Cloning the Schizosaccharomyces pombe lys2+ gene and construction of new molecular genetic tools.
Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2(+) gene, which is homologous to Saccharomyces cerevisiae LYS4(+). We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2(+)-based cloning vector pRH3. In addition, we deleted the S. pombe his7(+) gene with a lys2(+) -marked polymerase chain reaction (PCR) product and the S. pombe lys2(+) gene with a his7(+)-marked PCR product. Strains carrying these deletions of lys2(+) or his7(+) serve as relatively efficient hosts for the deletion of the ade6(+) gene by lys2(+)-- or his7(+)--marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2(+) as a selectable marker, along with improved strains for the use of his7(+) to mark gene deletions.
Hoffman
Richard L
RL
Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA.
Hoffman
Charles S
CS
D000483:Alleles; D003001:Cloning, Molecular; D017353:Gene Deletion; D005800:Genes, Fungal / Q000235:genetics*; D005822:Genetic Vectors; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics; D008239:Lysine / Q000096:biosynthesis / Q000235:genetics*; D009841:Oligonucleotides; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*
D009841:Oligonucleotides; D029702:Schizosaccharomyces pombe Proteins; D006639:Histidine; D008239:Lysine