Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

Abstract

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.