JP3773460B2 - Histone H3 and H4 single amino acid mutant proteins and mutant cells thereof, and uses thereof - Google Patents

Description

【００７４】
実施例４
実施例２で作成したヒストンH4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および図５に示したとおりである。図４では、第35位ArgをAlaに置換した変異型ヒストンH4を発現するR35A変異酵母細胞が、また図５ではR36A変異酵母、S47A変異酵母、G48A変異酵母、L49A変異酵母がSPT表現型を示している。
【００７５】
作製した96種類のH4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、野生型に対するSPT表現型の倍率（−Hisプレートでの生育の倍率）は表２に示したとおりである。作成した９６種類のヒストンH4変異酵母細胞のうち、表２に示した14種類の変異型ヒストンH4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。
【００７６】
これら14種類のヒストンH4変異酵母細胞は、変異型ヒストンH4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H4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H4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。
【００７７】
また図５に示したG48A変異酵母は、配列番号２の2番目から53番目までの領域においてカンジダヒストンH4のアミノ酸配列と同一であり、このG48A変異酵母が野生型に比べて1000倍のSPT表現型を示したことから、カンジダ型のヒストンH4変異酵母（G48A）はゲノムDNAの細胞内高次構造または染色体のクロマチン構造が野生型（またその22-53アミノ酸領域はヒト型）とは異なっていることが確認された。従って、このカンジダ型のヒストンH4変異酵母（G48A）は、野生型の出芽酵母（そして恐らくはヒト）には作用せず、カンジダ菌のみに作用する因子を特定するために使用可能であることが確認された。
【００７８】
【表２】

【００７９】
実施例５
実施例１および２で作成したヒストンH3変異酵母細胞およびヒストンH4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。
【００８０】
具体的には、実施例１および２で作成したヒストンH3変異酵母細胞、ヒストンH4変異酵母細胞、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。
【００８１】
その結果、試験した全てのヒストンH3変異酵母細胞では6AUに対して有意に高い感受性を示した変異酵母株は得られなかった。一方、ヒストンH4変異酵母細胞の中には、6AUに対して高い感受性を示す変異酵母株が２株観察された。その結果の例を図６に示す。97位LeuをAla残基に置換した変異型ヒストンH4（L97A）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。
【００８２】
この図６に例示されるようなH4変異酵母細胞のアミノ酸置換部位と、その6AU感受性増強の程度は表３に示したとおりである。これらの変異型ヒストンH4を発現する酵母細胞が、野生型酵母細胞に比べ３倍以上の6AU感受性を示した。
【００８３】
これらのヒストンH4変異酵母細胞は、変異型ヒストンH4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H4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、それらの変異型ヒストンH4またはそれら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。
【００８４】
【表３】

【００８５】
実施例６
実施例１で作成したヒストンH3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。
【００８６】
具体的には、実施例１で作成したヒストンH3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DMSO含有培地上に塗布した。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は左がDMSO含有培地、中がTBZ含有培地、右がBenomyl含有培地における生育状況の一例である。この図７に示した例では、41位TyrをAlaに置換した変異型ヒストンH3（Y41A）、42位LysをAlaに置換した変異型ヒストンH3（K42A）を発現する変異酵母細胞が、TBZおよびBenomylに対する感受性を増強させている。
【００８７】
作製した119種類のH3変異酵母細胞のうち、TBZおよび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４（TBZ）および表５（Benomyl）に示したとおりである。作成した119種類のヒストンH3変異酵母細胞のうち、表４および表５に示した７種類の変異型ヒストンH3を発現する酵母細胞が、野生型酵母細胞に比べ、TBZおよびBenomylに対して少なくとも３倍以上の感受性を示した。
【００８８】
これら７種類のヒストンH3変異酵母細胞は、変異型ヒストンH3の発現によって、TBZおよび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およびBenomylは細胞分裂阻害剤であることから、これらのヒストンH3変異酵母細胞は、TBZおよび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H3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。
【００８９】
【表４】

【００９０】
【表５】

【００９１】
実施例７
実施例２で作成したヒストンH4変異酵母細胞を使用し、実施例６と同様にしてTBZおよびBenomylに対する各細胞の感受性を調べた。その結果、図８に示した変異酵母株（97位LeuがAlaに置換された変異型ヒストンH4(L97A)を発現する酵母株）および99位GlyがAlaに置換された変異型ヒストンH4（G99A）を発現する酵母株が、TBZおよびBenomylに対して、野生型酵母株よりも300倍以上の感受性を示した（表６、７）。
【００９２】
この実施例７に代表されるヒストンH4変異酵母細胞は、TBZおよび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、それらの変異型ヒストンH4またはそれら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。
【００９３】
なお、この実施例７に代表されるヒストンH4変異酵母細胞は、実施例５で得られた6AUに対して高い感受性を有する変異酵母株と同一であり、これらのヒストンH4変異酵母細胞は、作用の異なる２種類の薬剤（6AUと、TBZおよびBenomyl）に対して感受性が増強した変異酵母株である。
【００９４】
【表６】

【００９５】
【表７】

【００９６】
実施例８
実施例１で作成したヒストンH3変異酵母細胞を使用して、各細胞の生育の程度を調べた。
【００９７】
具体的には、実施例１で作成したヒストンH3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（5FOA培地）上に塗布した。このプレートを30度で48時間保温し、培地上での生育状況を観察した。
【００９８】
作成した１１９種類のヒストンH3変異酵母細胞のうち、表８に示した５種類の変異型ヒストンH3を発現する酵母細胞が、致死または野生型細胞株に比べて生育不良を示した。
【００９９】
これら５種類のヒストンH3変異酵母細胞は、変異型ヒストンH3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H3変異酵母細胞は、細胞の生育（細胞増殖）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H3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（細胞増殖）を抑制することが可能であることが確認された。
【０１００】
【表８】

【０１０１】
実施例９
実施例２で作成したヒストンH4変異酵母細胞を使用し、実施例８と同様にして生育（細胞増殖）の程度を調べた。生育状況の一例は図９に示したとおりである。この図９に示した例では、100位PheをAlaに置換した変異型ヒストン（F100A）を発現する酵母株が良好に生育するのに対し、99位GlyをAlaに置換した変異型ヒストン（G99A）を発現するヒストンH4変異酵母細胞は生育不良（細胞増殖不良）を示した。
【０１０２】
作成した96種類のヒストンH4変異酵母細胞のうち、表９に示した２種類の変異型ヒストンH4を発現する酵母細胞が、致死または野生型細胞株に比べて生育不良（細胞増殖不良）を示した。
【０１０３】
これら２種類のヒストンH4変異酵母細胞は、変異型ヒストンH4の発現によって、生育（細胞増殖）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H4変異酵母細胞は、細胞増殖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H4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、細胞の増殖を抑制することが可能であることが確認された。
【０１０４】
【表９】

【０１０５】
【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１アミノ酸変異（１アミノ酸置換）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、ヒストンH3および／またはヒストンH4が関係するゲノムDNAを鋳型として起こる各種反応（クロマチン構造の維持、遺伝子の転写、細胞分裂、細胞増殖等）について新たな形質を獲得した新しい酵母細胞株が提供される。
【０１０６】
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アミノ酸レベルでの作用点をスクリーニングする方法が提供される。
【０１０７】
【配列表】

【図面の簡単な説明】
【図１】ヒストンH3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。sceはパン酵母（出芽酵母）、humはヒト、aspは真菌アスペルギウス、calは真菌カンジダ、tcrはトリパノゾーマである。
【図２】ヒストンH4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３におけるヒストンH3変異酵母細胞の94時間培養後の生育状況である。左図はロイシンを含有しない培地、右図はロイシンとヒスチ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。細胞は、1:DY2864株（SPTを示さないコントロール）、2:DY2864株esal-L327S（SPTを示すコントロール）、3,4:H3/H4野生型プラスミド導入株（SPTを示さないコントロール）、5,6:S57A変異酵母、7,8:T58A変異酵母、9,10:E59A変異酵母、11,12:L60A変異酵母である。
【図４】実施例４におけるヒストンH4変異酵母細胞の64時間培養後の生育状況である。左図はロイシンを含有しない培地、右図はロイシンとヒスチ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。細胞は、1-4は図３と同一であり、5,6：K31A変異酵母、7,8:P32A変異酵母、9,10:R35A変異酵母である。
【図５】実施例４におけるヒストンH4変異酵母細胞の64時間培養後の生育状況である。左図はロイシンを含有しない培地、右図はロイシンとヒスチ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。細胞は、1-4は図３と同一であり、5,6：R36A変異酵母、7,8:S47A変異酵母、9,10:G48A変異酵母、11,12:L49A変異酵母である。
【図６】実施例５におけるヒストンH4変異酵母細胞の64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。細胞は、1:W303株（野生型酵母株；6AU感受性を示さないコントロール）、2:W303-cia（6AU感受性を示すコントロール）、3,4: H3/H4野生型プラスミド導入株（6AU感受性を示さないコントロール）、5,6:Q93A変異酵母、7,8:R95A変異酵母、9,10:T96A変異酵母、11,12:L97A変異酵母である。
【図７】実施例６におけるヒストンH3変異酵母細胞の64時間培養後の生育状況である。左からDMSO含有培地、DMSO+TBZ(25μl/ml)含有培地、DMSO+Benomyl(10μl/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。細胞は、1:W303株（野生型酵母株；コントロール）、2:WTH-1D株（TBZおよびBenomylに感受性を示すコントロール）、3,4: H3/H4野生型プラスミド導入株（TBZおよびBenomylに感受性を示さないコントロール）、5,6:R40A変異酵母、7,8:Y41A変異酵母、9,10:K42A変異酵母、11,12:P43A変異酵母である。
【図８】実施例７におけるヒストンH4変異酵母細胞の64時間培養後の生育状況である。左からDMSO含有培地、DMSO+TBZ(25μl/ml)含有培地、DMSO+Benomyl(10μl/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。細胞は、1-4は図７と同一、5,6:Q93A変異酵母、7,8:R95A変異酵母、9,10:T96A変異酵母、11,12:L97A変異酵母である。
【図９】実施例９におけるヒストンH4変異酵母細胞の5FOA培地上での48時間培養後の生育（細胞増殖）の状況である。12時および３時方向はG99A変異酵母、６時および９時方向はF100A変異酵母である。[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a mutant yeast cell in which a nucleotide polymorphism is generated in a histone gene region and its use. In more detail, the invention of this application expresses mutant histone H3 or histone H4 in which one specific amino acid residue is substituted with Ala residue, and these mutant proteins, and screens for various useful substances, etc. The present invention relates to a mutant yeast cell useful for.
[0002]
[Prior art]
Histones are common in the nucleus of eukaryotic cells, ranging from multicellular organisms including humans to unicellular organisms such as fungi (molds and yeasts), and ionically bind to genomic DNA. It is a basic protein. Histones usually consist of five components (H1, H2A, H2B, H3 and H4) and are highly similar across species. For example, in the case of histone H3, 93% of amino acids are found 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) The sequences are identical and the difference is only 8 residues. In the case of histone H4, the budding yeast histone H4 (full length 102 amino acid sequence) and human histone H4 (full length 102 amino acid sequence) are 92% identical in amino acid sequence, and the difference is only 8 residues. Among natural proteins assumed to exist in tens of thousands in one organism, histone H4 is known to be the most highly conserved protein among eukaryotic 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 also have histones like budding yeast, and the amino acid sequences of histones H3 and H4 are the amino acid sequences of budding yeast. Although high similarity is shown, some differences are also seen (FIGS. 1 and 2).
[0003]
Genomic DNA is folded by regular bonds with histones, and the complex of both forms 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 in a yeast cell (Saccharomyces cerevisiae). Yes. In addition, since histone is a control target for various reactions (gene expression, DNA replication, DNA repair, etc.) that occur using genomic DNA as a template, it may be a target for various compounds such as drugs in vivo. In fact, in recent years, inhibitors to enzymes that deacetylate histones have been shown to have anticancer activity and are positioned as important candidates for drug discovery (Current Opinion in Oncology 13: 477-483, 2001; Anti -cancer Drugs 13: 1-13, 2002). However, the conventional technique has a problem that it is difficult to specify which surface site (amino acid residue) of a histone protein a physiologically active substance acting on histones exerts pharmacological activity.
[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 an amino acid point mutation into histone, which is a protein 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]
Another object of the invention of this application is to provide a screening method for various useful substances using the mutant cells, and further a method for narrowing the action point of these useful substances against histones to the level of one amino acid residue.
[0009]
[Means for Solving the Problems]
In this application, as a first invention for solving the above-mentioned problems, wild type histone genes H3 and H4 are deleted, and a Ty sequence or a δ sequence is inserted into an essential gene promoter region of a yeast cell genome. A yeast cell carrying a polynucleotide construct that expresses mutant histone H3 and wild type histone H4, wherein the mutant histone H3 is the second position Arg in SEQ ID NO: 1 showing the amino acid sequence of wild type histone H3 , 3rd Thr, 4th Lys, 40th Arg, 41st Tyr, 42nd Lys, 44th Gly, 45th Thr, 46th Val, 49th Arg, 50th Glu , 52nd Arg, 53rd Arg, 54th Phe, 56th Lys, 59th Glu, 61st Leu, 64th Lys, 66th Pro, 69th Arg, 72nd Arg , 84th Phe, 86th Ser, 94th Glu, 105th Glu, 115th Lys, 117th Val, 120th Gln, 122nd Lys, 125th Lys, 126th Leu 129th Arg or 133rd Glu Provided is a histone H3 mutant yeast cell that is a mutant histone H3 in which any one amino acid residue is substituted with an Ala residue and exhibits an SPT phenotype.
[0010]
This application provides, as a second invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H3 and H4 and expresses the mutant-type histone H3 and the wild-type histone H4. H3 is the 5th position Gln, 41st position Tyr, 42nd position Lys, 44th position Gly, 45th position Thr, 49th position Arg or 52nd position in SEQ ID NO: 1 showing the amino acid sequence of wild type histone H3 A mutant histone H3 in which any one amino acid residue of Arg is replaced with an Ala residue. Wild type yeast against Thiabendazole (TBZ) and Methyl 1- (butylcarbamoyl) -2-benzimidazolecarbamate (Benomyl) A histone H3 mutant yeast cell exhibiting higher sensitivity is provided.
[0011]
This application provides, as a third invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H3 and H4 and expresses the mutant-type histone H3 and the wild-type histone H4. H3 is the amino acid residue of any one of position 48 Leu, position 51 Ile, position 55 Gln, position 112 Ile or position 113 His in SEQ ID NO: 1 showing the amino acid sequence of wild-type histone H3 Is a mutant histone H3 substituted with an Ala residue, and provides a histone H3 mutant yeast cell having a lower degree of growth than wild-type yeast.
[0012]
In this application, as a fourth invention, the wild type histone genes H3 and H4 are deleted, and the Ty sequence or δ sequence is inserted in the essential gene promoter region of the yeast cell genome. A yeast cell having a polynucleotide construct that expresses histone H4, wherein the mutant histone H4 is the amino acid sequence of wild-type histone H4 at position 25 Asn, position 32 Pro, and position 35. Arg, 36th Arg, 47th Ser, 48th Gly, 49th Leu, 51st Tyr, 52nd Glu, 53rd Glu, 55th Arg, 59th Lys, 78th Provided is a histone H4 mutant yeast cell exhibiting an SPT phenotype, which is a mutant histone H4 in which any one amino acid residue at position Arg or position 99 Gly is substituted with an Ala residue.
[0013]
This application provides, as a fifth invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H3 and H4 and expresses the wild-type histone H3 and the mutant histone H4, H4 is a mutant histone H4 in which the 97th position Leu or the 99th position Gly in SEQ ID NO: 2 showing the amino acid sequence of wild type histone H4 is substituted with an Ala residue, and against 6 azauracil (6AU) Provided is a histone H4 mutant yeast cell that exhibits higher sensitivity than wild-type yeast.
[0014]
This application provides, as a sixth invention, a yeast cell having a polynucleotide construct that lacks the wild-type histone genes H3 and H4 and expresses the wild-type histone H3 and the mutant histone H4. H4 is a mutant histone H4 in which amino acid sequence of wild type histone H4 in SEQ ID NO: 2 in which SEQ ID NO: 2 is substituted with Ala residue at position 97 Leu or 99, and Thiabendazole (TBZ) and Methyl 1- Provided is a histone H4 mutant yeast cell having higher sensitivity to (butylcarbamoyl) -2-benzimidazolecarbamate (Benomyl) than wild-type yeast.
[0015]
This application provides, as a seventh invention, a yeast cell having a polynucleotide construct that lacks wild-type histone genes H3 and H4 and expresses wild-type histone H3 and mutant-type histone H4, H4 is a mutant histone H4 in which any one amino acid residue at position 23 Arg or position 99 Gly in SEQ ID NO: 2 showing the amino acid sequence of wild-type histone H4 is substituted with an Ala residue. Provided is a histone H4 mutant yeast cell having a lower degree of growth than wild type yeast.
[0016]
As the eighth invention, this application provides, as the eighth invention, the second Arg, the third Thr, the fourth Lys, the 40th Arg, the 41st Tyr, the first amino acid in SEQ ID NO: 1 showing the amino acid sequence of wild type histone H3, 42nd Lys, 44th Gly, 45th Thr, 46th Val, 49th Arg, 50th Glu, 52nd Arg, 53rd Arg, 54th Phe, 56th Lys, No. 59th Glu, 61st Leu, 64th Lys, 66th Pro, 69th Arg, 72nd Arg, 84th Phe, 86th Ser, 94th Glu, 105th Glu, 115th position Lys, 117th position Val, 120th position Gln, 122nd position Lys, 125th position Lys, 126th position Leu, 129th position Arg or 133rd position Glu Ala residue Provided is a mutant histone H3 substituted with a group.
[0017]
As the ninth invention, this application is directed to the fifth position Gln, position 41 Tyr, position 42 Lys, position 44 Gly, position 45 Thr, position No. 1 in SEQ ID NO: 1 showing the amino acid sequence of wild type histone H3. A mutant histone H3 in which any one amino acid residue at position 49 Arg or position 52 Arg is substituted with an Ala residue is provided.
[0018]
In this application, as the tenth invention, any of 48th Leu, 51st Ile, 55th Gln, 112th Ile and 113th His in SEQ ID NO: 1 showing the amino acid sequence of wild type histone H3 Or a mutant histone H3 in which one amino acid residue is replaced with an Ala residue.
[0019]
As the eleventh aspect of the present application, this application is directed to the 25th position Asn, the 32nd position Pro, the 35th position Arg, the 36th position Arg, the 47th position Ser, the 48th Gly, 49th Leu, 51st Tyr, 52nd Glu, 53rd Glu, 55th Arg, 59th Lys, 78th Arg, or 99th Gly amino acid Mutant histone H4 is provided in which the residue is replaced with an Ala residue.
[0020]
This application provides, as a twelfth invention, mutant histone H4 in which the 97th Leu or 99th Gly in SEQ ID NO: 2 showing the amino acid sequence of wild type histone H4 is substituted with an Ala residue.
[0021]
In this application, as the thirteenth invention, any one of the 23rd Arg or 99th Gly amino acid residues in SEQ ID NO: 2 showing the amino acid sequence of wild-type histone H4 is substituted with an Ala residue. Provide mutant histone H4.
[0022]
This application provides, as a fourteenth invention, a polynucleotide encoding the mutant histone H3 of the eighth invention.
[0023]
This application provides, as the fifteenth invention, a polynucleotide encoding the mutant histone H3 of the ninth invention.
[0024]
This application provides, as the sixteenth invention, a polynucleotide encoding the mutant histone H3 of the tenth invention.
[0025]
This application provides, as the seventeenth invention, a polynucleotide encoding the mutant histone H4 of the eleventh invention.
[0026]
This application provides, as an eighteenth invention, a polynucleotide encoding the mutant histone H4 of the twelfth invention.
[0027]
This application provides, as a nineteenth invention, a polynucleotide encoding the mutant histone H4 of the thirteenth invention.
[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 H3 of the eighth invention or the polymorph of the fourteenth 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 affecting the chromosomal chromatin structure or gene expression form of yeast cells or non-yeast cells, wherein the mutant histone H4 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.
[0030]
This application is, as a twenty-second invention, a method for enhancing the sensitivity of a yeast cell or non-yeast cell to an inhibitor of RNA transcription or DNA replication, the mutant histone H4 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 H3 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 provides, as a twenty-fourth invention, a method for enhancing the sensitivity of a yeast cell or a non-yeast cell to a mitotic inhibitor, wherein the mutant histone H4 of the twelfth invention or the polynucleotide of the eighteenth invention is used. There is provided a method comprising the step of introducing into a cell.
[0033]
This application is, as a twenty-fifth invention, a method for reducing the growth of yeast cells or non-yeast cells, wherein the mutant histone H3 of the tenth invention or the polynucleotide of the sixteenth invention is introduced into a cell. A method characterized by comprising:
[0034]
This application is, as a twenty-sixth invention, a method for reducing the growth of yeast cells or non-yeast cells, the step of introducing the mutant histone H4 of the thirteenth invention or the polynucleotide of the nineteenth invention into a cell. A method characterized by comprising:
[0035]
This application is, as a twenty-seventh invention, a method for screening factors affecting the chromosomal chromatin structure or gene expression form of a yeast cell or a non-yeast cell, wherein the mutant yeast cell of the first invention or the fourth invention is used. A method for identifying a candidate factor that alters the SPT phenotype of a mutant yeast cell as a target factor.
[0036]
This application is, as a twenty-eighth invention, a method for searching factors affecting RNA transcription or DNA replication in yeast cells or non-yeast cells, comprising the step of introducing a candidate factor into the mutant yeast cell of the fifth invention. And a candidate factor that alters the mortality of mutant yeast cells is identified as a target factor.
[0037]
This application is, as a twenty-ninth invention, a method for screening a factor that affects cell division of a yeast cell or a non-yeast cell, wherein the candidate factor is introduced into the mutant yeast cell of the second invention or the sixth invention. A candidate factor that changes the mortality of mutant yeast cells is identified as a target factor.
[0038]
This application is, as a thirtieth invention, a method for screening factors affecting the growth of yeast cells or non-yeast cells, wherein a candidate factor is introduced into the mutant yeast cell of the third invention or the seventh invention. A method comprising identifying a candidate factor that alters the growth of a mutant yeast cell as a target factor is provided.
[0039]
This application, as a thirty-first invention, is a collection of yeast cells 119 having a polynucleotide construct that lacks wild-type histone genes H3 and H4 and expresses mutant-type histone H3 and wild-type histone H4, Mutant histone H3 of each yeast cell is different in that amino acid residues other than Ala residues at positions 1 to 135 in SEQ ID NO: 1 showing the amino acid sequence of wild type histone H3 are respectively replaced with Ala residues. Provided is an assembly of histone H3 mutant yeast cells that are mutant histone H3.
[0040]
This application, as a thirty-second invention, is a collection of yeast cells 96 carrying a polynucleotide construct that lacks wild-type histone genes H3 and H4 and expresses wild-type histone H3 and mutant histone H4, Mutant histone H4 of each yeast cell is different in that amino acid residues other than the 1st to 102nd Ala residues in SEQ ID NO: 2 showing the amino acid sequence of wild type histone H4 are respectively replaced with Ala residues. Provided is an assembly of histone H4 mutant yeast cells that are mutant histone H4.
[0041]
This application provides, as a thirty-third invention, a method for screening an action point of a factor that acts on histone H3 on histone H3, wherein each histone H3 mutant yeast cell constituting the yeast cell aggregate of the thirty-first 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 H3 expressed in yeast cells in which the action of the factor is changed is identified as the site of action of the factor.
[0042]
This application is, as a thirty-fourth invention, a method for screening an action point for histone H4 of a factor that acts on histone H4, wherein each histone H4 mutant yeast cell constituting the yeast cell aggregate of the thirty-second invention is provided. And a substitution amino acid site of mutant histone H4 expressed by a yeast cell in which the action of the factor is changed is identified as a site of action of the factor.
[0043]
This application is, as a thirty-fifth invention, a method for screening a factor that affects chromosomal chromatin structure, gene expression form or cell proliferation of Candida, which does not act on human and yeast cells, and comprises a wild-type histone gene H3 and H4 are deleted, and Ty sequence or δ sequence is inserted in the essential gene promoter region of the yeast cell genome. The wild type histone H3 and the 48th position Gly in SEQ ID NO: 2 are replaced with Ala residues. A candidate factor that alters the SPT phenotype and cell growth activity of the mutant yeast cell, including the step of introducing the candidate factor into a histone H4 mutant yeast cell having a polynucleotide construct that expresses the mutant histone H4 A method characterized by identifying as follows.
[0044]
Hereinafter, embodiments of the present invention will be described in detail.
[0045]
DETAILED DESCRIPTION OF THE INVENTION
The mutant yeast cells of the first to fourth inventions are deficient in the original wild type histone H3 gene and histone H4 gene, respectively, and one amino acid residue of either histone H3 or histone H4 is an alanine residue. And possessing a polynucleotide construct (expression vector) that expresses the mutant histone H3 or histone H4 substituted in the above.
[0046]
Yeast cells deficient in wild-type histone H3 gene and histone H4 gene (hereinafter referred to as “H3 / H4-deficient yeast cells”, where “/” means “and”) are known target gene replacements. It can also be created by gene knockout using the method, but preferably, the genotype of MATa Δ (HHT1 HHF1) Δ (HHT2 HHF2) his4-912δ lys2-128δ leu2-3,112 ura3-52 pMS329 [URA3 HHT1 HHF1] Saccharomyces cerevisiae strain MSY748 (Santisteban MS et al., EMBO Journal 16: 2493-2506, 1997) can be used. This MSY748 strain lacks two types of wild-type histone H3 genes and two types of wild-type histone H4 genes in the genome, and possesses one histone H3 gene and one histone H4 gene, and pSAB6 plasmid carrying the URA3 gene. (Hereinafter referred to as “H3 / H4 wild-type plasmid”. In this description, “/” means “and”).
[0047]
A construct obtained by cloning a histone H3 protein containing an alanine point mutation or a polynucleotide encoding a histone H4 protein into such an H3 / H4-deficient yeast cell (specifically, a plasmid. Hereinafter, an “H3-H4 mutant plasmid”) In this description, “-” means “or”). The H3-H4 mutant plasmid carries the LEU2 gene. Since the MSY748 strain loses the function of the LEU2 gene, it cannot grow in a medium that does not contain leucine, but it can grow by possessing this H3-H4 mutant plasmid. The H3- / H4-deficient yeast strain into which this H3-H4 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 H3 / H4 wild type plasmid has the URA3 gene, so as long as the H3 / H4 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 the parent cell, but are not distributed to one daughter cell at a low frequency and drop off from the host cell. 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. B strain is deficient in the histone H3 gene and histone H4 gene in the genome and carries the H3-H4 mutant plasmid. That is, the B strain is established as a budding yeast strain in which only one specific amino acid of histone H3 or histone H4 is selectively substituted with alanine.
[0048]
A method of substituting one amino acid residue of histone H3 protein or H4 protein with an alanine residue is based on the Kunkel method (Kunkel, TA Proc. Natl. Acad. Sci. USA 82) based on hybridization of oligonucleotide and single-stranded DNA. : 488, 1985 and Kunkel, TA, et al. Methods in Enzymology 154: 367, 1987). Ie dut ^- , Ung ^- Escherichia coli (BW313, CJ236, etc.) represented by this genotype is deficient in dUTPase (Dut) and Uracil-DNA glycosylase (Ung). Therefore, DNA is synthesized in which part of thiamine (T) in the DNA is replaced by deoxyuracil (dU). Using this Escherichia coli as a host fungus, a single-stranded DNA of a plasmid into which a histone H3 or H4 gene targeted for mutation introduction has been cloned is prepared. The histone H3 gene and histone H4 gene can be obtained, for example, by screening yeast cDNA using an oligonucleotide probe synthesized based on known sequence information (H3: GenBank Accession No. Z35879; H4: GenBank Accession No. Z35878, etc.) It can be obtained by PCR amplification using oligonucleotide primers. Moreover, the well-known well-known clone can also be utilized.
[0049]
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, the mutation-introducing plasmid obtained by cloning the gene encoding the histone H3 protein or histone H4 protein containing the alanine point mutation can be obtained by selectively amplifying the mutation-introduced DNA strand.
[0050]
In addition, the mutant yeast cells of the first and fourth 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, for example, by the his4-912δ genotype. The budding yeast strain MSY748 also has this his4-912δ genotype. This means that the δ sequence has been inserted into the promoter region of the HIS4 gene in the genome and expression of the HIS4 gene no longer occurs. Such a strain cannot grow unless it contains histidine on the medium. However, when a quantitative or qualitative change in the expression of a gene occurs, a strain having the his4-912δ genotype can grow on a medium not containing histidine. 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 fourth inventions grow on a medium not containing histidine, it was confirmed that the mutant yeast cells showed 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 fourth inventions are useful as a system for examining how the specific amino acid point mutations of histones H3 and H4 affect chromosomal chromatin structure and gene expression form, respectively. . It is also useful as a system for searching for factors that affect the chromosomal chromatin structure and gene expression form (the 27th invention). Furthermore, mutant H3 protein and mutant H4 protein (8th invention, 11th invention) possessed by these mutant yeast cells, or polynucleotides encoding these mutant proteins (14th invention and 17th invention) It can be used to change the chromosomal chromatin enzyme or gene expression form of yeast or non-yeast cells (20th invention, 21st invention).
[0051]
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).
[0052]
On the other hand, the mutant yeast cells of the second to third inventions and the fifth to seventh inventions are yeast cells that exhibit phenotypes different from wild-type yeast cells, respectively, under specific conditions.
[0053]
The mutant yeast cell of the fifth invention was identified as a cell line 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 28th invention). A mutant H4 protein possessed by the mutant yeast cell (the 12th invention) or a polynucleotide encoding the mutant protein (the 18th invention) is used for an inhibitor of RNA transcription or DNA replication in yeast or non-yeast cells. It can be used to enhance sensitivity (22nd invention).
[0054]
The mutant yeast cells of the second and sixth inventions were identified as cell lines 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 affecting cell division, they are useful as a system for searching for these factors (the 29th invention). Furthermore, mutant H3 protein (9th invention) and mutant H4 protein (12th invention) possessed by these mutant yeast cells, or polynucleotides encoding these mutant proteins (15th invention, 18th invention) are: It can be used to enhance the sensitivity of yeast or non-yeast cells to inhibitors of cell division (23rd invention, 24th invention).
[0055]
The mutant yeast cells of the third and seventh inventions have been identified as cell lines that have a lower degree of growth than the wild type. Therefore, 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 30th invention). Furthermore, the mutant H3 protein (the 10th invention) and the mutant H4 protein (the 13th invention) possessed by these mutant yeast cells, or the polynucleotides encoding these mutant proteins (the 16th and 19th inventions) It can be used to reduce the growth of yeast or non-yeast cells (25th invention, 26th invention).
[0056]
The eighth to thirteenth inventions are a mutant histone H3 protein and a mutant histone H4 protein 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 (14th to 19th 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.).
[0057]
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.
[0058]
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.
[0059]
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.
[0060]
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.
[0061]
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.
[0062]
The mutant histones H3 and H4 obtained as described above and the mutant polynucleotide encoding them can be used in the twentieth to twenty-sixth 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.
[0063]
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.
[0064]
The thirty-first and thirty-second inventions are a collection of mutant yeast cells that express mutant histone H3 protein or mutant histone H4. Since these yeast cell populations express mutant proteins each having a different amino acid point mutation, factors acting on histone H3 or histone H4 (known factors or those identified in the aforementioned 27-30 inventions) It is useful as a system (33rd invention, 34th 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.
[0065]
A thirty-fifth aspect of the present invention is a method for screening a factor that does not act on human and yeast cells but affects Candida chromosomal chromatin structure, gene expression form or cell proliferation. In the thirty-fifth invention, a histone H4 mutant yeast cell that expresses a mutant histone H4 (G48A) in which the 48th Gly of SEQ ID NO: 2 is substituted with an Ala residue is used. That is, as shown in FIG. 2, the sequence of the 32nd to 53rd amino acids of histone H4 of wild-type budding yeast is identical to the amino acid sequence of the corresponding region of human histone H4. Therefore, the 32nd to 53rd 32 amino acid sequence region of wild-type budding yeast histone H4 (SEQ ID NO: 2) is not only budding yeast type but also human type. On the other hand, mutant histone H4 (G48A) in which Gly at position 48 is substituted with Ala is identical to the amino acid sequence of Candida histone H4 in the 2nd to 53rd regions. There is no Candida type. And this histone H4 mutant yeast (G48A) showed the SPT phenotype 1000 times compared with the wild type, as shown in Example 4 and Table 2. This means that the Candida histone H4 mutant yeast (G48A) has a different intracellular higher order structure of genomic DNA or chromosomal chromatin structure compared to human budding yeast (wild type strain) at least in the 22-53 region. It shows that. Therefore, by introducing candidate factors into this Candida-type histone H4 mutant yeast (G48A) and examining its SPT phenotype and cell growth activity, it does not act on humans and budding yeast, but only on Candida. Factors (eg killing Candida) can be identified.
[0066]
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.
[0067]
【Example】
Example 1
Has a polynucleotide construct that lacks wild-type histone genes H3 and H4, has a δ sequence inserted into the essential gene promoter region of the yeast cell genome, and expresses mutant histone H3 protein and wild-type histone H4 Histone H3 mutant yeast cells were prepared. The mutant histone H3 protein is a mutant histone H3 protein in which each amino acid residue of SEQ ID NO: 1 showing the amino acid sequence of the wild type histone H3 protein is substituted with an Ala residue.
[0068]
Specifically, according to the Kunkel method, a plasmid having a polynucleotide encoding a mutant histone H3 and a wild type histone H4 in which amino acid residues other than the alanine residue of SEQ ID NO. The cells were introduced into the MSY748 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 H3 / H4 wild type plasmid has the URA3 gene, so as long as the H3 / H4 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 the parent cell, but are not distributed to one daughter cell at a low frequency and drop off from the host cell. 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 H3 gene and histone H4 gene in the genome and possesses a plasmid in which a gene encoding a histone H3 protein containing an alanine point mutation and a wild type histone H4 protein is cloned.
[0069]
As described above, 119 histone H3 mutant yeast cells in which only one specific amino acid of histone H3 was selectively substituted with alanine were established.
Example 2
A histone having a polynucleotide construct that lacks the wild-type histone genes H3 and H4, has a δ sequence inserted into the essential gene promoter region of the yeast cell genome, and expresses wild-type histone H3 and mutant histone H4 H4 mutant yeast cells were created. Mutant histone H4 was prepared in the same manner as in Example 1 except that each amino acid residue other than the alanine residue of SEQ ID NO: 2 showing the amino acid sequence of wild type histone H4 was replaced with an alanine residue. .
Example 3
Using the histone H3 mutant yeast cells prepared in Example 1, changes in the SPT phenotype were examined.
[0070]
Specifically, the histone H3 mutant yeast cells and wild type budding yeast prepared in Example 1 are grown on a medium, respectively, and the strains from which the medium components have been removed by centrifugation and washing with water each contain histidine. It was spread on medium and medium without histidine. 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 H3 mutant yeast in which the 59th position of Glu is replaced with Ala (E59A) grows well even in a medium not containing histidine (right figure), indicating an SPT phenotype.
[0071]
Table 1 shows the amino acid substitution sites of the mutant yeast showing the SPT phenotype and the magnification of the SPT phenotype relative to the wild type (magnification of growth on the -His plate) among the 119 types of H3 mutant yeast cells produced. It is as follows. Among the prepared 119 histone H3 mutant yeast cells, the yeast cells expressing the 33 mutant histone H3 shown in Table 1 showed at least a three-fold change in SPT phenotype compared to wild-type yeast cells. showed that.
[0072]
These 33 types of histone H3 mutant yeast cells were confirmed to have altered gene expression by changing the nucleosome and chromosomal chromatin structure by the expression of mutant histone H3. Therefore, it was confirmed that by using these histone H3 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 the cells can be modified by introducing each mutant histone H3 or a polynucleotide encoding them individually into the cells.
[0073]
[Table 1]

[0074]
Example 4
Using the histone H4 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. 4 and FIG. In FIG. 4, R35A mutant yeast cells expressing mutant histone H4 in which Arg at position 35 is substituted with Ala, and in FIG. 5, R36A mutant yeast, S47A mutant yeast, G48A mutant yeast, and L49A mutant yeast have SPT phenotypes. Show.
[0075]
Table 96 shows the amino acid substitution site of the mutant yeast showing the SPT phenotype and the magnification of the SPT phenotype relative to the wild type (magnification of growth on the -His plate) among the 96 types of H4 mutant yeast cells produced. It is as follows. Among the 96 types of histone H4 mutant yeast cells that were prepared, the 14 types of mutant histone H4 yeast cells shown in Table 2 showed an SPT phenotype change that was at least 3 times greater than that of wild-type yeast cells. showed that.
[0076]
These 14 types of histone H4 mutant yeast cells were confirmed to have altered gene expression by changing the nucleosome and chromosomal chromatin structure by the expression of mutant histone H4. Therefore, it was confirmed that by using these histone H4 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 H4 or a polynucleotide encoding them individually into the cells.
[0077]
In addition, the G48A mutant yeast shown in FIG. 5 is identical to the amino acid sequence of Candida histone H4 in the 2nd to 53rd region of SEQ ID NO: 2, and this G48A mutant yeast has an SPT expression 1000 times that of the wild type. The Candida type histone H4 mutant yeast (G48A) is different from the wild type (and its 22-53 amino acid region is human type) in the intracellular higher order structure of genomic DNA or the chromatin structure of chromosome. It was confirmed that Therefore, this Candida-type histone H4 mutant yeast (G48A) does not act on wild-type budding yeast (and probably humans), but can be used to identify factors that act only on Candida. It was done.
[0078]
[Table 2]

[0079]
Example 5
Using the histone H3 mutant yeast cells and histone H4 mutant yeast cells prepared in Examples 1 and 2, the sensitivity of each cell to 6AU was examined.
[0080]
Specifically, the histone H3 mutant yeast cells, histone H4 mutant yeast cells, and wild-type yeast cells prepared in Examples 1 and 2 are grown on a medium, and the medium components are removed by centrifugation and washing with water. The strains were spread on a medium containing 6 AU at a concentration of 1 mg / ml and a medium containing no 6 AU. 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.
[0081]
As a result, none of the tested histone H3 mutant yeast cells yielded mutant yeast strains that showed significantly higher sensitivity to 6AU. On the other hand, in the histone H4 mutant yeast cells, two mutant yeast strains showing high sensitivity to 6AU were observed. An example of the result is shown in FIG. Mutant yeast cells expressing mutant histone H4 (L97A) in which the Leu at position 97 was replaced with an Ala residue showed significantly higher sensitivity to 6AU.
[0082]
The amino acid substitution sites of the H4 mutant yeast cells as exemplified in FIG. 6 and the degree of 6AU sensitivity enhancement are as shown in Table 3. Yeast cells expressing these mutant histone H4 showed 6 AU sensitivity more than 3 times that of wild type yeast cells.
[0083]
These histone H4 mutant yeast cells were confirmed to have enhanced sensitivity to 6AU due to the expression of mutant histone H4. Since 6AU is an inhibitor of cellular RNA transcription and DNA replication, it was confirmed that these histone H4 mutant yeast cells are useful as a system to search 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 these mutant histone H4s or polynucleotides encoding them into cells.
[0084]
[Table 3]

[0085]
Example 6
The histone H3 mutant yeast cells prepared in Example 1 were used to examine the sensitivity of each cell to TBZ or Benomyl.
[0086]
Specifically, the histone H3 mutant yeast cells and wild-type budding yeast prepared in Example 1 were grown on a medium, respectively, 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 DMSO-containing medium without 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 status in a DMSO-containing medium on the left, a TBZ-containing medium in the middle, and a Benomyl-containing medium on the right. In the example shown in FIG. 7, mutant yeast cells expressing mutant histone H3 (Y41A) in which position 41 Tyr is substituted with Ala and mutant histone H3 (K42A) in which position 42 Lys is replaced with Ala are expressed as TBZ and Increases sensitivity to Benomyl.
[0087]
Among the 119 types of H3 mutant yeast cells produced, the amino acid substitution sites and the degree of sensitivity enhancement of the mutant yeast with enhanced sensitivity to TBZ and Benomyl are shown in Table 4 (TBZ) and Table 5 (Benomyl). It is. Among the 119 types of histone H3 mutant yeast cells prepared, the yeast cells expressing the seven types of mutant histone H3 shown in Tables 4 and 5 were at least 3 compared to wild-type yeast cells against TBZ and Benomyl. It was more than twice as sensitive.
[0088]
These seven types of histone H3 mutant yeast cells were confirmed to have enhanced sensitivity to TBZ and Benomyl by expression of mutant histone H3. Since TBZ and Benomyl are cell division inhibitors, these histone H3 mutant yeast cells may be useful as a system for highly sensitive search for cell division inhibitors that have similar effects to TBZ and Benomyl. confirmed. It was also confirmed that the sensitivity to mitosis inhibitors could be enhanced by introducing each mutant histone H3 or a polynucleotide encoding them individually into cells.
[0089]
[Table 4]

[0090]
[Table 5]

[0091]
Example 7
Using the histone H4 mutant yeast cells prepared in Example 2, the sensitivity of each cell to TBZ and Benomyl was examined in the same manner as in Example 6. As a result, the mutant yeast strain shown in FIG. 8 (a yeast strain expressing a mutant histone H4 (L97A) in which the 97th position Leu was replaced with Ala) and a mutant histone H4 in which the 99th position Gly was replaced with Ala (G99A). ) Expressed 300 times more sensitive to TBZ and Benomyl than wild-type yeast strains (Tables 6 and 7).
[0092]
It was confirmed that the histone H4 mutant yeast cells represented by this Example 7 are useful as a system for searching for a cell division inhibitor having the same action as TBZ and Benomyl with high sensitivity. In addition, it was confirmed that the sensitivity to mitosis inhibitors can be enhanced by introducing the mutant histone H4 or the polynucleotide encoding them into cells.
[0093]
The histone H4 mutant yeast cells represented by this Example 7 are the same as the mutant yeast strain having high sensitivity to 6AU obtained in Example 5, and these histone H4 mutant yeast cells are effective. Mutant yeast strains with enhanced sensitivity to two different drugs (6AU, TBZ and Benomyl).
[0094]
[Table 6]

[0095]
[Table 7]

[0096]
Example 8
Using the histone H3 mutant yeast cells prepared in Example 1, the degree of growth of each cell was examined.
[0097]
Specifically, histone H3 mutant yeast cells and wild-type yeast cells prepared in Example 1 can be grown on a medium, and normal yeast can grow on a strain from which medium components have been removed by centrifugation and washing with water. It was applied on a nutrient rich agar medium (5FOA medium). This plate was incubated at 30 degrees for 48 hours, and the growth state on the medium was observed.
[0098]
Among the prepared 119 types of histone H3 mutant yeast cells, the yeast cells expressing the 5 types of mutant histone H3 shown in Table 8 showed a growth failure compared to lethal or wild type cell lines.
[0099]
These five types of histone H3 mutant yeast cells were confirmed to significantly reduce the degree of growth by the expression of mutant histone H3. Therefore, it was confirmed that these histone H3 mutant yeast cells are useful as a system for searching for factors that affect cell growth (cell proliferation) with high sensitivity. In addition, it was confirmed that cell growth (cell proliferation) can be suppressed by introducing each mutant histone H3 or a polynucleotide encoding them individually into cells.
[0100]
[Table 8]

[0101]
Example 9
Using the histone H4 mutant yeast cells prepared in Example 2, the degree of growth (cell proliferation) was examined in the same manner as in Example 8. An example of the growth situation is as shown in FIG. In the example shown in FIG. 9, a yeast strain expressing a mutant histone (F100A) in which the Phe at position 100 is substituted with Ala grows well, whereas a mutant histone (G99A in which the Gly at position 99 is substituted with Ala). ) Expressed histone H4 mutant yeast cells showed poor growth (cell growth failure).
[0102]
Among the 96 types of histone H4 mutant yeast cells that were prepared, the yeast cells that express the two types of mutant histone H4 shown in Table 9 showed poor growth (cell growth failure) compared to lethal or wild type cell lines. It was.
[0103]
It was confirmed that these two types of histone H4 mutant yeast cells significantly reduced the degree of growth (cell proliferation) due to the expression of mutant histone H4. Therefore, it was confirmed that these histone H4 mutant yeast cells are useful as a system for searching for factors that affect cell proliferation with high sensitivity. It was also confirmed that cell proliferation could be suppressed by introducing each mutant histone H4 or a polynucleotide encoding them individually into cells.
[0104]
[Table 9]

[0105]
【The invention's effect】
As described above in detail, according to the present invention, a single amino acid mutation (one amino acid substitution) is introduced into histone, which is one component of a chromosome, so that the gene encoded by genomic DNA and its transcript are not directly manipulated. New yeast cell lines that have acquired new traits for various reactions (maintaining chromatin structure, gene transcription, cell division, cell proliferation, etc.) that occur using genomic DNA related to histone H3 and / or histone H4 as a template are provided. The
[0106]
In addition, the invention of this application 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 points of action of these useful substances on the histones at the amino acid level. Is done.
[0107]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an amino acid sequence diagram showing sequence homology among species of histone H3. “.” 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. sce is baker's yeast (budding yeast), hum is human, asp is fungal aspergius, cal is fungal candida, and tcr is trypanosome.
FIG. 2 is an amino acid sequence diagram showing the sequence homology between histone H4 species as in FIG.
FIG. 3 shows the growth status of the histone H3 mutant yeast cells in Example 3 after culturing for 94 hours. The left figure shows the growth of yeast cells in a medium without leucine, and the right figure shows the growth of yeast cells in a medium without leucine and histidine. 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. Cells are 1: DY2864 strain (control not showing SPT), 2: DY2864 strain esal-L327S (control showing SPT), 3,4: H3 / H4 wild type plasmid introduced strain (control not showing SPT), 5 , 6: S57A mutant yeast, 7,8: T58A mutant yeast, 9,10: E59A mutant yeast, 11,12: L60A mutant yeast.
FIG. 4 shows the growth status of the histone H4 mutant yeast cells in Example 4 after culturing for 64 hours. The left figure shows the growth of yeast cells in a medium without leucine, and the right figure shows the growth of yeast cells in a medium without leucine and histidine. The number of cells (A to H) is the same as FIG. The cells 1-4 are the same as those in FIG. 3, and are 5,6: K31A mutant yeast, 7,8: P32A mutant yeast, and 9,10: R35A mutant yeast.
FIG. 5 shows the growth status of the histone H4 mutant yeast cells in Example 4 after culturing for 64 hours. The left figure shows the growth of yeast cells in a medium without leucine, and the right figure shows the growth of yeast cells in a medium without leucine and histidine. The number of cells (A to H) is the same as FIG. The cells 1-4 are the same as those in FIG. 3, and are 5,6: R36A mutant yeast, 7,8: S47A mutant yeast, 9,10: G48A mutant yeast, and 11,12: L49A mutant yeast.
FIG. 6 shows the growth of the histone H4 mutant yeast cells in Example 5 after culturing for 64 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. Cells are 1: W303 strain (wild type yeast strain; control not showing 6AU sensitivity), 2: W303-cia (control showing 6AU sensitivity), 3,4: H3 / H4 wild type plasmid introduced strain (6AU sensitivity Controls not shown), 5,6: Q93A mutant yeast, 7,8: R95A mutant yeast, 9,10: T96A mutant yeast, 11,12: L97A mutant yeast.
FIG. 7 shows the growth status of the histone H3 mutant yeast cells in Example 6 after culturing for 64 hours. From left, yeast cells are grown in a DMSO-containing medium, a DMSO + TBZ (25 μl / ml) -containing medium, and a DMSO + Benomyl (10 μl / ml) -containing medium. The number of cells (A to H) is the same as FIG. Cells are 1: W303 strain (wild type yeast strain; control), 2: WTH-1D strain (control sensitive to TBZ and Benomyl), 3,4: H3 / H4 wild type plasmid introduced strain (TBZ and Benomyl) Controls not showing sensitivity), 5,6: R40A mutant yeast, 7,8: Y41A mutant yeast, 9,10: K42A mutant yeast, 11,12: P43A mutant yeast.
FIG. 8 shows the growth status of the histone H4 mutant yeast cells in Example 7 after culturing for 64 hours. From left, yeast cells are grown in a DMSO-containing medium, a DMSO + TBZ (25 μl / ml) -containing medium, and a DMSO + Benomyl (10 μl / ml) -containing medium. The number of cells (A to H) is the same as FIG. The cells 1-4 are the same as in FIG. 7, and are 5,6: Q93A mutant yeast, 7,8: R95A mutant yeast, 9,10: T96A mutant yeast, and 11,12: L97A mutant yeast.
FIG. 9 shows the state of growth (cell proliferation) of the histone H4 mutant yeast cells in Example 9 after culturing on 5FOA medium for 48 hours. The 12 o'clock and 3 o'clock directions are G99A mutant yeast, and the 6 o'clock and 9 o'clock directions are F100A mutant yeast.

Claims (6)

  A yeast cell having a polynucleotide construct that lacks wild-type histone genes H3 and H4 and expresses wild-type histone H3 and mutant-type histone H4, wherein the mutant-type histone H4 is an amino acid sequence of wild-type histone H4. Is a mutant histone H4 in which the 97th Leu or 99th Gly in SEQ ID NO: 2 is replaced with an Ala residue, and is more than 3 times more sensitive to 6 azauracil (6AU) than wild type yeast Showing histone H4 mutant yeast cells.   A mutant histone H4 in which the 97th Leu or 99th Gly in SEQ ID NO: 2 showing the amino acid sequence of wild type histone H4 is substituted with an Ala residue.   A polynucleotide encoding the mutant histone H4 of claim 2. A method for enhancing the sensitivity of a yeast cell to an inhibitor of RNA transcription or DNA replication, comprising the step of introducing the mutant histone H4 of claim 2 or the polynucleotide of claim 3 into the cell. Method. A method for enhancing the sensitivity of a yeast cell to a mitotic inhibitor comprising the step of introducing the mutant histone H4 of claim 2 or the polynucleotide of claim 3 into a cell. A method of searching for factors that affect RNA transcription or DNA replication of yeast cells comprises introducing the candidate agent to the mutant yeast cell of claim 1, object candidate agent to alter the mortality mutant yeast cells A method characterized by identifying as a factor.

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