| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
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>.
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