FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl‐induced expression is dependent on Pdr3 transcriptional regulator

In this work we report the disruption of a Saccharomyces cerevisiae ORF YBR008c (FLR1 gene) within the context of EUROFAN (EUROpean Functional Analysis Network) six‐pack programme, using a PCR‐mediated gene replacement protocol as well as the results of the basic phenotypic analysis of a deletant strain and the construction of a disruption cassette for inactivation of this gene in any yeast strain. We also show results extending the knowledge of the range of compounds to which FLR1 gene confers resistance to the antimitotic systemic benzimidazole fungicide benomyl and the antitumor agent methotrexate, reinforcing the concept that the FLR1 gene is a multidrug resistance (MDR) determinant. Our conclusions were based on the higher susceptibility to these compounds of flr1Δ compared with wild‐type and on the increased resistance of both flr1Δ and wild‐type strains upon increased expression of FLR1 gene from a centromeric plasmid clone. The present study also provides, for the first time, evidence that the adaptation of yeast cells to growth in the presence of benomyl involves the dramatic activation of FLR1 gene expression during benomyl‐induced latency (up to 400‐fold). Results obtained using a FLR1–lacZ fusion in a plasmid indicate that the activation of FLR1 expression in benomyl‐stressed cells is under the control of the transcriptional regulator Pdr3p. Indeed, PDR3 deletion severely reduces benomyl‐induced activation of FLR1 gene expression (by 85%), while the homologous Pdr1p transcription factor is apparently not involved in this activation. Copyright © 1999 John Wiley & Sons, Ltd.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  K. Kashiwagi,et al.  Identification of a Gene for a Polyamine Transport Protein in Yeast* , 1999, The Journal of Biological Chemistry.

[3]  F Baganz,et al.  Systematic functional analysis of the yeast genome. , 1998, Trends in biotechnology.

[4]  S. Donnelly,et al.  Identification and Expression of Multidrug Transporters Responsible for Fluconazole Resistance in Candida dubliniensis , 1998, Antimicrobial Agents and Chemotherapy.

[5]  M H Saier,et al.  Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae , 1998, FEBS letters.

[6]  B. Pearson,et al.  Construction of PCR‐ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV , 1998, Yeast.

[7]  I. Paulsen,et al.  Major Facilitator Superfamily , 1998, Microbiology and Molecular Biology Reviews.

[8]  P. F. Almeida,et al.  The H+-ATPase in the Plasma Membrane ofSaccharomyces cerevisiae Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability , 1998, Applied and Environmental Microbiology.

[9]  K. Kuchler,et al.  The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators , 1997, FEBS letters.

[10]  H. Jungwirth,et al.  Diazaborine Resistance in the Yeast Saccharomyces cerevisiae Reveals a Link between YAP1 and the Pleiotropic Drug Resistance Genes PDR1 andPDR3 * , 1997, The Journal of Biological Chemistry.

[11]  A. Goffeau,et al.  Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes , 1997, Molecular and General Genetics MGG.

[12]  C. Jacq,et al.  Clustered amino acid substitutions in the yeast transcription regulator Pdr3p increase pleiotropic drug resistance and identify a new central regulatory domain , 1997, Molecular and General Genetics MGG.

[13]  A. Goffeau,et al.  Active efflux by multidrug transporters as one of the strategies to evade chemotherapy and novel practical implications of yeast pleiotropic drug resistance. , 1997, Pharmacology & therapeutics.

[14]  F. Galibert,et al.  Disruption of Six Novel Yeast Genes Reveals Three Genes Essential for Vegetative Growth and One Required for Growth at Low Temperature , 1997, Yeast.

[15]  C. Jacq,et al.  Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters , 1997, Molecular and cellular biology.

[16]  Rupert De Wachter,et al.  Classification of all putative permeases and other membrane multispanners of the Major Facilitator Superfamily encoded by the complete genome of Saccharomyces cerevisiae , 1997, German Conference on Bioinformatics.

[17]  M. Raymond,et al.  AP1-mediated Multidrug Resistance in Saccharomyces cerevisiae Requires FLR1 Encoding a Transporter of the Major Facilitator Superfamily* , 1997, The Journal of Biological Chemistry.

[18]  A. Driessen,et al.  Mechanisms of multidrug transporters. , 1997, FEMS microbiology reviews.

[19]  M H Saier,et al.  Multidrug‐Resistant Transport Proteins in Yeast: Complete Inventory and Phylogenetic Characterization of Yeast Open Reading Frames within the Major Facilitator Superfamily , 1997, Yeast.

[20]  I. Paulsen,et al.  Proton-dependent multidrug efflux systems , 1996, Microbiological reviews.

[21]  P. Roepe,et al.  Altered drug translocation mediated by the MDR protein: Direct, indirect, or both? , 1996, Journal of bioenergetics and biomembranes.

[22]  T. Hallstrom,et al.  Multiple Pdr1p/Pdr3p Binding Sites Are Essential for Normal Expression of the ATP Binding Cassette Transporter Protein-encoding Gene PDR5* , 1996, The Journal of Biological Chemistry.

[23]  S. Oliver A network approach to the systematic analysis of yeast gene function. , 1996, Trends in genetics : TIG.

[24]  K. Kuchler,et al.  The ATP‐binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3 , 1996, Molecular microbiology.

[25]  A. Wach PCR‐synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae , 1996, Yeast.

[26]  A. Goffeau,et al.  Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing , 1995, FEBS letters.

[27]  B. André,et al.  An overview of membrane transport proteins in Saccharomyces cerevisiae , 1995, Yeast.

[28]  M. Voet,et al.  Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[29]  C. Jacq,et al.  Positive autoregulation of the yeast transcription factor Pdr3p, which is involved in control of drug resistance , 1995, Molecular and cellular biology.

[30]  A. Goffeau,et al.  Yeast multidrug resistance: The PDR network , 1995, Journal of bioenergetics and biomembranes.

[31]  E. Georges,et al.  Benzimidazoles, potent anti-mitotic drugs: substrates for the P-glycoprotein transporter in multidrug-resistant cells. , 1994, Biochemical pharmacology.

[32]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[33]  A. Goffeau,et al.  Genetics and biochemistry of yeast multidrug resistance. , 1994, Biochimica et biophysica acta.

[34]  J. Becker,et al.  Candida albicans gene encoding resistance to benomyl and methotrexate is a multidrug resistance gene , 1994, Antimicrobial Agents and Chemotherapy.

[35]  A. Goffeau,et al.  Regulation of the expression of the H(+)-ATPase genes PMA1 and PMA2 during growth and effects of octanoic acid in Saccharomyces cerevisiae. , 1994, Biochimica et biophysica acta.

[36]  T. Bouwmeester,et al.  The FAR domain defines a new Xenopus laevis zinc finger protein subfamily with specific RNA homopolymer binding activity. , 1994, Biochimica et biophysica acta.

[37]  R. Schiestl,et al.  Improved method for high efficiency transformation of intact yeast cells. , 1992, Nucleic acids research.

[38]  M. Labouesse,et al.  A family of low and high copy replicative, integrative and single‐stranded S. cerevisiae/E. coli shuttle vectors , 1991, Yeast.

[39]  A. Goffeau,et al.  Multiple or pleiotropic drug resistance in yeast. , 1991, Biochimica et biophysica acta.

[40]  B. Dujon,et al.  The complete sequence of the 8·2 kb segment left of MAT on chromosome III reveals five ORFs, including a gene for a yeast ribokinase , 1990, Yeast.

[41]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[42]  L. C. Davidse,et al.  Benzimidazole Fungicides: Mechanism of Action and Biological Impact , 1986 .

[43]  A. Nachmias,et al.  Decreased permeability as a mechanism of resistance to methyl benzimadazol-2-yl carbamate (MBC) in Sporobolomyces roseus. , 1976, Journal of general microbiology.

[44]  R. Rothstein Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. , 1991, Methods in enzymology.