Vectors for the expression and analysis of DNA-binding proteins in yeast.
A series of 13 vectors is described. All are yeast centromere plasmids with the LEU2 gene for selection in yeast, and pUC19 sequences for growth in Escherichia coli. All contain the GAL1 promoter directing transcription into a multiple cloning site (MCS). For twelve of the plasmids, synthetic oligodeoxyribonucleotides create an ATG start codon, in a productive context for yeast, prior to the MCS. Spacing between the ATG and the MCS is variable, to facilitate the cloning of gene fragments in the appropriate reading frame. Nine of the plasmids also contain the strong transcriptional activator from the herpes simplex virus VP16 gene, joined downstream from the MCS. In these nine vectors, all possible combinations of reading frames are available. The suitability of these plasmids for the expression and analysis of DNA-binding domains is tested by cloning into them fragments of the yeast HSF1 gene, encoding the heat shock transcription factor (HSF). The regulation of reporter gene expression by the chimeric HSF-VP16 fusions is described, as is the utility of these vectors for other applications.
Bonner
J J
JJ
Department of Biology, Indiana University, Bloomington 47405.
D001483:Base Sequence; D002503:Centromere / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal; D005814:Genes, Viral; D005822:Genetic Vectors; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D003062:Codon; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors