Construction of plasmid vectors that facilitate subcloning and recovery of yeast and Escherichia coli DNA fragments

Louie Naumovski, Errol C. Friedberg

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

We have constructed a plasmid vector (pNF2) which is a derivative of the multicopy yeast cloning vehicle YEp24. This derivative contains a single BamHI site flanked immediately on each side by SalI sites. The latter site was selected because it appears to be infrequent in yeast nuclear DNA. Thus, DNA fragments produced by partial digestion with enzymes (such as Sau3A) that cut at frequent intervals and leave single-stranded ends that have sequence homology with BamHI sites, can be conveniently subcloned into this site. Such fragments can then be excised intact by digestion with SalI enzyme. Plasmid pNF2 also contains the kanamycin-resistance (kanR) gene derived from Tn903 and confers resistance in yeast to the antibiotic G418. pNF2 was converted into an integrating vector (pNF3) by deleting a 2.2-kb EcoRI fragment containing a sequence that determines autonomous replication in yeast. Further deletion of a HindIII fragment containing the yeast URA3 gene converts the plasmid into one containing only pBR322 sequences plus the kanR gene (pNF4).

Original languageEnglish (US)
Pages (from-to)203-209
Number of pages7
JournalGene
Volume22
Issue number2-3
DOIs
StatePublished - Jan 1 1983

Keywords

  • Recombinant DNA
  • Saccharomyces cerevisiae
  • kanamycin resistance gene
  • multicopy cloning vehicle
  • nuclear DNA
  • plasmid

ASJC Scopus subject areas

  • Genetics

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