YEAST VOL. 12: 999-1004 (1996)

0 0 0 xv % 0 Yeast Sequencing Reports 0 o o o o

Sequence of 29 kb around the PDRlO Locus on the Right Arm of Saccharomyces cerevisiae Chromosome XV: Similarityto Part of Chromosome I ANNE G. PARLE-McDERMOTT, NICHOLAS J. HAND?, SARAH E. GOULDING AND KENNETH H. WOLFE*

Department of Genetics, University of Dublin, Trinity College Dublin 2, Ireland

Received 12 January 1996; accepted 19 February 1996

We report a 29,445 bp sequence from the right arm of yeast chromosome XV. It contains the genes M Y 0 2 , SNC2, PDRIO, SCDS (also called FTBl), MIPI, VMA4, MRS2, ALAI , KRES, TEAI, and a homologue of YALO34c. Several discrepancies with previously published sequences were found. PDRIO encodes a protein highly similar to the pleiotropic drug resistance protein Pdr5p. This sequence contig forms part of a region of extended similarity to part of the left arm of chromosome I, which is a relic of an ancient duplicated chromosomal region.

KEY W O R D S - M Y O ~ ; SNC2; PDRlO; SCDS; FTBl: MIPI; VMA4; MRS2; ALAI; KRES; TEAl; YALO34c; duplicate chromosomal region; ABC transporter

INTRODUCTION We report the sequence of a 29 kb region from the right arm of Saccharomyces cerevisiae chromo- some XV, determined as part of the European collaborative project to sequence this chromo- some. The sequence is derived from parts of two cosmids: pUOAS02 and pEOA232. The ends of the contig reported here overlap with adjacent cosmids sequenced in the laboratories of M. Schweizer (Institute of Food Research, Norwich, UK; cosmid pEOA284; Pearson et al., 1996) and A. Goffeau (UniversitC Catholique de Louvain, Belgium; cosmid pEOA138; B. Purnelle et al., in preparation).

?Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. *Corresponding author: Kenneth H. Wolfe, Department of Genetics, University of Dublin, Trinity College, Dublin 2, Ireland. (Telephone +353-1-608-1253; Fax +353-1-679-8558; e-mail khwolfe@tcd.ie){end}

MATERIALS AND METHODS Cosmids pEOA232 and pUOA502 from chromo- some XV of Saccharomyces cerevisiae strain FY1679, isogenic to S288C, were provided by B. Dujon (Institut Pasteur). Cosmid pEOA232 was fragmented by sonication and subcloned into pB- luescript SK - (Stratagene). Randomly chosen subclones were sequenced from both ends using an Applied Biosystems model 373A automated DNA sequencer with dye-labelled primers (Genpak Ltd, Sussex, UK). Subclones of pOUA502 in pBlue- script SK were made after DNAase I or Sau3AI digestion and sequenced at random. When it was realized that the two cosmids overlap substan- tially, the more directed strategy of making exo- nuclease I11 deletion series of some subclones (Henikoff, 1987) was used to complete the se- quence of the non-overlapping region (positions 1-5836 in the contig). A total of 63.5 sequence reads were assembled into the contig, including data from 40 oligonucleotide primers used to close

CCC 0749-503X/96/100999-06 1996 by John Wiley & Sons Ltd

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SNCZ SCDS M I P l ALAl

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chromosome I (same scale)

SNCl YAL YAL YAL YAL 033 034A 035 037

Figure 1. Gene map of the sequenced region from chromosome XV, and comparison to part of the left arm of chromosome I (Bussey et al., 1995). Genes drawn above the horizontal line are transcribed from left to right. Vertical lines indicate homologous gene pairs. The dashed line indicates the presence of a YAL037 homologue in the region to the right of the sequenced contig

gaps. Sequence assembly was carried out using the Staden XBAP program on a Sun workstation. We have previously released the sequence of the PDRlO region to the EMBL(GenBank/DDBJ databases with accession numberfZ49821.

Protein sequence alignments were made using ClustalW (Thompson et al., 1994), which employs the neighbour-joining method of Saitou and Nei (1987) for phylogenetic tree construction. Trees were visualized using NJPLOT software (M. Gouy, unpublished).

RESULTS AND DISCUSSION A sequence of 29,445 nucleotides was determined, containing nine complete and two incomplete genes. The locations and orientations of genes in the region are indicated in Figure 1.

Nine old genes Nine of the 11 genes in this region have pre-

viously been identified by other laboratories and are summarized in Table 1. There are several conflicts between our sequence and those deter- mined previously by other groups, including frameshift differences in KRES and A L A I , mul- tiple amino acid replacements in MIPI, and more minor changes to SNC2, VMAI , MRS2 and TEAl proteins. We have checked our sequence carefully at all positions where it is in conflict with database entries (from GenBank release 92; December 1995). The differences in the MIPl sequence (ten amino acid replacements in 1254 residues) can

almost certainly be attributed to strain differences because the original clone of the MIPl gene, isolated by Foury (1989) from yeast strain C1378b, has been extensively re-sequenced in the course of analysis of mutant alleles generated by mutagen- esis of the cloned gene (Foury and Vanderstraeten, 1992; Hu et al., 1995).

A new gene: PDRIO An open reading frame (ORF) of 1564 codons is

predicted to be a member of the ABC (ATP- binding cassette) transporter family, with 12 transmembrane domains and two ATP-binding cassettes. A database search revealed that this gene is highly similar to the pleiotropic drug resistance gene PDR.5, which is also situated on yeast chro- mosome XV. The sequence identity between the two proteins is 68Yo over their entire length. The new gene reported here was designated as PDRlO by Balzi and Goffeau (1 994) on the basis of its high similarity to PDR.5; we hypothesize that it repre- sents a new pleiotropic drug resistance locus.

The ABC transporters form a large family with several hundred known members from prokary- otes and diverse eukaryotes (Dean and Allikmets, 1995). Within this family, PDRlO and several other fungal ABC transporters form a distinct group of related sequences that are similar even outside the ATP-binding cassettes that are the hallmark of the entire family. Database searches using the BLAST algorithm (Altschul et al., 1990) suggested that these fungal proteins form a group and are only distantly related to other ABC

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transporters from yeast such as STE6 and YCFl (for example, PDRlO shows greater similarity to the white gene of Drosophila than to YCFl).

Phylogenetic analysis of this subfamily of ABC transporters (Figure 2) groups PDRlO with PDRS and an uncharacterized ORF from chromosome IV (systematic name D9509.24) which has been termed PDRIS (Dietrich et al., 1995). Among these three, PDRS and PDRIS are slightly more closely related to each other than they are to PDRlO. The CDRl sequence from Candida albi- cans (Prasad et al., 1995) falls outside the three Succharomyces sequences (Figure 2), which sug- gests that gene duplication has occurred twice in Succhuromyces after its divergence from Candida, although it is possible that more closely related genes from Candida exist but have not yet been cloned.

Four other Saccharomyces ABC transporter genes (SNQ2 and three ORFs identified by genome projects) can be placed in this subfamily (Figure 2). Two of these ORFs (OR26.01 and YILO13c, which is also known as PDRII) are particularly closely related (70% protein sequence identity). The sub- family also includes a brefeldin A resistance gene from the fission yeast, Schizosaccharomyces pombe (Turi and Rose, 1995; Nagao et al., 1995), which is approximately equidistantly related to the two groups of Saccharomyces genes (the PDRI CDRl group and the SNQ2ILPEl4cIYILO13cI OR26 01 group). This indicates that this subset of ABC transporters is very old, predating the SuccharomyceslSchizosacchuromyces split.

hba2 (Schizosaccharomyces pombe) X82891

0.527 o'130 YILO13c (PDRl1) (IX) 247047

655 0.396

PDRS was independently isolated by four lab- oratories and named PDRS (Balzi et al., 1994), STSl (Bissinger and Kuchler, 1994), YDRl (Hirata et al., 1994) or LEMl (Krdlli et a/., 1995). The Pdr5p protein is consequently the best charac- terized drug resistance ABC transporter in yeast. Overexpression of PDRS produces resistance to multiple drugs including cycloheximide, spori- desmin, sulfometuron methyl, and other struc- turally unrelated drugs. Disruption of this gene results in drug hypersensitivity.

Expression of PDRS is controlled by two tran- scription factors, Pdrlp and Pdr3p, and at least one of these factors is required for PDRS-mediated cycloheximide resistance (Meyers et al., 1992; Balzi et al., 1994; Katzmann et al., 1994; Dexter et al., 1994). Overproduction of Pdrlp and Pdr3p also gives rise to resistance to other drugs such as oligomycin, but the resistance to oligomycin is not due to PDRS (Meyers et al., 1992; Katzmann et al., 1994), which indicates that Pdrlp and Pdr3p mediate this resistance via another gene. PDRlO is an obvious candidate, and Balzi and Goffeau (1995) have proposed that PDRlO may be regu- lated by Pdrlp. The PDRlO promoter sequence does not, however, contain the palindromic octa- nucleotide TCCGCGGA found at Pdr3p binding sites in the promoters of PDRS and PDRS itself (Katzmann et al., 1994; Delahodde et al., 1995). The binding site specificity of Pdrlp is not so well characterized, though it may be identical to that of Pdr3p (Balzi and Goffeau, 1995; Delahodde et al., 1995).

29 kb FROM RIGHT ARM OF CHROMOSOME XV

The other new gene discovered in this study is a homologue of YALO34c, a gene of unknown func- tion on chromosome I (Bussey et al., 1995). Our sequence of this gene is substantially incomplete so it will not be discussed here; the complete gene has been sequenced and analysed in the laboratory of A. Goffeau (B. Purnelle et al., in preparation).

Similarity to part of chromosome I Three genes in this portion of the right arm of

chromosome XV (MYO2, SNC2 and the YALO34c homologue) have homologues on the left arm of chromosome I (MY04 , SNCl and YALO34c; Bussey et al., 1995). The homologous genes on the two chromosomes are arranged in the same rela- tive order and transcriptional orientation, but they are interspersed with genes that are unique to one or other chromosome (Figure 1). The situation must have arisen through duplication of a large chromosomal region (>30 kb), followed by diver- gence of gene content through loss of redundant duplicates from one or other chromosome, or by transposition of other genes into one of the daugh- ter regions formed by the duplication. SNCl con- tains an intron but SNC2 does not, so an intron must have been lost or gained in one of these genes subsequent to the duplication.

The similarity between chromosomes XV and I extends beyond the sequence contig reported in this paper. In the region immediately to the left of M Y 0 2 (Pearson et al., 1996) there are two genes (06159 and PMT3) that are homologues of genes to the left of M Y 0 4 on chromosome I (YAL028w and PMT2). Continuing leftwards, the similarity to chromosome I ends and instead a region of extended similarity to chromosome XI11 is found (Pearson et al., 1996). Our preliminary shotgun sequence data from the region further to the right of the contig reported here indicates that a homo- logue of the chromosome I gene YAL037w is present. The region of similarity therefore includes at least six duplicated genes, out of a 16-gene interval on chromosome I (PMT2 to YAL037w) and a 216-gene interval on chromosome XV (PMT3 to the YAL037w homologue).

This “mosaic’ pattern of duplicate chromosomal regions has been seen elsewhere in the yeast genome. We have previously found a smaller re- gional similarity around the centromeres of chro- mosomes I1 and IV (Wolfe and Lohan, 1994); there is also similarity between centromere regions of chromosomes 111 and XIV (Lalo et al., 1993), and a large region is duplicated between chromo-

1003

somes I and VIII (Johnston et al., 1994; Bussey et al., 1995). We are currently conducting a systematic analysis of this phenomenon.

ACKNOWLEDGEMENTS We are grateful to David Noone and Niamh Nolan of the TCDINPBC DNA sequencing fa- cility for technical assistance; Andrew Lloyd and INCBI for computing assistance; Amanda Lohan for moral support and the final three nucleotides; Andre Goffeau, Karl Kuchler and Sandy Lemmon for discussions; and our neighbours on chromo- some XV (Michael Schweizer and Benedicte Purnelle) for sharing unpublished data.

REFERENCES Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and

Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403410.

Balzi, E. and Goffeau, A. (1994). Genetics and biochem- istry of yeast multidrug resistance. Biochim. Biophys.

Balzi, E. and Goffeau, A. (1995). Yeast multidrug resist- ance: the PDR network. J. Bioenerg. Biomembr. 27, 71-76.

Balzi, E., Wang, M., Leterme, S., Van Dyck, L. and Goffeau, A. (1994). PDRS, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDRI. J. Biol. Chem. 269, 2206-22 14.

Bissinger, P. H. and Kuchler, K. (1994). Molecular cloning and expression of the Saccharomyces cere- visiae STSl gene product. J. Biol. Chem. 269, 4180- 4186.

Bussey, H., Kaback, D. B., Zhong, W., et ul. (1995). The nucleotide sequence of chromosome I from Saccharo- myces cerevisiae. Proc. Natl. Acad. Sci. USA 92, 3809-38 13.

Dean, M. and Allikmets, R. (1995). Evolution of ATP- binding cassette transporter genes. Curr. Opin. Genet. Devel. 5, 779-785.

Delahodde, A,, Delaveau, T. and Jacq, C. (1995). Posi- tive autoregulation of the yeast transcription factor Pdr3p, which is involved in control of drug resistance. Mol. Cell. Biol. 15, 40434051.

Dexter, D., Moye-Rowley, W. S., Wu, A. and Golin, J. (1994). Mutations in the yeast PDR3, PDRI, PDR7 and PDRY pleiotropic (multiple) drug resistance loci affect the transcript level of an ATP binding cassette transporter encoding gene, PDRS. Genetics 136, 505-515.

Dietrich, F. S., Mulligan, J., Allen, E., et al. (1995). The sequence of Saccharomyces cerevisiae chromosome IV right. EMBL/GenBank/DDBJ accession number U32274.

Acta 1187, 152-162.

1004 A. G. PARLE-McDERMOTT ET AL.

Nagao, K., Taguchi. Y.. Arioka, M.. rt 01. (1995). bfrl +, a novel gene of Sc/ri-osacc/iciroi~i~ces yombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily. J. Bucteriol. 177. 1536-1543.

Nelson, K. K., Holmer, M. and Lemmon, S. K. (1996). SCDS, an essential gene, encodes a novel protein involved in vesicular transport at a late stage of the secretory pathway. Mol. Biol. Cdl , in press.

Pearson, B. M.. Hernando, Y., Payne, J.. Wolf, S. S.. Kalogeropoulos, A. and Schweizer, M. (1996). Se- quencing of 35,710 kb DNA segment on the right arm of yeast chroniosonie XV reveals a region of extended similarity to chromosome XIII, 1 1 novel open reading frames. two of which contain putative introns. Yeast, manuscript submitted.

Prasad, R., de Wergifosse, P., Balzi, E. and Goffeau, A. (1995). Molecular cloning and characterization of a novel gene of Crirididci nlbicans, CDRI, conferring multiple resistance to drugs and antifungals. Ciirr. Grtier. 27. 320-329.

Protopopov, V., Govindan, B., Novick, P. and Gerst. J. E. (1993). Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiiie. Cdl 74. 855-861.

Ripmaster. T. L.. Shiba. K. and Schimmel, P. (1995). Wide cross-species aminoacyl-tRNA synthetase re- placement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proc. Nirtl. Acad. Sci. USA 92, 49324936.

Saitou. N . and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylo- genetic trees. Mol. Biol. E i d . 4. 306425.

Song, J. M., Cheung, E. and Rabinowitz, J. C. (1996). Cloning and characterization of the Saccliiironijces i~crevisicir FTBI gene. GenBank/EMBL/DDBJ acces- sion number U42227.

Thompson, J. D., Higgins, D. G. and Gibson. T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through se- quence weighting, position specific gap penalties and weight matrix choice. Nucl. Acic1.T Rrs. 22. 46734680.

Turi. T. G. and Rose, J. K. (1995). Characterization of a novel Schi-osacchnr.otiz~ces pottzbe niultidrug resist- ance transporter conferring brefeldin A resistance. Biocheni. Bioplijx Res. Cotnrtiiin. 213, 4 1 0 4 1 8.

Wiesenberger. G., Waldherr, M. and Schweyen, R. J. (1992). The nuclear gene MRS2 is essential for the excision of group I1 introns from yeast mitochondrial transcripts in vivo. J. Biol. Clreni. 267, 6963-6969.

Wolfe, K. H. and Lohan. A. J. E. (1994). Sequence around the centromere of Sncclraroniycrs c.crevisiar chromosome 11: similarity of CEN3 to CEN4. Yeast 10, S41-S46

Foury, F. (1989). Cloning and sequencing of the nuclear gene MIPl encoding the catalytic subunit of the yeast hitochondrial DNA polymerase. J. Bid. Chcm 264, 20552-20560.

Fo~iry. F. (1990). The 31-kDa polypeptide is an essential subunit of the vacuolar ATPase in Sr/cc/ziirorii~.i.es c.cwviJiae. J . Biol. Clzetti. 265, 18554-18560.

Foury, F. and Vanderstraeten, S. (1992). Yeast mito- chondrial DNA mutators with deficient proofreading exonucleolytic activity. EMBO J. 11, 271 7-2726.

Gray, W. and Fassler. J. S. (1995). Isolation and analysis of the yeast TEA1 gene encoding a zinc cluster Ty enhancer binding protein. EMBL/GenBank/DDBJ accession number U37696.

Henikof. S. ( 1987). Unidirectional digestion with exonticlease I11 i n DNA sequence analysis. Meth. Eri:ytiu/. 155, 156-165.

Hirata, D.. Yano, K., Miyahara. K. and Miyakawa, T. ( 1994). Succ~lznror?i~~c.e.c cuevisiur YDRI. which en- codes a member of the ATP-binding cassette (ABC) superfamily. is required for inultidrug resistance. Cwr. Gcnrt. 26, 285-294.

Hu. J. P., Vanderstraeten. S. and Foury, F. (1995). Isolation and characterization of ten mutator alleles of the mitochondrial DNA polymerase-cncoding MIPI gene from Scicr~raroni~.c.r.~ cwwisiae. Getir 160,

Johnston. G. C.. Prendergast. J. A. and Sinser, R. A. ( 199 I ). The Sac.c/rirror?ij~ces ceiwi.siae M Y 0 3 gene encodes an essential myosin for vectorial transport of vesicles. J. Cd1. Biol. 113, 539-55 1.

Johnston. M., Andrews. S.. Brinknian. R. , rr id. (1994). Complete sequence of Scicchcirorii~~i,es cwcvisi(ii> chromosome VIII. Scierzcr 265. 2077-2082.

Katzmann, D. J., Burnett. P. E.. Colin, J., Mahe, Y. and Moye-Rowley, W. S. (1994). Transcriptional control of the yeast PDR.5 gene by the PDR3 gene product. Mid Cell. Biol. 14. 4653466 1.

Kralli. A, , Bohen, S. P. and Yamamoto, K. R. (1995). L E M I . an ATP-binding-cassette transporter. selec- tively modulates the biological potency of steroid hormones. Proc. Nrrtl. Acacl. Sci. USA 92. 47014705.

Lalo, D., Stettler, S.. Mariotte, S., Slonimski. P. P. and Thuriaux, P. (1993). Two yeast chromosomes are related by a fossil duplication of their centromeric regions. C. R. .1c.ird. Sci. Paris 316, 367-373.

Meaden, P.. Hill. K., Wagner, J.. Slipetz. D., Sonimer, S. S. and Bussey. H. (1990). The yeast KRE5 gene encodes a probable endoplasniic reticulum protein required for ( I -.6)-b-D-glucan synthesis and normal cell growth. Mol. C d . Biol. 10, 3013-3019,

Meyers. S.. Schauer. W., Balzi, E.. Wagner, M., Gofeau, A. and Colin, J . (1992). Interaction of the yeast pleiotropic drug resistance genes PDRl and PDRS. Ciirr. GmcJt. 21, 431436.

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