Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions

Abstract

We report yeast/ Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZgene of E. coli. The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII. The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene. All the restriction sites have been phased to three different reading frames. Two series of vectors have been constructed. The first series (YEp) has two origins of replication ( ori), i.e., of the yeast 2μ. circle and of the ColEl plasmid of E. coli, and can therefore replicate autonomously in both organisms. These shuttle vectors also have the Ap R gene of E. coli and either the yeast LEU2 or URA3 genes to allow for selection of both E. coli and yeast transformants. The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2μ. ori. The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA. None of the vectors express β-galactosidase (βGal) in yeast or E. coli in the absence of inserted yeast promoter sequences. The 5′-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors. In-frame gene fusions can be detected by βGal activity when either yeast or E. coli clones are plated on media containing XGal indicator. Quantitative determinations of promoter activity were made by colorimetric assay of βGal activity in whole cells. Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.