The mapping of chromosomes in Saccharomyces cerevisiae: I. A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome

Abstract

A series of vectors for cosmid cloning in yeast has been derived from cosmid pHC79. Vectors pMT4 through pMT6 contain two tandemly arranged cohesive end sites ( cos) from the genome of bacteriophage γ Their design allows the rapid and simple preparation of cosmid arms by linearizing a vector at the unique PvwII-restriction site located between the two cos-sequences and then cutting the linearized molecule at one of its unique cloning sites for BamHI, ClaI, PvuI, SalI or Scal. Cosmids generated with arms from the most advanced vector, pMT6, carry the origin of replication ( ori) and the Ap R gene from pBR322 and the TRP1/ARS1 and URA1 genes from Saccharomyces cerevisiae. A yeast genomic DNA library was established by packaging in vitro, into bacteriophage λ preheads, of partially restricted yeast DNA fragments ligated to cosmid arms of vector pMT6. About 80% of the clones thus obtained comprise inserts of contiguous genomic DNA over 30 kb in length. Unique DNA probes for the yeast genes CDC10, CDC39, HIS4, LEU2, and PGK1 have successfully been applied when testing for completeness of this library by isolating a series of overlapping cosmid clones that carry the respective genes. The library will thus be useful for the selection of cosmid clones which carry CDC genes from yeast by complementing first, with the vectorial yeast gene URA1, the pyrimidine auxotrophy of most cdc-strains and then, with the respective CDC wild-type genes, of the temperature-sensitive mutant alleles. Most CDC clones thus obtained will provide unique DNA probes which serve as randomly distributed start sequences within the yeast genome for overlap hybridization screening in chromosome mapping studies.