Expression of lacZ gene fusions affects downstream transcription in yeast.
Chimeric genes containing Escherichia coli lacZ sequences are often used to characterize gene expression in yeast cells. By Northern analysis, we found that such genes produce multiple transcripts due to inefficient 3'-end formation. The same transcript pattern was found for two related chimeric genes when these genes were cloned separately into the commonly used vector, YIp5, and integrated into the yeast genome at two different locations. Each chimeric gene was composed of promoter and N-terminal coding regions from the yeast SSA1 or SSA2 genes fused in-frame to the lac operon. Transcripts were shown to initiate within the yeast promoter fragment, but transcript size indicated that 3' ends were localized to three different regions: within the lac operon near the 3' end of the lacZ gene; near a terminator region previously identified upstream of the URA3 gene in YIp5; and at the URA3 terminator region. Readthrough transcription of the URA3 promoter from upstream lac sequences decreased the basal activity of the URA3 promoter, although induced URA3 transcription levels were unaffected. This readthrough transcription also resulted in a novel, longer URA3 transcript.
Barnes
C A
CA
Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada.
Johnston
G C
GC
Singer
R A
RA
D001483:Base Sequence; D015152:Blotting, Northern; D002678:Chimera; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D006360:Heat-Shock Proteins; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase