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Studies on the factors affecting gene expression in yeast.

酵母基因表达影响因素的研究。

基本信息

批准号：
61580225
负责人：
KIKUCHI Yoshiko
金额：
$ 1.02万
依托单位：
Toho University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

Many factors should be involved in gene expression in yeast. In this project, we focused on the effiect of the stability of vectore. We have isolated two types of nuclear mutants of Saccharomyces cerevisiae, which affect the maintenance of plasmid and mini-chromosomes.1. map1; We have isolated host mutants in which the 2<micro>m plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1. Minichromosomes carrying chromosomal replication oringin and a centromere were also affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS ot a set of 2<micro>m inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.2. gst1; A new temperature-sensitive mutant, gst1 was isolated. At non-permissive temperature the mutant cells with large buds accumulated and DNA synthesis was substantially arrested. From the reciprocal experiment of temperature-shift ands mating factor treatment, it was shown that the execution point was post'START'. This suggested that the mutation affected the G1-to-S phase transition in the cell cycle. A DNA clone complementing the gst-1 mutation was isolated from gene library, and gst1 was mapped in chr 4R, by Southern blotting of cloned sequence to the individual yeast chromosome DNA by OFAGE system and by genetic analysis. The gene product was tentatively assigned from DNA sequencing analysis, as a protein of MW 76565 which contained consensus sequences for a target site of cAMP-dependent protein kinase and for GTPase with extensive homology to polypeptide chain elongation factor EF1<alpha>.

酵母中基因的表达涉及许多因素。在本项目中，我们重点研究了矢量稳定性的影响。我们分离了两种酿酒酵母核突变体，它们影响质粒和小染色体的维持。map 1;我们已经分离出宿主突变体，其中2 <micro>m质粒维持不良。所有测试的突变体构成一个互补组，指定为map 1。携带染色体复制oringin和着丝粒的微小染色体也受到影响的突变体。体内和体外产生的两种类型的杂交质粒似乎可以补偿突变，并且具有含有多个ARS或一组2 <micro>m反向重复序列的DNA区域。这些结果表明，质粒的维持能力差是由于低水平的复制，可能是在复制的起始. gst 1;分离到一个新的温度敏感突变体gst 1。在非允许的温度下，突变体细胞与大芽积累和DNA合成基本上被阻止。温度转换与交配因子处理的交互试验表明，温度转换的执行点在“启动”后。这表明该突变影响了细胞周期中的G1至S期转变。从基因文库中分离出一个互补gst-1突变的DNA克隆，通过OFAGE系统将克隆序列与单个酵母染色体DNA进行Southern印迹，并通过遗传分析，将gst 1定位在chr 4 R中。基因产物从DNA测序分析中被暂时指定为MW 76565的蛋白质，其含有cAMP依赖性蛋白激酶的靶位点和与多肽链延伸因子EF 1具有广泛同源性的GT α的共有序列<alpha>。