Annual Report

29. A Mutation in the Largest (Catalytic) Subunit of RNA Polymerase II and Its Relation to the Arrest of the Cell Cycle in G1 Phase

Kimihiko Sugaya, Shun-ichi Sasanuma, Peter R. Cook*, and Kazuei Mita (*The Sir William Dunn School of Pathology, Oxford Univ., UK)

Keywords: RNA polymerase II, temperature-sensitive mutant, cell cycle, hemizygosity, sequencing

Transcriptional activity of RNA polymerase II is modulated during the cell cycle. We previously identified a temperature-sensitive mutation in the largest (catalytic) subunit of RNA polymerase II (RPB1) which causes cell-cycle arrest and genome instability. We have characterized a different cell line that has a temperature-sensitive defect in cell cycle progression, and found that it also has a mutation in RPB1. The temperature-sensitive mutant, tsAF8, of the Syrian hamster cell line, BHK21, arrests at the non-permissive temperature in the mid-G1 phase. The RPB1 in tsAF8, which is found exclusively in the nucleus at the permissive temperature, is also found in the cytoplasm at the non-permissive temperature. Comparison of the DNA sequences of the RPB1 gene in the wild-type and mutant shows the phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one RPB1 allele; this gives rise to an ala-to-asp substitution at residue 315 in the protein. Aligning the amino acid sequences from various species reveals that ala315 is highly conserved in eukaryotes.

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