Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells

Abstract

We have cloned the human genomic DNA and the corresponding cDNA for the gene which complements the mutation of tsBN51, a temperature-sensitive (Ts) cell cycle mutant of BHK cells which is blocked in G1 at the nonpermissive temperature. After transfecting human DNA into TsBN51 cells and selecting for growth at 39.5 degrees C, Ts+ transformants were identified by their content of human AluI repetitive DNA sequences. Following two additional rounds of transfection, a genomic library was constructed from a tertiary Ts+ transformant and a recombinant phage containing the complementing gene isolated by screening for human AluI sequences. A genomic probe from this clone recognized a 2-kilobase mRNA in human and tertiary transformant cell lines, and this probe was used to isolate a biologically active cDNA from the Okayama-Berg cDNA expression library. Sequencing of this cDNA revealed a single open reading frame encoding a polypeptide of 395 amino acids. The deduced BN51 gene product has a high proportion of acidic and basic amino acids which are clustered in four hydrophilic domains spaced at 60- to 80-amino-acid intervals. These domains have strong sequence homology to each other. Thus, the tsBN51 protein consists of periodic repetitive clusters of acidic and basic amino acids.