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

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Cloning

by

Gene Amplification of

Two

Loci Conferring Multiple Drug

Resistance in Saccharomyces

Gregory Leppert,* Raymond McDevitt?

S. Carl Falco,? Tina K. Van Dyk? Marion B. Ficke* and

John Golin*’

*Institute for Biomolecular Studies, Department of Biology, Catholic University of America, Washington, D.C. 20064, and +E. I . Dupont and Company, Experimental Station, Wilmington, Delaware 19898

Manuscript received October 24, 1989 Accepted for publication January 27, 1990

ABSTRACT

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 PDRS (for pleiotropic drug resistant). PDR4 was mapped to chromosome XIZZ, 3 1.5 cM from LYS7 and 9 cM from the centromere. PDRS was mapped

to chromosome XV between ADE2 and HlS3. 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.

I

N mammalian cells, amplification of specific se- quences in no fewer than three genes can result in resistance to a large number of structurally and me- chanistically unrelated inhibitors (HOWELL et al. 1984; RONINSON et al. 1984; VARSHAVSKY 1984; KARNER et al. 1985; RICHERT et al. 1985; STARK 1986). For example, in Chinese hamster cell lines, amplification is associated with resistance to Adriamycin and Col- chemid. When the duplicated regions are subse- quently lost by culturing in media without inhibitor, there is loss of resistance. Often, the multiple drug resistance phenotype is associated with an increase in the amount of a high molecular weight plasma mem- brane glycoprotein. Recent studies also suggest that the heightened resistance is due to an ATP dependent transport system which actively pumps inhibitors out of the cell (SKORSGAARD 1978; FOJO et al. 1985; GROS, CROOP and HOUSMAN 1986; WILLINCHAM et a l . 1987).

Multiple drug resistance is not restricted to mam- malian cells. It has been suggested that similar genes exist in the malaria parasite resulting in resistance to many of the common antimalarial agents (KROGSTAD et al. 1987). There is also striking homology between some of the mammalian mdr genes and those involved with energy mediated bacterial transport such as the malk locus of Escherichia coli (FERRO-LUZZI AMES 1986 for review). Furthermore, several such loci have been identified in yeast. T h e P D R l locus has been the

To whom correspondence and reprint requests should be addressed. Genetics 1 2 5 13-20 (May, 1990)

subject of considerable genetic analysis (RANK and BECH-HANSEN 1973; SAUNDERS and RANK 1982). Mu- tations at this locus can be dominant or recessive, and are sometimes unstable or suppressible. In addition, alterations in the P D R l locus result in resistance to no fewer than fifteen different inhibitors. Mutations at a second site (FALCO et a l . 1986), initially designated SMR3, but now called PDR2, produce resistance to at least three different compounds, cycloheximide, oli- gomycin and sulfometuron methyl. Yet a third gene (PDR3) has been located on chromosome

ZZ

(SUBIK, ULSAZEWSKI and GOFFEAU 1986). Recently, Mc-

GARTH and VARSHAVSKY (1989) demonstrated that the yeast gene, STE6, shows striking homology to the mammalian gene that codes for the P glycoprotein. Interestingly enough, STE6 does not appear to me- diate multiple drug resistance, but mediates mating factor transport.

T h e PDRl locus has recently been cloned and se- quenced (BALZI et al. 1987). It specifies a 3192 nu- cleotide open reading frame. T h e deduced amino acid sequence contains zinc fingers suggesting that the encoded peptide might bind to DNA.

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TABLE 1

Yeast strains and plasmids

S t I . . k Genotype Origin

mata ade2 his7-1 l e u l - c trp5-c metl3-c cyh2, tyrl-2, lys2-2, his7-1, ura3-1

mata ade5 smr3-37 his7 trpl ura3 FALCO and DUMAS (1985)

mata ade2 ade5 pdr2-33, leu2, ura3 FALCO and DUMAS (1 985)

mata ade2 pdr2-33 (smr3-33) FALCO and DUMAS (1 985)

mata ade2 PDR2-73 (smr3-73) FALCO and DUMAS ( 1 985)

mata a spol I ura3 can1 cyh2 ade2 his7 hom3 KLAPHOLZ and EASTON-ESPOSITO ( 1 982)

mata a spol 1 ura3 adel his1 leu2 lys7 met3 trp5 KLAPHOLZ and EASTON-ESPOSITO ( 1 982)

mata his4 ABC ura3-50 gcn4 FALCO and BOTSTEIN (1983)

mata cir" his3 ura3-52 t r p l canl-100 pdrl mata ura3 ade2

pCL706 (URA3) integrated into chromosome X V in strain JG 273 pGL706 integrated into strain JG272

mata ura3 leu2 his3 PDR5:Tn5

pBT208 integrated into strain DBY 947

mata met5 leu2 ura3-52 mak71 KRBI Cir" mata leu2 ura3 lys7

REED WICKNER

5.5-kb pair yeast integrating vector

7.7-kb pair yeast autonomously replicating plasmid YEp24 plus an 8-kbp insert containing PDR4

YEp24 plus an 14.7 kbp insert containing PDR5

A 6.4-kbp KpnI deletion of pDR3.3

T h e 7-kbp Sal1 fragment of pDR3.1 placed in Y l p 5

phenotype.

PDR5

is tightly linked to two other genes identified by drug-resistance mutations.

MATERIALS AND METHODS

Strains, ,plasmids and media: The strains and plasmids used in thls study are listed in Table 1. T h e recipes for media and the conditions for culturing yeast cells are de- scribed elsewhere (FALCON and DUMAS 1985). Strains con- taining plasmids were maintained under selective conditions (medium lacking uracil).

DNA preparation: The methods for the preparation of yeast chromosomal DNA and plasmid DNA were described previously (GOLIN, FALCO and MARCOLSKEE 1986).

Transformation: Transformation of yeast with plasmid DNA was done according to the procedure of HINNEN, HICKS and FINK (1978). Bacterial transformation was per- formed using the calcium chloride procedure as described by MANIATIS, FRITSCH and SAMBROOK (1 982).

Restriction enzyme analysis: Restriction enzymes and buffers were purchased from International Biotechnologies Inc. Restriction enzyme analysis was carried out as described by MANIATIS, FRITSCH and SAMBROOK (1 982).

Nick translation of probes and DNA hybridization: Nick translation of probes was carried out using a nick translation reagent kit (BRL, Bethesda, Maryland). DNA hybridization was carried out by the method of SOUTHERN (1975). The filters containing the fragments of DNA were treated for thirty minutes at 42" in a solution of 50% formamide, 5X SSPE, and 1% Sarkosyl, before hybridiza- tion. The DNA was hybridized to heat denatured, nick translated probe in the same solution at 42" for 15 hr before washing four times (30 min, 50') in 0.1 X SSPE, 0.1 % SDS. The washed filter was dried and exposed to Kodak X-O- Mat R film with DuPont Cronex Hi-Plus intensifying screens at -70".

Colony hybridization: Colony hybridization was per- formed as described by ROTHSTEIN (1 985).

Inhibitors: Cycloheximide, antimycin, oligomycin, and nystatin were purchased from Sigma (St. Louis, Missouri). Sulfometuron methyl was obtained from the Agricultural Chemical Department of E. I. duPont de Nemours (Wil- mington, Delaware). Inhibitors were dissolved in dimethyl sulfoxide.

Quantitative measurement of drug resistance: T o deter- mine the effect of a particular inhibitor, 2-ml cultures of the strains to be studied were grown overnight to saturation. A volume of 0.2 ml of cells was suspended in 3 ml of 1 % agar and spread on the appropriate medium. T h e agar was allowed to harden before placing a 6-mm disc of filter paper containing the desired concentration of inhibitor in the center. T h e relative degree of drug resistance was deter- mined by comparing the diameter of the zone of inhibition at 48 hr. The values reported represent the average of two or more samples.

Genetic analysis: Random spore and tetrad analysis were carried out as described by SHERMAN, FINK and LAWRENCE (1974).

Construction of chromosomal insertion mutations: T o determine whether the PDR4 or PDR5 genes are essential for growth, chromosomal insertion mutations were created using the T n 5 bearing plasmids described in the results. A disruption in the PDR5 gene was constructed as follows. T h e plasmid carrying the Tn5-5 insertion in pDR3.3 (Figure

1) was linearized with Sac11 and used to transform a Ura- diploid strain (DBY 703 X RW 2802). This diploid lacks the 2-wm circle plasmid and therefore cannot support autono- mous replication of the plasmid. Integrative transformants were obtained in which one chromosome contained the wild

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pDR3.3

6

++++#+u

+.

E I’ E I’ E sa S c C P K E S

FIGURE 1 .-Location of restriction enzyme sa h‘ sites and T n 5 insertion mutations in pDR3.3

I 4 p B T Z 0 8

mosomal exchange between the direct repeats (and loss of one copy) were obtained by replica plating several thousand colonies to uracil emission medium and identifying Ura- segregants. The recombinants that were heterozygous for the Tn5 insertion were identified by Southern hybridiza- tion. T h e interruption ofPDR4 was carried out in analogous fashion, however the Tn5-F knockout mutation was first subcloned ino a YIp5 vector (BOTSTEIN et al. 1979). Follow- ing transformation of a ura3 diploid, Ura- segregants were selected on a 5-fluoro-orotic acid as described by BOEKE, LACROUTE and FINK (1984). T n 5 insertion heterozygotes were identified by SOUTHERN (1 975) hybridization and spor- ulated for subsequent analysis.

RESULTS

Cloning multidrug resistance genes by DNA am- plification: T h e strategy used to clone multidrug resistance loci was similar to that initially described by RINE et al. (1983). They showed that when cells are transformed with a high copy number plasmid con- taining the gene coding for yeast UDP-N-acetyl-glu- cosamine- 1 -p-transferase, resistance to tunicamycin is conferred. A similar approach was used by FALCO and DUMAS (1 985) to clone the yeast ZLV2 gene. Overpro- duction of acetolactate synthase, the product of the locus, results in resistance to the herbicide sulfomet- uron methyl, an inhibitor of this enzyme.

T o identify loci that confer multidrug resistance as a result of gene amplification, we screened a library that was generated by insertion of Sau3A partially digested yeast genomic DNA into the BamHI site of the high copy number, extrachromosomally replicat- ing plasmid, YEp24 (BOTSTEIN et al. 1979). Yeast cells (strain FY 138) containing this clone bank, selected as Ura+ transformants, were plated on cycloheximide medium. Resistant colonies appeared after 48 hr. One hundred were then screened for resistance to sulfo- meturon methyl. Six cross-resistant colonies were found.

T o determine whether the cross resistance was plas- mid mediated, the six strains were subcultured in nonselective (YEPD) medium. Under such conditions, the plasmid is lost from some cells in the culture and, after many generations, Ura- colonies derived from such cells were identified. In five of the six cases, all

and pDR3.1. T h e restriction sites indicated are as follows: EcoRI, E; PuuII, P; SalI, Sa; ClaI, C; Sacll, Sc; XhoI, X ; K p n I , K. T n 5 mutations that inactivate gene function are indicated by a V. T n 5 mutations that fail to inactivate the gene are indicated by V. T h e insert found in pDR3.3 is 14.7 kb, whereas that found in pDR3.1 is 8.0

kb.

-

1 kb

Ura- segregants were also sensitive to both cyclohex- imide and sulfometuron methyl. One strain remained drug resistant and was not analyzed further.

Structural analysis of the cloned DNA fragments: DNA was prepared from each of the five yeast clones and used to transform E . coli. Restriction enzyme analysis was performed on plasmid DNA extracted from each of the five E . coli transformants to deter- mine whether the clones contained different DNA fragments. T h e patterns produced from two clones, pDR3.3 and pDR2.5, were related, but unlike those from pDR3.1, pDR3.2 and pDR3.4. T h e latter group gave identical profiles with all enzymes used. Since yeast cells containing the YEp24 clone bank were grown in culture for many generations prior to plating on cycloheximide medium, the latter group of three almost certainly represents the repeated selection of the same plasmid insertion.

T h e plasmids pDR3.1 and pDR3.3 were chosen for further study since they carried two unrelated DNA fragments. Reintroduction of these plasmids into strain FY 138 resulted in cycloheximide and sulfomet- uron methyl resistance, confirming the presence of resistance determinants on the plasmids. Restriction enzyme maps of pDR3.1 and pDR3.3 are shown in Figure 1. T h e sizes of the DNA fragments cloned in the YEp24 vector were 8.0 and 14.7 kb pairs, respec- tively. Southern blot hybridization indicated that each of these fragments was present once in the yeast genome (not shown).

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TABLE 2

Zones of inhibition (cm) produced by various inhibitors

St!-ain

Sulfometuron- Antimycin Oligomycin Nystatin Cycloheximide methyl

FY 138 + YEp24 3.3 1.7 3.3 5.7 6.0

3.3 1.4 3.2 5.7 6.5

3.4 1.6 3.5 5.5 6.4

3.3 1.5

3.3 1.4

Average 3.3 1.5 3.3 5.6 6 . 3

FY 138

+

pDK3.1 3.4 1.8 3.2 3.4 6.0

3.4 1.1 3.3 3.5 6.0

3.6 1.8 3.2 3.2 6.0

3.4 1.5

Average 3.5 1.5 3 . 3 3.4 6.0

FY138

+

pDR3.3 3.6 1.3 3.2 5.0 5.1

3.5 1.3 3.2 4.8 5.2

FY 138

+

pDR3.3 1.3 3.2 4.5 5.2

Average 3.6 1.3 3.2 4.7 5.2

Samples of 10 mg/ml of each inhibitor were dissolved in dimethyl sulfoxide and 100 ng were applied to a 0.6-cm disc.

effect of the insertion. T h e T n 5 insertions defined a 0.7-kb region essential for the high copy-mediated resistance of pDR3.1 and an approximately 3-kb re- gion in pDR3.3 (Figure 1). These values represent a minimum estimate of the critical region. T h e results of the T n 5 analysis were further verified in the case of pDR3.3 by constructing a 6.5-kb KpnI deletion in pDR3.3 (named pGL706, see Figure 1). When present in high copy, pGL706 confers drug resistance at levels identical to pDR3.3. pGL706 was used in most of the subsequent experiments. T h e pleiotropic drug resist- ance genes of plasmids pDR3.1 and pDR3.3 have been named PDR4 and PDRS, respectively.

Phenotypic characterization of PDR4 and PDR5 genes: The drug resistance phenotypes of strain FY138 bearing plasmids pDR3.1 (PDR4), pDR3.3

(PDR5) or YEp24 are compared in Table 2. Five structurally and functionally unrelated inhibitors were tested: cycloheximide, sulfometuron methyl, nystatin, antimycin and oligomycin. T h e plasmids pDR3.1 and pDR3.3 conferred resistance to cycloheximide and sulfometuron methyl, as expected since these were used to select clones, but not to any of the other inhibitors. An ANOVA statistical test on the data was ambigous with regard to whether pDR3.1 increased resistance to sulfometuron methyl. It may be that our choice of dose was not sensitive enough since the resistance phenotype is easily detectable by replica plating to medium containing 1 pg/ml of this inhibi- tor.

T o determine if the multidrug resistance phenotype associated with the cloned PDR5 gene requires ampli- fication, pGL706 was introduced into FY 138 cells in both circular and linear form. When introduced in circular form, this plasmid replicates autonomously and in high copy number. Linearizing the plasmid

with Sac11 prior to transformation resulted in integra- tion of the plasmid into the chromosome by homolo- gous recombination. Integrative transformants (low copy) were distinguished from the extrachromosomal variety by the markedly increased stability of the Ura+ phenotype and confirmed by Southern hybridization. While transformants containing the high copy number autonomously replicating plasmid were resistant to both cycloheximide and sulfometuron methyl, inte- grative transformants were sensitive (Table 3).

T h e PDR4 gene was tested for its high and low copy phenotype in a slightly different manner. T h e high copy effect was monitored in strain DBY947 by trans- forming it with the original plasmid pDR3.1. T o obtain integrative (low copy) transformants, the PDR4

gene was subcloned into YIp5 (BOTSTEIN et al. 1979) to produce the plasmid pBT208. Since this plasmid contains no yeast origin of replication, transformants are obtained only when the plasmid is integrated into the chromosome by homologous recombination. T h e cycloheximide resistance phenotypes of these trans- formants are also found in Table 3. T h e integrative transformants, which bears PDR4 in two copies, is slightly more resistant than the untransformed con- trol, while the presence of the gene in high copy number leads to a considerably more resistant phe- notype. Thus, as in the case of PDR5, it is increased copy number of PDR4, rather than allelic variation in the cloned DNA that is responsible for drug resist- ance.

To determine whether the PDR4 and PDRS genes are essential for growth, chromosomal insertion mu- tations were created as described in MATERIALS AND METHODS. Each of the two insertion heterozygotes was

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TABLE 3

Phenotypes of integrative transformants

Zones of inhibition (cm)

Sulfometuron Cycloheximide methyl Strain (.io ng) (.io ng)

FY I38 3.8 4.5

FY 138

+

pGL706 4.3 5.0

FY 138

+

pGL706 4.0 4.7

FY I38

+

pGL706 3.3 1.6

DBY947

+

YEp24 4.4

DBY947

+

pBT208 4.1

DBY947

+

pDR3.I 3.3

(untransformed)

(integrant #2)

(integrant #8)

(autonomous high copy)

(integrant # I )

(autonomous high copy)

loids bearing either of the insertion mutations exhib- ited high spore viability with no indication of a 2:2 segregation pattern characteristic of a lethal mutation. Furthermore, in each case, Southern blot hybridiza- tion indicated that spores containing the integrated T n 5 element were viable (not shown). This evidence strongly suggests that these genes are not essential. However, the possibility that the insertion mutations do not completely inactivate the genes cannot be ruled out at this point.

T h e cycloheximide drug resistance phenotypes of segregants bearing a T n 5 insertion mutation were compared to wild-type controls. Replacement of the wild type PDR4 gene with the Tn5-F disruption did

not result in any phenotypic change (data not shown). In contrast, however, disruption of PDR5 produced a semidominant hypersensitive phenotype. This is shown in Figure 2. Cells containing the insertion mutation failed to grow after 72-96 hr on media containing low doses (0.5 pg/ml) of cycloheximide that are normally not inhibitory.

T h e segregation of the drug hypersensitivity phe- notype was monitored by tetrad analysis of a hetero- zygous diploid. Fifty-two tetrads were replicated to a medium containing 0.5 pg/ml cycloheximide; 2:2 seg- regation for hypersensitivity was observed in each tetrad. T o determine whether hypersensitivity co- segregated with the T n 5 insertion mutation, colony hybridization was carried out on 16 of the tetrads. Radiolabeled pBR322 containing a T n 5 transposon was used as a probe. All the spores showing the hypersensitive phenotype contained the T n 5 element, and all spores showing wild type levels of resistance did not, indicating that the hypersensitivity was due to the T n 5 insertion in PDR5.

Genetic mapping of PDR4 and PDRS. T h e chro- mosomal locations of the PDR4 and PDRS genes

Wild type

-

Tn5 insertion

-

" mutation

Heterozygote

-

FIGURE 2.-Hypersensitivity of T n 5 insertion mutations in PDR5. The photograph shows the results of replica plating strains grown on YPD medium containing 0.5 pg/ml cycloheximide. The plates were photographed after 72 hr. Top: wild-type (sensitive) strain. Middle: Tn5 hypersensitive mutation. Bottom: heterozygous diploid. All strains grow vigorously on nutrient medium (left). The sensitive strain grew on cycloheximide media, while the T n 5 inser- tion mutation renders a cell unable to grow at this low dose. The heterozygous diploid exhibits an intermediate phenotype.

were found using two genetic mapping techniques. First, the linkage group assignment was made by the rapid mitotic mapping procedure of FALCO and BOT- STEIN (1 983). Then, tetrad analysis was used to deter- mine the location of each region relative to other known genes. Both methods require integration of the plasmids pDR3.1 and pGL706 plasmids into the chromosome by homologous recombination as de- scribed above. Integrative transformants were crossed to a series of genetically marked mapping stocks

(KLAPHOLZ and EASTON-ESPOSITO 1982). Because the

integrated plasmid still retains a copy of the 2-pm circle inverted repeat sequences, the chromosome containing the insertion is subject to heightened mi- totic recombination and/or chromosome loss. As a result, recessive markers originally present in hetero- zygous configuration are rendered homozygous. T h e change in phenotype for genes on a particular chro- mosome indicates the presence of the 2 pm plasmid in that homolog. T h e results of this analysis indicated that the PDR4 gene present in pDR3.1 mapped to chromosome XZIZ while the PDR5 gene present in pGL706 was located on chromosome XV between ADE2 and HZS3.

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TABLE 4

Genetic mapping of pDR3.1 (PDRI)

Cross Pertinent genotype LYS7-PDR3" LEUP-PDR3'

JG447 X FY201 LEU2 pBT208 (PDR#::URA3) LYS7 N P D P D TT N P D P D TT

leu2 lys7 2 8 1 33 23 15 13

The map distance of 3 1.5 cM is computed from the formula

cM = 6 (NPD)

+

TT

2 total asci .

*

Centromere linkage is approximately 9 cM. When data are corrected for the distance of LEU2 to the centromere (4 cM), the remaining tetratypes are an indication of a crossover between PDR4 and its centromere.

T h e location of the integrated pGL706 plasmid raised the possibility that PDR5 was allelic to SMR3, alleles of which are known to confer multiple drug resistance (FALCO et al. 1986). The SMR? locus was defined previously by several independently isolated sulfometuron methyl resistant mutants which were shown to map on chromosome XV between ADE2 and HZS3. Complementation tests revealed two comple- mentation groups indicating the presence of two tightly linked genes or alternatively, intragenic com- plementation (FALCO and DUMAS 1985). Three mu- tations in the first complementation group (repre- sented by smr3-37) lead to resistance to sulfometuron methyl only. Four mutations in the second group (represented by smr3-33) cause a multidrug resistance phenotype (FALCO et al. 1986). T h e genetic relation- ships between the mutations, smr3-33, smr3-37 and SMR3-73 (a dominant multidrug resistance mutation mapping in this region), and the PDRS gene cloned on pDR3.3 were determined by complementation tests and tetrad analysis.

T h e first experiment explored the genetic linkage between mutations assigned to the two complemen- tation groups by crossing the smr3-33 and smr3-37 mutants. If the two mutants were separated by ex- change, tetratype and nonparental ditype asci would have one and two sensitive spores, respectively. T h e data, found in Table 5 show a map distance of 6.4 cM indicating that these mutations are in two different genes. The dominant multidrug resistant mutation SMR3-73 was tightly linked to the recessive multidrug resistance mutation smr3-33, suggesting that these mutations are allelic. Thus, by the criteria of comple- mentation and crossing over, two genes are defined by the mutation smr3-37 and the pair smr3-33 and SMR3-73. T h e first retains the name SMR3, and the second has been designated P D R 2 (as previously sug- gested by BALZI et al. 1987). Thus, the smr3-33 and SMR3-73 alleles have been renamed pdr2-33 and

T o determine whether the multidrug resistance gene cloned in pDR3.3 was allelic to either of the two PDR2-73.

genes described above, two approaches were taken. In the first experiments, the pDR3.3 subclone,

pGL706, was integrated into ura3, pdr2-33 (previ- ously smr3-33) and ura3, smr3-37 mutant strains. Such transformants remained drug resistant, indicating that the DNA fragment on pGL706 could not complement either of these recessive mutants. In the second ex- periment, the URA3 on the plasmid served as a marker to allow pGL706 (and thus the drug resistance gene that served as its site of integration) to be followed in subsequent crosses, while the sulfometuron methyl resistance was used to monitor either of the two mutant alleles. If the plasmid-borne multidrug resist- ance gene was not allelic to either smr3-37 or pdr2- 33, sulfometuron resistant, Ura- and sulfometuron methyl sensitive, Ura+ recombinants would be found. Such recombinants were observed in both crosses (Table 5) indicating that the plasmid-borne PDR5 gene was distinct from each of these loci. PDRS was located 5.8 cM from pdr2-33 and 10.9 cM from smr3- 3 7.

In a third experiment, the cycloheximide hypersen- sitivity of the T n 5 insertion mutation was used as a genetic marker. A haploid containing the T n 5 inser- tion (JG334) was mated to a strain bearing the pdr2- 33 mutation (JG277). Tetrads were screened for cy- cloheximide hypersensitivity and sulfometuron methyl resistance. T h e two phenotypes were found to be separable by recombination. In thirty tetrads ana- lyzed, there were three tetratype asci indicating a distance of about 5 cM, which is consistent with that determined using the URA3 gene in pGL706 as a marker (Table 5).

In summary, the genetic experiments described above define three loci by the criteria of complemen- tation and crossing over: a gene represented by smr3- 3 7 , a second gene represented by pdr2-33 (and PDR2- 7 3 ) and a third gene, P D R 5 , carried on pGL706.

DISCUSSION

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TABLE

Genetic analysis of the SMR3 region

~~

Tetrads scored

Cross Pertinent genotype P NPD TT Map distance

JG277 X JG272 pdr2-33 41 0 6 6.4

JG279 X JG273 PDR2-73 44 0 0

JG327 X JG253 pCL706 (PDR5:URA3),pdr2-33 46 0 6 5.8

JG328 X JG28I pGL706 (PDR5:URA3),smr3-37 54 0 15 10.9

JG334 X JG277 pdr2-33 27 0 3 5.0

smr3-37

pdr2-33

PDR2

SMR3

PDR5:Tn5

The pdr2-33 and PDR2-73 mutations were originally designated as smr2-33 and SMR2-73.

multidrug resistant phenotype when they are present as part of an autonomously replicating high copy plasmid. Both genes, designated PDR4 and PDR5, are present in single copy in the yeast genome of wild- type sensitive cells. PDR4 is located on chromosome XZZI, 3 1.5 cM from LYS7 and 9 cM from the centro- mere. PDRS is on chromosome XV between ADE2 and H l S 3 ; it is tightly linked to, but separable from, two other drug resistance genes, SMR3 and PDR2. Inac- tivation of either the PDR4 or PDRS sites by insertion mutation is not lethal to the cell. However, a marked drug hypersensitivity is observed for the latter. Mild hypersensitivity was also obtained when deletions of the PDRl locus were analyzed (BALZI et al. 1987).

T h e clustering of loci involved in drug resistance on chromosome XV was unexpected. T h e genetic mapping of the PDRS gene to chromosome XV be- tween ADE2 and HIS3 initially suggested that the cloned gene was the wild type allele of the previously identified drug resistance gene SMR3 (FALCO and DUMAS 1985). T h e simplest interpretation of the ge- netic experiments described in this report is that there are, in fact, three different genes located between ADE2 and HIS3 involved in the cellular response to environmental chemicals. Other explanations, for ex- ample a single complex gene with large introns, are possible. Such regions have been found in animal cells, but they are unprecedented in yeast. In addition, the restriction map of T n 5 insertion mutations that inac- tivate the PDRS high copy resistance gene suggests that a single continuous 3-kb region is necessary and sufficient for function. Furthermore, trans-splicing or intragenic complementation would be required to ex- plain the complementation results. Sequencing of PDRS should further aid in determining the exact organization of this region as well as the possible relation to mammalian drug resistance genes.

It is interesting to note that mammalian multiple drug resistance genes are also clustered (STARK 1986).

Furthermore, the P D R l locus of yeast is complex and may be composed of several linked genes. Mutations that map to this region on chromosome VZZ show varying linkage to other markers (MORTIMER and SCHILD 1980), wide differences in drug resistance phenotype (SAUNDERS and RANK 1982) and comple- mentation (FALCO and DUMAS 1985). In addition to the P D R l gene, another gene, PMA1, which encodes a plasma membrane ATPase, has been cloned and mapped to this region (SERRANO, KIELLAND-BRANDT and FINK 1986). Mutations in PMAl also lead to a multidrug resistant phenotype (ULASZEWSKI, GREN-

SON and GOFFEAU 1983). Thus, at least two genes,

wherein mutations leading to a multidrug resistance phenotype have been found, are tightly linked on chromosome VZZ. A similar situation also exists with regard to the PDR3 locus on chromosome

ZZ

(SUBIK, ULSAZEWSKI and GOFFEAU 1986). T h e nucleotide se- quences of the two genes do not show any obvious similarity, nor do the deduced amino acid sequences of the encoded polypeptides suggest any functional relatedness. Possible interactions between various PDR genes are being explored by making combina- tions of mutant alleles and by searching for suppressor mutations.

Amplification of the PDR4 or PDRS genes imparts resistance to cycloheximide and sulfometuron methyl, agents that inhibit protein synthesis and isoleucine and valine production, respectively. T h e chemical structures of these two compounds do not show ob- vious similarities, other than the presence of an imide group. A multiple drug resistance phenotype to un- related inhibitors could be due to at least two different mechanisms already documented in the literature. Resistance could be brought about by changes in the membrane structure so that inhibitors are excluded. For example, various Pseudomonad mutants (HAN-

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multidrug resistant lines have a more efficient, energy- dependent export of inhibitors (SKOSGAARD 1978; FOJO et al. 1985). At present no biochemical experi- ments have been done to reveal which of these, if either, is the mechanism operating in yeast. The as- sociation of a resistance phenotype with amplification of the PDRS gene and hypersensitivity with disruption of the gene is consistent with it encoding a “pump” which exports the inhibitors. Alternatively, the PDRS gene product might be a positive activator of the Pump.

Supported by grant GM41875 from the National Institute of General Medical Sciences.

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Figure

TABLE 1 Yeast  strains  and  plasmids
TABLE 2
TABLE 3
TABLE 5 Genetic analysis of the SMR3 region

References

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