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Isolation and characterization of sporulation‐specific promoters in the yeast Saccharomyces cerevisiae
Author(s) -
Coe J. G. S.,
Murray L. E.,
Kennedy C. J.,
Dawes L W.
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb00839.x
Subject(s) - biology , plasmid , promoter , lac operon , saccharomyces cerevisiae , shuttle vector , genomic library , microbiology and biotechnology , yeast , dna , gene , genetics , genomic dna , gene expression , vector (molecular biology) , recombinant dna , peptide sequence
Summary A library of random yeast genomic DNA: lacZ fusions has been constructed using an episomal yeast‐ Escherichia coli shuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an inframe ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast. Yeast genomic DNA fragments of 4–6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 × 10 4 individual E. coli transformants. This library was screened to identify promoter‐ lacZ fusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited β‐galactosidase activity, two were found to express the lacZ gene in a sporulation‐specific manner. This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11–21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4–14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.