ALTERATION OF THE LARGEST SUBUNIT OF RNA-POLYMERASE-II AND ITS EFFECTON CHROMOSOME STABILITY IN SCHIZOSACCHAROMYCES-POMBE

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previou sly been described the CHO-K1 cell mutant tsTM4. To elucidate the mole cular basis of this effect we introduced the mutation into the homolog ous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpI I LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA s ynthesis in cells arrested in S phase at the nonpermissive temperature , we focussed on the study of growth, the cell cycle, and chromosome s tability at various temperatures. First, we examined the effects of th e mutation on haploid yeast cells. The mutant showed slower growth tha n the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive tem perature, an accumulation of cells in G1 and/or GO was observed. Tetra d analysis suggested that these phenotypes were associated with the mu tation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FAGS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These o bservations demonstrate that the S. pombe rpb1 mutant exhibits a pheno type very similar to that of the CHO-KI cell mutant tsTM4.