in which position 123 Lys in SEQ ID NO: 2 showing the region amino acid sequence is substituted with an Ala residue.   4th position Lys, 5th position Gly, 26th position Phe, 29th position Gly, 46th position Ser, 47th position Gly, 66th position in SEQ ID NO: 1 showing the N-terminal region amino acid sequence of wild type histone H2A Leu, 90th Asn, 91st Asp, 92nd Asp, 94th Leu, 99th Gly, 103rd Ile, 107th Gly or 115th Asn A mutant histone H2A in which is replaced with an Ala residue.   65th Glu, 66th Leu, 73rd Asp, 74th Asn, 86th Leu, 93rd Glu, 94th position in SEQ ID NO: 1 showing the amino acid sequence of the N-terminal region of wild type histone H2A Leu, mutant histone H2A in which any one amino acid residue at position 103 Ile, position 112 Ile, position 113 His or position Leu is replaced with an Ala residue.   65th position Glu, 93rd position Glu, 112th position Ile, 113th position His, 115th position Asn, 116th position Leu or 117th position in SEQ ID NO: 1 showing the amino acid sequence of the N-terminal region of wild type histone H2A Mutant histone H2A in which any one amino acid residue of Leu is replaced with Ala residue.   Mutant histone H2A in which any one amino acid residue at position 93 Glu or position 94 Leu in SEQ ID NO: 1 showing the N-terminal region amino acid sequence of wild-type histone H2A is substituted with an Ala residue. 1st position Ser , 5th position Glu, 6th position Lys, 7th position Lys, 8th position Pro, 10th position Ser, 90th position in SEQ ID NO: 2 showing the N-terminal region amino acid sequence of wild type histone H2B Ser, 92nd Ile, 93rd Ser, 105th Leu, 106th Pro, 107th Gly, 108th Glu, 111th Lys, 114th Val, 117th Gly, 118th A mutant histone H2B in which any one amino acid residue of Thr, position 121 Val, position 122 Thr or position Ser is substituted with an Ala residue.   A mutant histone H2B in which the 123rd position Lys in SEQ ID NO: 2 showing the amino acid sequence of the N-terminal region of wild type histone H2B is substituted with an Ala residue.   A polynucleotide encoding the mutant histone H2A of claim 7.   A polynucleotide encoding the mutant histone H2A of claim 8.   A polynucleotide encoding the mutant histone H2A of claim 9.   A polynucleotide encoding the mutant histone H2A of claim 10.   A polynucleotide encoding the mutant histone H2B of claim 11.   A polynucleotide encoding the mutant histone H2B of claim 12. Of yeast cells RNA Transcription or DNA A method for enhancing sensitivity to an inhibitor of replication comprising the variant histone of claim 8 H2A Or introducing the polynucleotide of claim 14 into a cell. Of yeast cells RNA Transcription or DNA 13. A method for enhancing sensitivity to an inhibitor of replication comprising the variant histone of claim 12 H2B Or introducing the polynucleotide of claim 18 into a cell. A method for enhancing the sensitivity of a yeast cell to a mitotic inhibitor, comprising the mutant histone of claim 9 H2A Or a method comprising introducing the polynucleotide of claim 15 into a cell. A method for reducing the growth of yeast cells, comprising the mutant histone of claim 10 H2A Or introducing the polynucleotide of claim 16 into a cell. Of yeast cells RNA Transcription or DNA A method for searching for a factor affecting replication, comprising the step of introducing a candidate factor into the mutant yeast cell according to claim 2 or 6, wherein the candidate factor that changes the mortality of the mutant yeast cell is identified as a target factor A method characterized by: A method for screening a factor that affects cell division of a yeast cell, comprising the step of introducing the candidate factor into the mutant yeast cell of claim 3, wherein the candidate factor that changes the mortality of the mutant yeast cell is used as a target factor. A method characterized by identifying. A method for screening a factor that affects the growth of a yeast cell, comprising the step of introducing a candidate factor into the mutant yeast cell of claim 4, wherein the candidate factor that alters the growth of the mutant yeast cell is identified as a target factor. A method characterized by that. Wild type histone gene H2A and H2B Mutated histone H2A And wild-type histones H2B Yeast cells harboring a polynucleotide construct that expresses 112 A mutant histone of each yeast cell H2A But wild-type histones H2A of N SEQ ID NO: 1 showing the amino acid sequence of the end region 1 From the top 131 Place Ala Amino acid residues other than residues Ala Different mutant histones substituted with residues H2A Histone H2A A collection of mutant yeast cells. Wild type histone gene H2A and H2B A wild type histone H2A And mutant histones H2B Yeast cells harboring a polynucleotide construct that expresses 112 Set of And mutant histones in each yeast cell H2B But wild-type histones H2B of N In SEQ ID NO: 2 showing the amino acid sequence of the end region 1 First 130 Place Ala Amino acid residues other than residues Ala Different mutant histones substituted with residues H2B Histone H2B A collection of mutant yeast cells.

Images