Yeast Sequencing Report Molecular and functional analysis of a MIG1 homologue from the yeast Schwanniomyces occidentalis Teresa A. Carmona, Patricia Barrado, Antonio Jime ´nez and Marı ´a Ferna ´ndez Lobato* Centro de Biologı ´a Molecular ‘Severo Ochoa’, Departamento de Biologı ´a Molecular (CSIC-UAM), Universidad Auto ´noma Madrid, Cantoblanco, 28049 Madrid, Spain * Correspondence to: M. Ferna ´ndez Lobato, Centro de Biologı ´a Molecular ‘Severo Ochoa’ (CSIC/UAM), Departamento de Biologı ´a Molecular, Universidad Auto ´noma de Madrid, Cantoblanco, 28049 Madrid, Spain. E-mail: mfernandez@cbm.uam.es Received: 3 October 2001 Accepted: 13 November 2001 Abstract A putative glucose repressor MIG1-homologue (SoMIG1) was isolated from the amylolytic yeast Schwanniomyces occidentalis. Degenerate primers were designed from the conserved zinc finger regions of Mig1 and CreA proteins from different organisms. PCR using these primers and S. occidentalis genomic DNA as template yielded a single 128 bp product. This fragment was used as a DNA probe to screen a S. occidentalis genomic library. Analysis of the positive clones led to the isolation by PCR of a DNA fragment, which contained an open reading frame (ORF) that would encode a 458 amino acid polypeptide. The DNA binding and effector domains of this putative protein showed an identity of 71% and 15%, respectively, to those of the Mig1 protein from Saccharomyces cerevisiae. The SoMIG1 gene complemented a mig1 mutant of this yeast, which suggests that in S. occidentalis SoMIG1 is a glucose repressor. The Accession No. is AJ417892. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: Schwanniomyces occidentalis; Mig1; CreA; glucose repression Introduction In yeast cell cultures, glucose represses the expres- sion of a variety of genes that are required for metabolism of alternative carbohydrates, gluco- neogenesis and mitochondrial functions. This pheno- menon is called glucose repression and its regulatory mechanisms have been widely investigated in Saccharomyces cerevisiae (Ronne, 1995; Gancedo, 1998; Carlson, 1998). Mig1 protein is a key com- ponent of this regulatory pathway (Nehlin and Ronne, 1990). It contains two C 2 H 2 zinc fingers which bind to well-characterized GC-rich sites in the promoter of many glucose repressed genes, including SUC2, GAL, MAL, MEL, FBP1 and HAP4 (Lundin et al., 1994). In the presence of glucose, Mig1p represses transcription by recruiting the Ssn6–Tup1 complex to the target promoter (Treitel and Carlson, 1995). Schwanniomyces occidentalis (Debaromyces occi- dentalis) is an amylolytic yeast able to use soluble starch as a carbon source. As in S. cerevisiae, expression of S. occidentalis genes involved in carbohydrate degradation, such as AMY, GAM1, INV and SWA2, is repressed in the presence of glucose (Abarca et al., 1991; Wang et al., 1999). Analysis of the SWA2 gene showed a Mig1-binding GC box located between positions x110 and x115 (Abarca et al., 1991). These data suggested that an analogue of Mig1 may be present and, probably, involved in glucose repression in S. occidentalis. In this paper, we report the isolation and sequencing of a S. occidentalis MIG1 homologue and that its encoded protein (SoMig1p) is functional in S. cerevisiae. Materials and methods Strains, plasmids, culture conditions and DNA methodology S. cerevisiae strain W303-1A (MATa SUC2 ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) was from our collection. S. cerevisiae strain H190 (MATa SUC2 ade2-1 can1-100 his3-11,15 leu2- 3,112 trp1-1 ura3-1 mig1 x d2::LEU2) (Nehlin and .Yeast Yeast 2002; 19: 459–465. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / yea.846 Copyright # 2002 John Wiley & Sons, Ltd.