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 PYKI 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 pykl-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 PYKI 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 PYKI 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, PYKI, 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 PYKI were only -7.5 kb apart, although the genetic map distance between them is -13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to -3 kb, the region between CDC24 and PYKI appears to undergo meiotic recombination at an unusually high frequency.

Meiotic Recombination at the Ends of Chromosomes inSaccharomyces cerevisiae

“…Of the 26 ends that recombined at higher-than-average rates, 10 contained intervals that underwent recombination at .1 cM/kb while another 9 contained intervals that exhibited unusually high recombination rates (1.5-3.6 cM/kb) and appear to define new hot spots ( Table 2). The rates in these hot spots were equal to or greater than those observed for previously identified recombination hot spots (Coleman et al 1986;Kaback et al 1989;Nicolas et al 1989;Malone et al 1994;Fan et al 1995;Lichten and Goldman 1995;Cherry et al 1997). For four of the new hot spots-VL, VIIL, VIIIR, and XR-high recombination rates were confirmed with multiple crosses utilizing adjacent open reading frames.…”

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

“…To verify that the newly cloned sequences were derived from chromosome I, gene dosage Southern blot experiments with the 32P-labeled probes used for overlap hybridization were performed as described previously (11,22 60 ,ug of DNA from the S. cerevisiae-YCp5O recombinant DNA library. More than 6,000 Ura+ colonies were selected on synthetic defined medium lacking uracil at the permissive temperature (25°C).…”

“…The discrepancy between the number of transcription units and the number of genetically defined loci has been referred to as the gene number paradox (31) and has been observed in extensively studied genomic regions of other organisms (4 To analyze the relative contributions of these factors to the gene number paradox and to determine whether there are more essential genes on chromosome I, we began a study whose goals were to clone all the DNA from chromosome I on recombinant DNA molecules, to localize transcribed sequences, to identify the physical locations of already known genes, to determine which genes are duplicated, and to determine which genes are essential for growth on rich medium. We recently reported the isolation and the location of transcribed regions on the 18 kb of chromosome I DNA surrounding the CDC24 and PYKI genes (11). Here we report the isolation and analysis of the centromeric region from this chromosome.…”

“…In addition to the transcribed regions corresponding to the ADEI and CDC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Tyl transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA. Physical studies suggest that 5,000 to 6,000 genes are expressed under conditions of vegetative growth in the yeast Saccharomyces cerevisiae (11,28). In contrast, far fewer genes have been characterized genetically.…”

“…260 kilobases (kb) long (8,44). Since there is, on average, one transcribed region per 2.2 to 2.8 kb of DNA in the S. cerevisiae genome, we estimated that chromosome I contains approximately 100 genes (11).…”