Studies on the factors affecting gene expression in yeast.

酵母的基因表达涉及多种因素。在这个项目中，我们主要研究向量稳定性的影响。我们分离到了两种影响质粒和微染色体维持的酿酒酵母核突变体。map1;我们已经分离出2<微>m质粒维持不良的宿主突变体。所有被测突变体组成一个互补组，命名为map1。携带染色体复制起始蛋白和着丝粒的小染色体也在突变体中受到影响。在体内和体外产生的两种杂交质粒似乎弥补了突变，并且在一组2<微>m的反向重复序列中含有多个ARS的DNA区域。这些结果表明，质粒的维持能力较差是由于低水平的复制，可能在复制开始时。gst1;分离到一个新的温度敏感突变体gst1。在不允许的温度下，具有大芽的突变细胞积累起来，DNA合成基本上被阻止。从温度变化和配合因子处理的倒数实验中可以看出，执行点在“start”后。这表明该突变影响了细胞周期中g1到s的相变。从基因文库中分离到gst-1突变的DNA克隆，利用OFAGE系统对克隆序列进行Southern blotting和遗传分析，将gst1定位于酵母个体染色体DNA的chr 4R中。该基因产物初步由DNA测序分析确定为mw76565蛋白，该蛋白包含camp依赖性蛋白激酶靶位点和GTPase的一致序列，与多肽链延伸因子EF1< α >具有广泛的同源性。

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>.