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

Abstract

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CENI-ADEI-CDC1S region from chromosome I was isolated and characterized. Starting with the previously cloned ADEI gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant A bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CENI). The CDC15 gene was located by isolating plasmids from a YCp5O S. cerevisiae genomic library that complemented the cdcl5-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdcl5-1-complementing plasmids all shared o,nly one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CENI to CDC15. 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.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the genes in the FUN38-MAK16-SPO7 region

“…In addition, the already characterized parts of chromosome I were 78% covered with transcribed regions (7,9,15,16,21,56). Sequence analysis has so far indicated that chromosomes I and III have similar gene densities, with ORFs comprising approximately 85% of the DNA (43,45).…”

“…Molecular studies have now shown that the cause of this result was neither a lack of genes nor a lack of essential genes (7,15,16,56 (18). If the remainder of chromosome I behaves similarly, we predict that there should be approximately 41 additional transcribed regions, of which 7 to 10 will prove essential.…”

“…A nearly contiguous set of nonoverlapping 1-to 3-kb DNA fragments from plasmid YCpSO-2C (64) and bacteriophages XK25c (56), XL4 (56), and XM7 (28) were subcloned into plasmid pBluescript-KS+ ( Fig. 1).…”

An ordered clone bank for chromosome I of Saccharomyces cerevisiae

“…1 (16), and one copy of a Ty element which was not genetically mapped on this chromosome was found. Thus, we correlated the genetic map of chromosome I with our physical map as shown in Fig.…”

“…By determining the nucleotide sequences of the boundaries of the duplicated regions on the two chromosomes, we may be able to obtain clues as to how this duplication happened. Attempts at physical dissection of S. cerevisiae chromosome I have been performed previously by Kaback and associates (4,8,16,17,22) and by Diel and Pringle (6). When these reports are taken together, a physical map amounting to about 75% of chromosome I, containing the cleavage sites of restriction enzymes BamHI, EcoRI, HindIII, PstI, PvuII, and XhoI, is available (different enzymes were used in different reports by these authors, and, therefore, the physical map data are not available for all of these restriction enzymes).…”