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Sensitivity to Saccharomyces Cerevisiae (Coxsb/Hypoxlc Gee/Aerobk Repression/High Mobiity Group Box) JAMES R

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Sensitivity to Saccharomyces Cerevisiae (Coxsb/Hypoxlc Gee/Aerobk Repression/High Mobiity Group Box) JAMES R Proc. Nati. Acad. Sci. USA Vol. 91, pp. 7345-7349, July 1994 Genetics The ORDI gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae (COXSb/hypoxlc gee/aerobk repressIon/hIgh mobiity group box) JAMES R. LAMBERT, VIRGINIA W. BILANCHONE, AND MICHAEL G. CUMSKY* Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 Communicated by Stephen J. Lippard, March 18, 1994 (receivedfor review December 23, 1993) ABSTRACT The yeast COX5a and COXSb genes encode gen-dependent processes (respiration, sterol synthesis, oxida- isoforms of subunit Va of the mitochondrial inner membrane tive damage repair), and several, like the COXS genes, exist as protein complex cytochrome c oxidase. These genes have been pairs inversely regulated by oxygen and heme (8, 9). shown to be inversely regulated at the level oftranscription by Several upstream elements that regulate the expression of oxygen, which functions through the metabolic coeffector the COX5b gene have been identified (5). These include two heme. In earlier studies we identified several regulatory ele- sites of positive control (activation elements or UASs) and ments that control tnscriptional activation and aerobic re- three sites ofnegative control (repression elements or URSs) pression of one of these genes, COX5b. Here, we report the that mediate aerobic repression. Two of the repression ele- isolation of trans-acting mutants that are defective in the ments contain the consensus sequence ATTGTTCT, which aerobic repression of COXSb transcription. The mutants fall is found upstream of most hypoxic genes and appears to be into two complementation groups. One group species ROXI, the binding site for the ROXI repressor protein (8, 10). The which encodes a product reported to be involved in transcrip- third repression element contains a 13-bp sequence TCGT- tional repression. The other group idented the gene we have TCGTTGCCT, which is also found upstream of several desiguated ORDI. Mutations in ORDI cause overexpression of hypoxic genes (5, 10). COX5b aerobically but do not affect the expression of the In we hypoxic genes CYC7, HEM13, andANBI. ORDI mutations also this study searched for trans-acting factors that are do not affect the expression ofthe aerobic genes COX5a, CYCI, involved in the aerobic repression ofCOX5b. Using a genetic ROXI, ROX3, and TIF51A. The yeast genome contains a single approach, we isolated mutants in which COX5b was over- ORDI gene that resides on chromosome XI. Strains caring expressed under aerobic growth conditions. The mutants fell chromosomal deletions ofthe ORDI locus are viable andexbit into two complementation groups. (i) The first corresponds to phenotypes similar to, but less severe than, that of the original the previously identified ROXI gene. (ii) The second defines mutant. The nucleotide sequence of ORD! revealed that It is another gene, which we call ORD]. The ORD) gene was identical to IXRI, a yeast gene whose product contains two high cloned, and its DNA sequence was determined; it specifies a mobility group boxes, binds to platinated DNA, and confers polypeptide of 67.2 kDa that contains two high mobility sensitivit to the antitumor drug cisplatin. Consistent with the group-boxes and several polyglutamine tracts. Surprisingly, latter observations, we found that the ORD1 product could we show that ORD] is identical to a recently described gene, bind to both the upstem region of COXSb and to DNA IXRJ, which has been shown to confer sensitivity to the modified with cisplatin. antitumor drug cisplatin (11).t Cytochrome c oxidase is a heterooligomeric protein complex MATERIALS AND METHODS located in the mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, the cytochrome oxidase complex Yeast Strains and Growth Conditions. The Saccharomyces is composed of 12 nonidentical subunits (1). The genes cerevisiae strains constructed in this study were as follows: COXSa and COXSb specify functionally interchangeable, yet JM43-GD5a/ord1 (MATa his4-580 trp)-289 leu2-3, 112 ura3-S2 structurally distinct, forms of subunit V of the yeast enzyme coxfaA::URA3 ordlA::LEU2) and JL1-20c (MATa his4-580 (refs. 2, 3; referred to as subunits Va and Vb, respectively). leu2-3, 112 ura3-52 cox aA::hisG ord -1). Additional strains Previous work has shown that the yeast COXS genes are used included JM43, JM43-GD5a, JM43-GD5ab, JM6, inversely regulated at the level of transcription by oxygen, BMH281, CT39-7B, and CT149-3D, which have been de- which exerts its effect through a metabolic coeffector, heme scribed (2, 3, 6, 7, 12). Yeast strains were grown aerobically (4-7). Specifically, during aerobic growth the presence of at 300C in either yeast extract/peptone/dextrose (YPD), yeast oxygen permits heme biosynthesis; heme then interacts with extract/peptone/glycerol/ethanol (YPGE), or SD medium one or more regulatory proteins to activate the transcription of (supplemented with amino acids as necessary; ref. 13). Res- COXSa and repress the transcription of COXSb (4-7). Under piratory proficiency was tested with YPGE medium, which is anaerobic or hypoxic conditions (low oxygen tension), when nonfermentable. heme is limiting, COXSa is not transcribed, whereas transcrip- Genetic Methods and the Isolation of Mutants That Over- tion of COXSb derepresses 5- to 20-fold (4, 5). It has become express COX5b Aerobically. Strain construction, matings, clear that the COXS genes are part of a family of physiologi- and tetrad standard cally important yeast genes whose expression is controlled sporulation, analysis were done by using either positively or negatively by oxygen and/or heme. These yeast genetic techniques (13). Mutants overexpressing genes share several common features: most function in oxy- Abbreviations: UAS, upstream activation site; URS, upstream re- pression site; HMG, high mobility group. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper was previously deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (under IXRI; accession no. L16900). 7345 Downloaded by guest on September 24, 2021 7346 Genetics: Lambert et al. Proc. Natl. Acad. Sci. USA 91 (1994) COXSb aerobically were selected by the ability ofyeast strain ofboth subunit V genes; because aformofsubunit V is essential JM43-GD5ab(YCp5b) to grow on YPGE plates (see Results). for cytochrome oxidase activity, which is, in turn, essential for Approximately 100 spontaneous mutants were selected; respiration, this strain cannot grow on nonfermentable sub- these displayed a wide variety of growth rates on YPGE. strates (4). JM43-GD5ab strains expressing COXSb from cen- Mutants displaying the best growth rates on YPGE were tromeric or low-copy plasmids, like YCpSb, also fail to grow subjected to a cis/trans test that consisted of curing the under these conditions due to the low level of aerobic COXSb mutant of its plasmid and then retransforming the plasmid- expression (4). Thus, these strains must overexpress COXSb to free isolate with a fresh preparation of vector YCp5b. Those grow on nonfermentable substrates. mutants retaining the ability to grow on YPGE medium after Spontaneous mutants were identified by their ability to retransformation were considered to carry trans-acting, chro- grow on YPGE medium. Initially, >100 prospective mutants mosomal mutations. Trans-acting mutants were then mated were characterized. From these, five strains carrying reces- to yeast strain BMH281 to determine whether the mutations sive, trans-acting mutations were chosen for further study. they carried were dominant or recessive. Recessive muta- Each of these mutants was stable, grew rapidly on YPGE tions are complemented in the diploid, which results in a medium, and, from the results ofNorthern blot analysis, had respiratory-deficient phenotype observed as the lack of elevated levels of COX5b mRNA when grown aerobically growth on YPGE medium. Five recessive, trans-acting mu- (data not shown). Each mutant was also analyzed by South- tants were ultimately chosen for further study. For comple- ern blotting, which confirmed that centromere function on mentation analysis, appropriately marked derivatives ofeach the plasmid was normal and that the COXSb gene had not mutant were constructed through standard genetic tech- niques. The complementation test consisted of mating each been amplified (data not shown). mutant in pairwise combinations and scoring the diploids for Appropriately marked derivatives of each mutant were growth on YPGE medium. Mutants that produced diploids mated in pairwise combinations to test for complementation, capable of growth on this medium were considered to be in which was defined as the failure ofthe resulting diploid strain the same complementation group. to grow on YPGE medium. Two complementation groups Cloning the ORDB Structural Gene. The ORD) gene was were identified. Because yeast strains with mutations in either cloned by functional complementation ofthe ordl-I-encoded the ROXI, ROX3, ROX4 (also called TUPI andAER2), orthe phenotype (in JL1-20c) using a yeast genomic library in the REOM genes might also be expected to cause overexpression vector YCp5O (14). Ura+ transformants were first selected on of COX5b (4, 6, 8, 12), we tested whether
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