Nucleic Acids Research, Vol 24, Issue 10 1822-1828, Copyright © 1996 by Oxford University Press
P Herrero, M Ramirez, C Martinez-Campa and F Moreno
A well-defined set of isogenic yeast strains has been constructed whereby
each strain contains a different HXK2::lacZ gene fusion integrated at the
URA3 locus. These HXK2::lacZ fusions differ in the amount of the HXK2 gene
(encoding hexokinase 2 isoenzyme) that is fused to the lacZ reporter gene.
Comparison of the beta-galactosidase activities of each strain during
growth on glucose or ethanol revealed that some part of the coding region
between +39 and +404 bp is involved in repressing gene expression in a
carbon source dependent manner. A series of deletions of this HXK2 coding
region were constructed and fused upstream of a minimal CYC1::lacZ
promoter. beta-Galactosidase activities on glucose or ethanol growth yeast
calls revealed that two different regulatory elements are present in this
DNA region. Gel mobility shift analysis and in vitro DNase I footprinting
have shown that proteins bind specifically to two downstream repressor
sequences (DRS1 located from +140 to +163 and DRS2 located between +231 and
+251) that influence the rate of HXK2 transcription when ethanol is used as
carbon source by Saccharomyces cerevisiae. We identified and partially
purified a 18 kDa protein that binds specifically to synthetic double-
stranded oligonucleotides containing the (A/C)(A/G)GAAAT box sequence. Our
data suggest that p18 synthesis is under the control of genes involved in
glucose repression (MIG1 = CAT4) and glucose derepression (SNF1 = CAT1).
ARTICLES
Identification and characterisation of two transcriptional repressor elements within the coding sequence of the Saccharomyces cerevisiae HXK2 gene
Instituto Universitario de Biotecnologia de Asturias (IUBA), Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo, Spain.
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