NEW GENE CHL-15 CONTROLLING CHROMOSOME-REPLICATION IN SACCHAROMYCES YEASTS - CLONING, PHYSICAL MAPPING, SEQUENCING, AND FUNCTIONAL-ANALYSIS

Gene CHL15 was identified by us recently upon screening mutant yeasts with increased loss of chromosome III and artificial circular and line ar chromosomes during mitosis. Mutations in the CHL15 gene lead to a 1 00-fold increase in the frequency of spontaneous chromosome loss durin g mitosis and to a 200-fold increase in the frequency of spontaneous m itotic recombinations. Analysis of segregation of artificial chromosom es in mitosis allowed us to suggest that the main reason of chromosome destabilization in the chl15-1 mutant is associated with the loss of one of the sister chromatids (segregation type 1:0). Gene CHL15 was cl oned from the yeast genome library by complementation of the appropria te mutation. The use of the cloned fragment allowed us to map gene CHL 15 in the right arm of chromosome XVI at a distance of 175 kbp from th e telomere. Nucleotide sequence analysis of the genomic fragment conta ining gene CHL15 demonstrated the presence of an open reading frame (O RF) of 2.8 kbp, which encodes a polypeptide of mol. mass 105 kDa. In t he 5' nontranslated region of gene CHL15 (position - 122) we found a d egenerate repeat ACGCGT (motif MluI) which is typical of all known yea st genes involved in DNA synthesis. Regions potentially capable of for ming DNA-binding domains of the ''zinc finger'' type were found in the protein N-terminal region. The C-terminal region of the CHL15 gene pr oduct displayed a similarity to the ''helix-loop-helix'' motif (HLH) f ound in a number of factors involved in development, cell growth, and transformation. Cells with gene CHL15 deletions cease growth at differ ent stages of the cell cycle including the G2 stage. The CHL15 mutant demonstrated a significantly decreased division rate upon deletion in the RAD52 gene, whose product is involved in DNA synthesis, and also i n processes of recombination and repair of double-strand breaks. All t he obtained results indicate that gene CHL15 belongs to the genes cont rolling chromosome replication in yeasts.