Chemical specificity of the PDR5 multidrug resistance gene product of Saccharomyces cerevisiae based on studies with tri-n-alkyltin chlorides.

To understand the chemical basis of action for the PDR5-encoded multidrug resistance transporter of Saccharomyces cerevisiae, we compared the relative hypersensitivities of the wild-type (RW2802) and null mutant strains toward a series of tri-n-alkyltin compounds. These compounds differ from each other in a systematic fashion-either by hydrocarbon chain length or by anion composition. Using zone-of-inhibition and fixed-concentration assays, we found that the ethyl, propyl, and butyl compounds are strong PDR5 substrates, whereas the methyl and pentyl compounds are weak. We conclude that hydrophobicity and anion makeup are relatively unimportant factors in determining whether a tri-n-alkyltin compound is a good PDR5 substrate but that the dissociation of the compound and the molecular size are significant.

A PDR5-independent pathway of multi-drug resistance regulated by the SIN4 gene product.

The SIN4 locus encodes a global transcriptional regulator of various yeast genes. In this report, we demonstrate that loss of function mutations in SIN4 create a multi-drug hypersensitive phenotype that is independent of PDR5 mediated resistance. Thus, double sin4, pdr5 mutants are more sensitive than single mutants. Furthermore, SIN4 does not regulate the PDR5 locus. These observations establish that yeast cells have two genetically distinct pathways conferring resistance towards similar substrates.

The unusual inheritance of multidrug-resistance factors in Saccharomyces.

The yeast PDR5 locus encodes a 160-kDa member of the ABC family of transport proteins. Strains bearing a deletion of this locus are drug hypersensitive. Resistant revertants arise when cells are plated on cycloheximide medium. About one-third of these are cross resistant to other agents, including oligomycin, fluconazole and sulfometuron methyl. Most of the revertants exhibit linkage to the PDR5 locus and map in three locations. Curiously, the multi-drug resistance is not due to a single mutation. Most of the revertants behave as though they contained several tightly linked resistance factors.

Analysis of second-site mutations that suppress the multiple drug resistance phenotype of the yeast PDR1-7 allele.

The yeast PDR1 locus encodes a member of the C6 zinc cluster family of transcriptional regulatory proteins. Among the targets of PDR1 is the yeast PDR5 locus. The product of this gene is a member of the ATP-binding cassette (ABC) transmembrane protein family and plays a major role in inhibitor efflux. Mutations in PDR1 affect the relative level of PDR5 transcript and can therefore result in increased or decreased drug resistance. We isolated three second-site suppressors of a PDR1-7 semidominant hyper-resistant mutation. These mutants were drug hypersensitive, as compared with isogenic controls. Two of the three mutations contained alterations in a putative DNA-binding domain. Significantly, the mutant proteins exhibited reduced DNA-binding capacity.

Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

Loss of function mutation in the yeast multiple drug resistance gene PDR5 causes a reduction in chloramphenicol efflux.

The yeast (Saccharomyces cerevisiae) PDR5 gene product encodes a 160-kDa protein related to the large ABC family of transporters, including the human MDR1 multidrug resistance p-glycoprotein. Loss of function mutations in PDR5 result in chloramphenicol hypersensitivity. A pdr5::Tn5 loss of function mutant exhibits a markedly impaired efflux of chloramphenicol compared with that of an isogenic PDR5 (wild-type) control.

Transcriptional control of the yeast PDR5 gene by the PDR3 gene product.

Saccharomyces cerevisiae cells possess the ability to simultaneously acquire resistance to an array of drugs with different cytotoxic activities. The genes involved in this acquisition are referred to as pleiotropic drug resistant (PDR) genes. Several semidominant, drug resistance-encoding PDR mutations have been found that map near the centromere on chromosome II, including PDR3-1 and PDR4-1. DNA sequencing of chromosome II identified a potential open reading frame, designated YBL03-23, that has the potential to encode a protein with strong sequence similarity to the product of the PDR1 gene, a zinc finger-containing transcription factor. Here we show that YBL03-23 is allelic with PDR3. The presence of a functional copy of either PDR1 or PDR3 is essential for drug resistance and expression of a putative membrane transporter-encoding gene, PDR5. Deletion mapping of the PDR5 promoter identified a region from -360 to -112 that is essential for expression of this gene. DNase I footprinting analysis using bacterially expressed Pdr3p showed specific recognition by this protein of at least one site in the -360/-112 interval in the PDR5 promoter. A high-copy-number plasmid carrying the PDR3 gene elevated resistance to both oligomycin and cycloheximide. Increasing the number of PDR3 gene copies in a delta pdr5 strain increased oligomycin resistance but was not able to correct the cycloheximide hypersensitivity that results from loss of PDR5. These data are consistent with the notion that PDR3 acts to increase cycloheximide resistance by elevating the level of PDR5 transcription, while PDR3-mediated oligomycin resistance acts through some other target gene.

Long-tract mitotic gene conversion in yeast: evidence for a triparental contribution during spontaneous recombination.

In Saccharomyces cerevisiae, spontaneous mitotic gene conversion at one site is statistically correlated with recombination at other loci. In general, coincident conversion frequencies are highest for tightly linked markers and decline as a function of intermarker distance. Paradoxically, a significant fraction of mitotic gene convertants exhibits concomitant nonreciprocal segregation for multiple and widely spaced markers. We have undertaken a detailed genetic analysis of this class of mitotic recombinants. Our results indicate that mitotic gene conversion in yeast is frequently associated with nonreciprocal segregation of markers centromere-distal to the selected site of conversion. In addition, distal markers are often found to be mosaic within the product colonies. These observations, and others described here, suggest that a percentage of gene conversion in vegetative yeast cells is coupled to a chromosome break and repair mechanism. This hypothesis was further tested using a strain trisomic for chromosome VII which was specially marked to detect homolog-dependent repair events. An association between mitotic gene conversion events and the production of broken chromosomes which are repaired by a homologous-pairing-copy mechanism was supported.

Mutations in the yeast PDR3, PDR4, PDR7 and PDR9 pleiotropic (multiple) drug resistance loci affect the transcript level of an ATP binding cassette transporter encoding gene, PDR5.

The yeast pleiotropic (multiple drug) resistance gene PDR5 encodes a product with homology to a large number of membrane transport proteins including the mammalian multiple drug resistance family. In this study, we identified four genes on chromosome II that affect the steady-state level of PDR5 transcript in addition to a previously identified positive regulator, PDR1. The genes in question are PDR3, PDR4, PDR7 and PDR9. We also analyzed the interaction between PDR5 and YAP1. YAP1 encodes a positive regulator with a leucine zipper motif that causes pleiotropic drug resistance when overproduced. YAP1-mediated pleiotropic drug resistance is not dependent on the presence of PDR5 and must act through other genes.

Interaction of the yeast pleiotropic drug resistance genes PDR1 and PDR5.

The network of genes which mediates multiple drug resistance in yeast includes, among others, the PDR1 gene, which encodes a putative regulator of gene expression, and PDR5, a locus whose amplification leads to resistance. We demonstrate that disruption of PDR5 causes marked hypersensitivity not only to cycloheximide but also to sulphometuron methyl and the mitochondrial inhibitors chloramphenicol, lincomycin, erythromycin and antimycin. Genetic analysis of double mutants containing an insertion in PDR5 (pdr5:Tn5), which renders cells hypersensitive to cycloheximide, and a pdr1 mutation, which confers resistance to this inhibitor, indicates that the expression of resistance requires a functional PDR5 gene. The same interdependency is observed for chloramphenicol, but not for oligomycin, lincomycin, erythromycin or sulphometuron methyl. Northern analysis of PDR1 and PDR5 transcripts reveals that the 5.2 kbp PDR5 transcript is overexpressed in pdr1 (resistant) mutants, but underexpressed in a disruption of PDR1. These observations provide strong experimental support for our former proposal that the PDR5 gene is a target for regulation by the PDR1 gene product.

Chromosomal rearrangement in Candida stellatoidea results in a positive effect on phenotype.

When type I Candida stellatoidea is plated onto sucrose agar at levels in excess of 10(8) cells, some isolates spontaneously form sucrose-positive colonies. These isolates do not display typical type I phenotypes but instead exhibit phenotypes intermediate between type I C. stellatoidea and C. albicans. Also, this phenotypic change only occurs in conjunction with a chromosomal rearrangement. These rearrangements have been studied in a strain naturally marked for methionine auxotrophy. Chromosome-size DNA bands separated by pulsed-field gel electrophoresis were probed with genes cloned from C. albicans. The hybridization pattern indicated that the genes on several chromosomes underwent extensive rearrangement.

Cloning by gene amplification of two loci conferring multiple drug resistance in Saccharomyces.

Yeast DNA fragments that confer multiple drug resistance when amplified were isolated. Cells containing a yeast genomic library cloned in the high copy autonomously replicating vector, YEp24, were plated on medium containing cycloheximide. Five out of 100 cycloheximide-resistant colonies were cross-resistant to the unrelated inhibitor, sulfometuron methyl, due to a plasmid-borne resistance determinant. The plasmids isolated from these resistant clones contained two nonoverlapping regions in the yeast genome now designated PDR4 and PDR5 (for pleiotropic drug resistant). PDR4 was mapped to chromosome XIII, 31.5 cM from LYS7 and 9 cM from the centromere. PDR4 was mapped to chromosome XV between ADE2 and H1S3. Genetic analysis demonstrated that at least three tightly linked genes (PDR5, PDR2 and SMR3) that mediate resistance to inhibitors are located in this region. Insertion mutations in the either PDR4 or PDR5 genes are not lethal, but the insertion in PDR5 results in a drug-hypersensitive phenotype.

Coincident recombination during mitosis in saccharomyces: distance-dependent and -independent components.

In mitosis, coincident recombination events between widely separated markers occur more frequently than expected for two independent acts. Several different mechanisms have been proposed to account for this phenomenon. It has been argued that coincident recombination could be due to either an extensive region of heteroduplex DNA or some other distance-dependent mechanism. Alternately, it has been suggested that at least some is due to subpopulations of cells which undergo recombination at very high frequencies. The purpose of these experiments is to evaluate the possible contribution of distance-dependent and distance-independent components. By comparing the coincident recombination frequencies for markers on the same homolog as well as pairs of unlinked sites, we show that there is a strong distance-dependent component for at least 8.8-35-kbp, depending on the type of recombination event (conversion or intrachromosomal exchange). For larger distances separating sites, a distance-independent mechanism(s) results in higher than expected frequencies.

The behavior of insertions near a site of mitotic gene conversion in yeast.

In yeast, coincident gene conversion events involving the LEU1 and TRP5 loci (16 cM apart) occur at frequencies that are far greater than is expected for two independent acts of recombination. When a large plasmid (pJM53) is placed between these genes so that a direct repeat is produced, there is frequent loss of the insert among coincident convertants. Previous results strongly suggest that this is due to a separate, intrachromosomal exchange between the direct repeats rather than to excision from an extensive region of heteroduplex DNA. In this paper, we extend our genetic and molecular analysis to a plasmid insertion (pKSH) which replaces rather than duplicates the chromosomal material. The relative stabilities of pKSH and pJM53 are compared among coincident Leu+Trp+ convertants and convertants involving only one locus (LEU1). The pKSH insertion is significantly more stable in the latter which constitute a large majority of the selectable recombinants. In the former, both insertions are lost with high frequency. These results are used to argue that, while most mitotic conversion does not result from long intermediates, coincident convertants may arise from either multiple intermediates or extensive heteroduplex regions.

Coincident gene conversion events in yeast that involve a large insertion.

In yeast, spontaneous gene conversion events involving sites that are far apart (16 cM) occur 1000 times more frequently in mitotic cells than is expected for two independent acts of recombination. It has been proposed that a major portion of these could be due to a long, continuous heteroduplex intermediate. We have examined this possibility in further detail by introducing, via transformation, a large plasmid insertion between the LEU1 and TRP5 loci and studying its behavior among coincident convertants involving the flanking sites. Among such convertants, there is frequent loss of the plasmid when it is present in hemizygous or homozygous configuration. Our results could support the long heteroduplex model for coincident recombination events, but only if novel assumptions regarding the formation and fate of mismatched DNA are made. Therefore, an alternative model that proposes multiple, concerted recombination events is discussed.

Coincident gene conversion during mitosis in saccharomyces.

During mitosis, gene conversion events at the TRP5 locus on chromosome VII are coupled with conversion events at LEU1, a locus 18 cM away, 1200 times more frequently than would be expected for two independent acts of recombination. Such coincident conversion events that occur over relatively long distances could be due to several mechanisms. We discuss these possibilities and describe an experiment that indicates that a portion of coincident events is due to extensive heteroduplexes. The phenomenon of coincident gene conversion is discussed in relation to our earlier evidence that spontaneous recombination between homologues occurs prereplicationally in mitosis.

Mitotic recombination: mismatch correction and replicational resolution of Holliday structures formed at the two strand stage in Saccharomyces.

In a preliminary report (Esposito 1978), evidence was presented which showed that heteroallelic recombination resulting in prototrophic colonies occurs at the 2-strand stage. A model utilizing replicative resolution of Holliday structures was proposed to explain how gene conversion at the 2-strand stage can result in exchange of outside markers. The object of the experiments reported herein was to present detailed genetic evidence for 2-strand recombination. In addition, we examined the features of mitotic recombination with respect to symmetry, length and polarity of heteroduplexes in wild type strains (REM1/REM1) and in strains bearing the hyper-recombination mutation rem1-1. To do this, we constructed strains so that prototrophs arising from heteroallelic recombination and recombinant for outside markers were detected by visual inspection. By analyzing these colonies genetically, we have inferred several features of mitotic recombination which distinguish it from its meiotic counterpart. Firstly, mitotic heteroduplexes are often symmetric while meiotic heteroduplexes are almost exclusively asymmetric. Secondly, heteroduplexes tend to be longer in mitosis that in meiosis. Thirdly, unlike meiotic conversion, mitotic conversion does not show strong polarity. Recombination in strains homozygous for the rem1-1 mutation also takes place at the 2-strand stage. The rem1-1 mutation, however, appears to alter the features of mismatch correction.

Mitotic versus meiotic recombination in Saccharomyces cerevisiae.

As part of a comparative analysis of spontaneous mitotic and meiotic recombination we have compared the mitotic and meiotic maps of the wild type and yeast hybrids homozygous for reml-l, a mitosis-specific hyper-rec mutation (Golin and Esposito, 1977; Golin, 1979). In wild type yeast strains recombination in centromere proximal intervals occurs relatively more frequently in mitosis than in meiosis. In reml-1/rem1-1 hybrids the distribution of mitotic exchange events is more similar to the distribution observed in meiosis.

Evidence for joint genic control of spontaneous mutation and genetic recombination during mitosis in Saccharomyces.

A procedure for detection of mutants exhibiting either enhanced or reduced spontaneous mutation during mitosis and/or meiosis has been developed to probe the joint genic control of spontaneous mutation and recombination in yeast. A semidominant mutator, rem1-1, recovered by this technique, exhibits enhanced spontaneous mutation,intragenic recombination, and intergenic recombination during mitosis. Diploids homozygous for rem1-1 exhibit normal levels of meiotic intragenic and intergenic recombination and diminished ascospore viability.