Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae

Abstract

Two sets of plasmids, each carrying a Saccharomyces ceret'isiae gene and a portion or all of the yeast 2-,im circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. ceretisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-jim

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae

“…The cloning vehicle was pJZ1 (constructed by J. Zeissler; Fig. 1); this is a typical yeast shuttle vector expressing Leu2+, ampicillin resistance (Ap'), and tetracycline resistance (Tc) (35). Growth and transformation of S. cerevisiae and E. coli were as previously described (27).…”

The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number

“…In transformed cells containing such a recombinant plasmid in addition to the endogenous plasmid, this generally leads to segregation and loss of the recombinant plasmid. Depending on the type of vector and the DNA inserted into it, the observed instability varies between 1 and 5% loss per generation (3,13,15,23,35,36).…”

The herpes simplex virus thymidine kinase gene is not transcribed in Saccharomyces cerevisiae.

“…The HSV tk gene, carried on a BamHI fragment that had been cloned in pBR322, was transferred in both orientations to the chimeric yeast plasmid cloning vehicle, pYF91 (28), by in vitro BamHI digestion and ligation. The plasmids thus constructed (pGY14, pGY15) contain a 3.6-kilobase pair (kbp) EcoRI fragment of the yeast 2-p,m circle, a 6.0-kbp BamHI-HindIII fragment of the Saccharomayces cerevisiae LEU2 gene, almost the entire E. coli plasmid pBR322, and the 3.4-kbp BamHI fragment carrying the HSV tk gene.…”