A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization.
While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non-glucose carbon source than in glucose-grown cells (5-10-fold higher on galactose and up to 40-fold higher on ethanol). Furthermore, expression increased 30-fold upon shifting from a high-glucose to a low-glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5' region is short. We determined that the cis-elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence.
Pauwels
K
K
Department of Microbiology, Vrije Universiteit Brussels and Vlaams Interuniversitair Instituut voor Biotechnologie, c/o Research Institute, COOVI, 1 E. Gryson av., B-1070 Brussels, Belgium.
Abadjieva
A
A
Hilven
P
P
Crabeel
M
M
D016477:Artifacts; D002244:Carbon / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D006133:Growth Substances; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D006133:Growth Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; C101833:ORT1 protein, S cerevisiae; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; C085797:auxin regulated proteins, Vigna radiata; D002244:Carbon; D005947:Glucose