Isolation, heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis

Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B-2, Slovak Republic.
Current Genetics (Impact Factor: 2.68). 11/2002; 42(1):21-6. DOI: 10.1007/s00294-002-0327-6
Source: PubMed


By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.

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    • "Twenty-microgram aliquots of total RNA samples were separated per lane on a 1.2% agarose gel and blotted onto a nylon membrane using the LKB2016 VacuGene vacuum blotting system (Amersham Pharmacia Biotech Inc., Uppsala, Sweden). Hybridization was performed with an a-[ 32 P]dCTPlabeled DNA probe specific for KlPDR5 generated by random priming using Rediprime II Random Prime labeling system, (GE Healthcare Bio-sciences Corp., Vienna, Austria) (Takacova et al., 2002). After hybridization, blots were washed and exposed to autoradiography film. "
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    ABSTRACT: In Saccharomyces cerevisiae, the simultaneous resistance to various cytotoxic compounds known as multidrug resistance (MDR) is caused by overexpression of membrane efflux pumps under the control of two main transcriptional activators Pdr1p and Pdr3p. In this work we describe the results of functional analysis of a single Kluyveromyces lactis homolog of the PDR1 gene, which encodes a zinc finger Zn(2)Cys(6)-containing transcription factor. The KlPDR1 deletion generated a strain hypersusceptible to oligomycin, antimycin A and azole antifungals. Overexpression of KlPDR1 from a multicopy plasmid in the Klpdr1Delta mutant strain increased the tolerance of transformants to all the drugs tested (oligomycin, antimycin A and azole antifungals). The plasmid-borne KlPDR1 gene was able to complement drug hypersensitivity of the S. cerevisiae pdr1Deltapdr3Delta mutant strain. The KlPDR1 was found to be necessary for upregulation of the ATP-binding cassette transporter encoded by the KlPDR5 gene and rhodamine 6G efflux out of the cells. The KlPDR5 and some other K. lactis pleiotropic drug resistance (PDR) orthologues were found to contain putative PDR-responsive elements in their promoters. These results demonstrate that KlPdr1p is involved in K. lactis MDR and is required for cell's tolerance to various cytotoxic compounds.
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    • "According to the published data concerning glucose metabolism and its regulation in K. lactis, it seems that the redundancy of genes encoding hexose transporters and glycolytic enzymes that exists in S. cerevisiae is absent in K. lactis (Wesolowski-Louvel et al. 1992; Prior et al. 1993; Bianchi et al. 1996; Billard et al. 1996; Blaisonneau et al. 1997). Until now, only the drug efflux ABC transporter gene KlPDR5 has been isolated from the budding yeast K. lactis (Chen 2001; Takacova et al. 2002) and the encoded KlPdr5p was found to be posttranslationally regulated by the type 2A-related Ser/Thr protein phosphatase, Sit4p (Chen 2001). In this work, we describe the isolation, molecular cloning and characterization of the first K. lactis multidrug permease, as a prelude to studying the molecular mechanisms which led to the development of multidrug resistance in this biotechnologically important microorganism. "
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    ABSTRACT: Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells. In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene. The isolated gene exhibited 48.9% identity with the S. cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline- N-oxide and encoded a protein of 553 amino acids. When present in multicopy, it efficiently complemented the phenotype associated with the Delta pdr5 or Delta pdr1Delta pdr3 mutations in S. cerevisiae. Overexpression of the KNQ1 gene in K. lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline- N-oxide, 3-aminotriazole, bifonazole and ketoconazole. The gene was assigned to K. lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide. Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.
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    ABSTRACT: Kluyveromyces lactis KlPDR5, the drug eux ABC transporter gene, and KlRPL28, a gene encoding the large subunit ribosomal protein, were isolated in our laboratory recently. When overexpressed in Saccharomyces cerevisiae host strains, both genes confer an increased resistance to cycloheximide. In this work the chromosomal origin of the cloned genes was determined by Southern hybridization to contour-clamped homogenous electric eld (CHEF) separated chromosomal DNA. Both genes were mapped to K. lactis chromo- some VI.
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