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Isolation and characterization of transcription factors of ribosoma DNA in lower eukaryotes

低等真核生物核糖体 DNA 转录因子的分离和表征

基本信息

批准号：
05680597
负责人：
NOGI Yasuhisa
金额：
$ 1.34万
依托单位：
Sitama Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680597/
关键词：
Budding yeast Fission yeast Transcription factor Ribosomal DNA

项目摘要

1.We have cloned three genes, RRN3, RRN6, and RRN7 involved in the transcription of 35S rRNA genes by RNA polymerase I in Saccharomyces cerevisiae, From deduced amino acid sequences, RRN3 appcars to encode a protein of 627 amino acids, RRN6 to encode a protein of 894 amino acids, and RRN7 to encode a protein of 514 amino acids. Standard gene disruption experiments showed that RRN3, RRN6, and RRN7 are essential genes. By use of immunoaffinity purification and biochemical fractionation, we obtained a highly purified preparation (Rrn6/Rrn7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparet molecular weight of 66,000. In vitro transcription experiments showed the Rrn6/Rrn7 complex is required for the formation of a transcription -competent preinitiation complex. The role of Rrn3p in the transcription process ia still unclear. However, a overproduction of Rrn3p in S.cerevisiae inhibits cell growth, suggesting that Rrn3p traps some essential protein.2.Schizosaccha … More romyces pombe cDNA bank in S.cerevisiae expression vector and Kluveroyces lactis genomic DNA bank, respectively, were used to complement S.cerevisiae rrn3, rrn6, and rrn7 mutants to isolate functional homologues. We have found SRN31 and SRN32 obtained from S.pombe cDNA bank can complement temperature-sensitive rrn3 mutation, determined the complete nucleotide sequence of SRN31 and found that SRN31 encodes a protein of 434 amino acids with a calculated molecular weight of 50,200. There is no apparent homology between Rrn3p and Srn31p. SRN33 derived from K.lactic can also complement temperature-sensitive rrn3. We have not analyzed SRN32 and SRN33 yet.3.We attempted to transcribe the functional 35S ribosomalRNA from RNA polymerase II promoter in S.pombe, We fused nmt1 promoter to one unit of rDNA to construct a nmt1-35S rDNA fusion gene. The fusion gene on a multicopy vector was transformed into a temperature-sensitive RNA polymerase I mutant (nuc1) of S.pombe. However, the growth of transformants was not observed at a high temperature, indicating that the amount rRNA supplied from nmt1 promoter is not enough to complement nuc1 mutation. Less

1.我们克隆了酿酒酵母菌35S rRNA基因被RNA聚合酶I转录的三个基因RRN3、RRN6和RRN7，从推导的氨基酸序列来看，RRN3适用于编码627个氨基酸的蛋白，RRN6适用于编码894个氨基酸的蛋白，RRN7适用于编码514个氨基酸的蛋白。标准基因破坏实验表明，RRN3、RRN6和RRN7是必需基因。通过免疫亲和纯化和生化分离，我们获得了由Rrn6p、Rrn7p和另一种表观分子量为66,000的蛋白组成的高纯度制剂（Rrn6/Rrn7复合物）。体外转录实验表明，rn6/ rn7复合体是转录起始前复合体形成所必需的。rn3p在转录过程中的作用尚不清楚。然而，酿酒酵母中过量产生的Rrn3p抑制了细胞生长，这表明Rrn3p捕获了一些必需的蛋白质。利用酿酒酵母表达载体中的Schizosaccha . More romyces pombe cDNA库和klluveroyces lactis基因组DNA库，分别对酿酒酵母rn3、rn6和rn7突变体进行互补，分离出功能同源物。我们发现从S.pombe cDNA库中获得的SRN31和SRN32可以对温度敏感的rrn3突变进行互补，确定了SRN31的完整核苷酸序列，发现SRN31编码一个434个氨基酸的蛋白，计算分子量为50,200。Rrn3p和Srn31p之间没有明显的同源性。来源于k.l乳酸的SRN33也可以补充温度敏感的rrn3。我们还没有分析SRN32和SRN33。我们尝试从S.pombe的RNA聚合酶II启动子中转录35S核糖体RNA，并将nmt1启动子与一个rDNA单元融合，构建了nmt1-35S rDNA融合基因。将多拷贝载体上的融合基因转化为S.pombe的温度敏感RNA聚合酶I突变体（nuc1）。然而，在高温下没有观察到转化子的生长，这表明nmt1启动子提供的rRNA数量不足以补充nuc1突变。少