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VOL. 7: 881-888 (1991)

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The MAT Locus Revisited within a 9.8 kb Fragment of Chromosome I11 Containing BUDS and Two New Open Reading Frames MICHEL JACQUET*, JEAN MARIE BUHLER?, FRANCOIS IBORRA, MARIE CLAUDE FRANCINGUES-GAILLARD AND CHRISTINE SOUSTELLE

Institut de Ge'ne'tiqueet Microbiologie UR.41354, Brit 4 0 M n i v e r s i t C Paris-Sud, 91405 Orsay and tService de Biochimie CEA Sacluy, France Received 30 May 1991; revised 25 June 1991

This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III. The sequenced DNA contains the MATa locus. The new sequence of the MATa locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC2.5 and CDC25. A large ORF of 1399 codons is found on the opposite side of MATa (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF , YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.

KEY WORDS-Chromosome 111; Succharomyces cerevisiue; mating type; HML; BUDS.

INTRODUCTION Within the framework of the European BAF' program of sequencing chromosome 111, we have sequenced 10 kb of a DNA fragment which encompasses the MAT locus. This fragment is present in a lambda clone (hPM5240) containing a 20 kb insert from a genomic library produced by M.V. Olson. This fragment is a contig of the right arm of chromosome 111. On the basis of the averaged ratio between genetic and physical distance, it was originally mapped 20-30 cM apart from the MAT locus (Mortimer et al., 1989) but as shown in this paper, this fragment encompasses the MAT locus. We thus report the sequence and DNA *Addressee for correspondence.

0749-503X/9 1/08088 148$05.00 01991 by John Wiley & Sons Ltd

analysis of 9.8 kb of the left part of this fragment (toward the centromere) which contains a gene independently identified as BUDS,the MAT locus and two new genes YCR724 and YCR72.5. Sequence and DNA analysis of the other part of the insert 3LPM5240 which overlaps the insert of the clone hPM5239 will be presented separately (Grivell et al., 1991). The left part partially overlaps the sequence previously reported by Thieny et al. (1990). In addition to the sequence of M A T a , in which we find discrepancies with previously reported sequences, we have also re-examined the sequence of the W fragment of HML.

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MATERIALS AND METHODS Strains, DNA and vectors Escherichia coli strains used for cloning experiments were DHSa, JMlOl and XL1 blue. The DNA hPM5240 is derived from a gene bank constructed by M. V. Olson from the yeast strain AB972 (isogenic to S2886). The 5.2 kb EcoRI fragment containing HML is derived from h Charon 4A p78 and was cloned from the strain A364A. These fragments were provided to us by S . Oliver. Cloning and subcloning experiments were performed for sequencing in plasmids bluescript KS+, and in filamentous phages M13mp18 and M13mp19 (Messing, 1983).

DNA Sequencing

We used an Applied Biosystem DNA synthesizer to synthesize oligonucleotides. These oligonucleotides were used to promote DNA synthesis for the sequencing of some DNA segments, thus allowing complete coverage of both strands as well as resolution of sequence in regions presenting compressions. The d-aza-cytidine kit from Pharmacia was also used in some instances to solve ambiguous readings.

DNA sequence analysis Analysis of DNA sequences and their open reading frames (ORFs) was performed using the DNA Strider program (Marck, 1988). Comparisons of the sequences with data banks were performed on VAX using on line connection to the MIPS facilities (Mewes). RESULTS AND DISCUSSION

DNA was sequenced by the dideoxy-termination procedure (Sanger et al., 1977) on single- and doublestrand DNA using the Klenow fragment of the E . coli DNA polymerase and/or the 'I7 DNA polymerase (Pharmacia kit). The DNA was labelled with [35S]dNTP.

Sequencing strategy The complete nucleotide sequence of a 9.8 kb DNA fragment starting at the Smal site of the hPM5240 insert and ending at the second XhoI site was performed

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YCR724 Figure 1 Sequencing strategies. At the top is presented the restriction map of the sequenced fragment of 1PM5240. The restriction sites used for subcloning experiments are indicated. Sequence analyses are represented by arrows indicating length and reading orientation. When a synthetic oligonucleotide was used as primer a rectangle is shown. The six reading frames are indicated below : the large bars represent stop codons and the small bars AUG codons. (This figure was drawn with the DNA Strider program.) The five ORFs are represented by heavy arrows from the centromere (CEN) to the telomere side (TEL).

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SEQUENCE OF SEGMENT OF 9.8 KB OF CHROMOSOME Ill

passes the M A T a locus. It contains five ORFs larger than 100 codons respectively named YCR721 to YCR72.5. Two ORFs, YCR722 and 723 correspond to the previously described genes of the mating type cassette, the a1 and a 2 genes (Astell et al., 1981). One corresponds to BUDS, a gene independently isolated and characterized by Chant et al. (1991). The last two ORFs correspond to new genes YCR724 and YCR72.5. Since four out of the five ORFs are in the same orientation, there is a possibility that some belong to the same transcription unit and each ORF would represent an exon of the same gene, but the search for the splicing consensus sequence TACTAAC was negative and therefore they are most likely to correspond to different genes. The sequence TTTTATATTTT, which matches the ARS consensus sequence(A/”)T’ITAT(A/G)TTT(A/T) (Newlon, 1988), is present at position 4755. This sequence is present within an ORF but has not been analysed for its capacity to promote DNA replication.

on both directions using strategies schematized in Figure 1. After determination of the restriction map, the DNA fragment was split into smaller fragments. These fragments were sequenced from subcloned DNA using either double-stranded or single-stranded DNA. Primed DNA synthesis using synthetic oligonucleotides was also performed to complete the sequence on both strands. Some ambiguities due to compressions found in the MAT and HML loci were resolved using d-aza-CTP sequencing in HML. The W segment of H M L present on a plasmid was also sequenced using synthetic oligonucleotide primed DNA synthesis. The sequence on the centromeric side of our fragment up to the EcoRZ site overlaps the sequence determined by Thieny et al. (1990). The sequence on the other side overlaps with the sequence of the hPM5239. We have sequenced the overlapping region, and the sequence of the other part of the hPM5240 will be published separately by Grivell et al. (1991). As shown in Figures 1 and 2, this sequence encom-

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TAAGACTCTACCCAGAT W--> TTGTATTAGACGAGGGACGAGTGATTTTTGTGTTTGTTTTTATTAATTGT~ATAGGATAGTA~~CTTffiA~GAGCATTGTCAGTTGTC~GTCT CTGAAGTTAAGTAGTMGTTTGCGGAGT~~ATGGCTTTT~CATTTGT~~GTT~~~~AT~TATT~TAGA~~TATTCTGAAGACCTCTTGTA~CATCA TCCATACT~GTAIIRTCGTCCTGTCCCATTACGAGCTGTATTA~GC~T~CCCTCTGTATATTTACGTTGC~~GAAffiTAA~GATATTTTGATACMTTCCTGAGTT GCATGTTffiATTGAGTTTACGAAGGGTCGCCAGAC~A~CCTCCAffiC~GTTAA~CTMTAATAC~TCCATGTTT~TCAGCGCC~GCCTATAC~GTCACTGAGT AGACGTTTTCTTGCTCTTTTTATGTCCTGACTTCTTTTGACGA~ffiCATTCTCTAGAGACACA~AGTTGCTTCCAGCMCTGCGGTACGGCCGTTCTUT TCTTGTTCAAGAAGGTAGGCGAffiACATTATETBTCAGTTAC~CATCTTA~AGTGTCTGAGGA~ffiGTTGATTGTTTATG~GC 2400 GAAATATATATATATATATTCTACACA-mGTTTTTCGGGC ~ T T C T T T C T T C T T T G C C A G A G G C T ~ C C G C T U L A G A G G T T T GTTTTTTCTTTTCTTCTTCTATTCGAAACCCAGTTTTTGATTTGAATGC~GATI\AACT~AT~TT~TTAGATTC~TAGGCCCTT~ATCTAGATAT~TTCTC~TGTTCTTT G~CCMCTTTCTAGTATTCGWLCATTTTCTTTTGT~CCffiTGTCC~TGTMffiTTTAGTA~TTTGTTTATCATATCTTGAGTTAC~~TTI\AATAC~CCCATCC~CGAT TTATTTTTCTGTGTI\AGTTGATAATTACTTCTATCGTTTTCTATGCT~~TTTCTTTGAGTAATACAGT~TffiTA~AGTGAGTTGAGATGTTGTTT~ULACTTCTTCTCCTCA TCACTAATCTTACGGTTTTTGTTGGCCCTAGATM~TCCTAATATATCCCTTMTT~CTTCTTCTTCT~TGTTACACTCTCTGGTMCTTAffiT~TTACAG~TA-G AGCTTTTTATT W Y - > TATGTCTAGTATGCTGGATTTlUACTCATCTGTGATTTGT~TTTlUAAGGTCTTTAATGffiTATTTTATTUT ATGTTTACTTC GAAGCCTGCTTTC~UAATTI\AGAACAAAGCATC~T~TACAG~~CAGC~~~TTT~~TGI\AAG~C~CTAG~GA~ATGT~~ULAGCTGCTTCAATATTAT T C G A C C A C T W G I U L A G A T C C A G A T T C C T G T T C C T T C C T C T C G A T T T T T ~ T A A A A T C ~ T T C A ~ ~ T A ~ ~ C T f f i A A G T ~ T A C T C A G T T T C G A ~ G T T C3600 AAT~GAC ATCTATAAAATCTTC~GI\AATATTTAAACTCATTTAT~TTTAGA~TATTACTCACAGTTT~TCCGGTGTAAAA~T~CTTGTCTTCT~GCTCGCTG~GAATGGCA

CGCGGACAAAATGCAGCACGGAATATGGGACTACTTCG Y/Zl--> CGCAACAGTATMTTTTAT~CCCTGGTTTTGGTTTTGTAGA~GGTTGACGAATAATTATGCTGAAGTACGTGGTGACGG ATATTGGGAAGATGTGTTTGTACATTTGGCCTTATU AGTGTGGTCGTGGCGGAffiTTGTTTATCTTTCGAGTA~~TGTTGTCAGTATA~TATC~ATTT~CTCCC~TCGTC

TTGCTCTTGTTCCWTGTTTGTTTATACACTCATAT 21/22--> GG~TACCCTTATCTACTTGCCTCTTTTGTTTATGTCTATGTATTTGTATlUAATATGATATTACTCAGACTCMGC~CA ATCAA .nd of UAT TTCTTAGCATCATTCTTTGTTCTTATC ~AACCATAI\ACGAATCTTGATGTGACTTTTGTAATTTGAACGAATTffi~ATACGG~CGGAT~C~TGCACCATTACTCTAGGT TGTTGTTGGATCTTAACACGTARAGGTAAACTGCCCATGCGGTTCACATGACTTTTGACTTTCCTTTGTTTGCTAGTTACCTTCGGCTTCAULATTTGTTTTTCCACTTTTCTAACA

Figure 2 Nucleotide sequence. Final DNA sequence presenting the different O m s and the segments of the MAT locus. Putative TATA boxes and signals of transcription termination (Zarret and Sherman, 1986) are underlined with full and dotted lines respectively . The ARS consensus is underlined with a double line.

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CCTTTGCCAAAGTACGTTAAGAGCGCTTTCACTGCCTACTG 9600 TTGCCCCRAARATTTCTTGGCTTATCTTGGCACAC~CTTTTGCATTTAGTTCTATACATACAGAAAGAGCGIGTG~TAAACAGRATGTGATAACGGT~GAA AATCAAAAATRRACAATACATATATAAC~CATGATAGTAAACAGGTGAATTAAG~GTACAATTGTCTATTACCCCAATAGGCATAATCCTCGAG

Figure 2 continued

The MAT and HML sequences revisited Because we were unaware of its presence within our DNA fragment, the complete sequence of the mating type locus MATa was determined again independently of the known sequence (Astell et al., 1981). Our data (Figure 2) confirm most of the previously published sequence. We found 100% identity in sequences X, Ya, Z1 and 22. Unexpectedly, six differences were found in the W segment which is common to MAT and H M L . In order to confirm that the differences found between our results and the previously published sequences were not due to allelic polymorphism, we also sequenced the W portion of the H M L locus coming from a different gene bank. The six changes of the difference in sequence are found in both MATa and HML. As illustrated in Figure 3, some correspond to sequence elements which have a strong tendency to form compression on sequencing gels. Our initial results were ambiguous at these positions and several experiments were required

to resolve these ambiguities. We use oligo-primed DNA synthesis with different primers in both directions as well as d-aza-CTP to obtain clean data. Therefore, we conclude that, at least in the two strains we used and most likely in all strains, the W segment differs from the sequence present in the data bank. The corrected sequence of MAT presents now a large ORF which overlaps the adjacent DNA. The same ORF is found in H M L , but in contrast to the MAT W ORF which extends in the flanking DNA, this ORF ends one codon after the junction between H M L and the flanking D N A . Allelic differences in W between the H M L a locus and the MAT a locus have been noticed previously (Astell et al., 1981). We observe at these two positions the same sequence in MATa as in MATa, different from the sequence in H M L . This result demonstrates that allelic differences are characteristic of the loci rather than of the mating type and, as expected, further supports the idea that W and X are not duplicated simultaneously with Y.

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Figure 3 The W segment revisited. ( I ) Resolution of the compressions around position 2100. (a) Sequencing with dCTP: (b) sequencing with d-am-CTP. ( I 1 and 111) Two of the other corrections at positions I Y 4 4 and 2265.

A gene (YCR721 = BUDS) overlaps the W segment of’MAT On the left of the MAT locus, the ORF named YCR721 contains 538 codons. The DNA fragment containing this gene overlaps the DNA sequenced by Thierry et 01. (1990). They named this ORF YCR.526. Two differences in nucleotide sequence were found at positions 266 and 377. They might reflect allelic differences between the two DNAs obtained from different strains. The sequence provided to us by Chant and Herskowitz is identical to our sequence (Chant et al., 1991). They discovered this gene due to the effect of mutations first believed to be within MAT but later identified as mutations of a new gene closely linked to MAT. This gene, required for proper orientation of the bud, was

called BUDS. Gene disruption is not lethal but leads to random localization of the bud in both a, a and a/a cells (Chant et al., 1991). It is interesting to note that in an analysis of the transcripts of chromosome 111, Yoshikawa and Isono (1990) have detected a transcript of 2.1 kb, with the use of a probe containing the W segment of MAT. The corresponding gene, numbered 8, has been localized by these authors on the left of HML. The presence of a transcript of 2100 nucleotides within the IJML locus is very unlikely. First, it is known that this region is under transcriptional repression by cis-regulatory elements acting as silencers (Nasmyth, 1982) and second, our sequencing data give an ORF of only 190 bp. In contrast, the coding sequence of BUDS, which requires at least 1614 nucleotides for the ORF,

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is more likely included in a 2100 nucleotide transcript. Therefore the transcript no. 8 in Isono's analysis, which is detected with a probe containing the W segment of MAT and/or HML, is most likely the product of BUDS. It can then be concluded from the data of Yoshikawa and Isono that this gene is transcribed at low to moderate level in growing cells. The ORF of BUDS encodes a putative polypeptide of molecular weight 62,850. As shown in Figure 4 , the predicted amino acid sequence of BUDS presents homologies with the C-terminal part of the ORF of CDC25 and SDC2.5 (Camonis et al., 1986; BoyMarcotte et al., 1989; Damak et al., 1991). The highest degree of identity between BUDS and the other proteins lies in the most conserved sequences between CDC25 and SDC25. This strongly suggests some structural and functional conservation since this part of the CDC25 and SDC25 genes encodes the active part of these molecules. The product of the C-terminal domain of SCD25 has been shown to act in vitro as an exchange factor for ras proteins (Crkhet et al., 1990) and the product of the CDC25 is the biological activator of the RASI and RAS2 gene products in Saccharomyces cerevisiae. Another related gene, Ste6+, has been described in Schizosaccharomyces pomhe. This gene might also be an activator for a ras protein because its defect (steb-) can be suppressed by an 'oncogene'-like mutation in the rasl protein (gene product of SteS)

(Hughes et al., 1990). Since RSRl and CDC42 are both small GTP binding proteins also involved in the control of bud polarity (Johnson and Pringle, 1990; Adams et al., 1990; Bender and Pringle, 1989), it is possible that the product of BUD5 acts as an exchange factor for one of the small GTP binding proteins involved in the establishment of cell polarity.

A large ORF encodes a new gene YCR724 Flanking the right of the MAT locus is a large ORF, YCR724, which corresponds to a new gene (Figures 1 and 2). The ORF has the same orientation as BUDS and MATa2. There are several putative TATA boxes upstream of the initiation codon. A putative polyadenylation sequence can be seen at a position before the beginning of the MAT cassette. This gene contains an ORF of 1399 codons which should be encoded by a RNA molecule larger than 4200 nucleotides. A RNA molecule of 4400 nucleotides numbered 95 has been identified by Yoshikawa and Isono (1990) using DNA fragments from this region. This RNA belongs to the class of transcripts expressed at moderate level. Thus the gene YCR724 is most likely transcribed during growth. It is capable of encoding a putative polypeptide of molecular weight 160,000. The predicted amino acid sequence displays characteristic clusters of acidic residues (Figure 5). Similar clusters of acidic

SEQL ENCE OF SEGMEhT OF 9.8 K B OF CHROMOSOME I11

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Figure 5 Predicted amino acid sequence of YCR7-74 with a distribution of acidic and basic residues, The distribution of acidic ( A ) and basic ( B ) residues was generated hy a DNA Strider program.

residues have been described in several proteins such as nucleoline, but no other part of this molecule is similar to protein harbouring acidic clusters. YCR725 oncodrs u sniall O R F An ORF of 175 amino acids, Y C R 7 2 5 . is present on the right part of the DNA segment (Figures I and 2). This ORF could correspond to the RNA number 96 identified by Yoshikawa and lsono (1990) in their analysis. I t is an RNA of 600 nucleotides in length and was detected with probes containing the same DNA fragment. From the ORF, the length should be at least of 525 nucleotides. This RNA, which belongs to class 3 of abundance. could be produced at a relatively high level. The predicted amino acid sequence presents a strong hydrophobic region, which could form a transniembrane domain. Searches for related sequences in the data banks were unsuccessful.

value for the ratio between physical and genetic distances (Olson, personal communication). The fact that this fragment contains the MAT locus clearly illustrates the bias in the evaluation of physical distance by the efficiency of recombination. The large genetic distance between M A T and THRJ (>50 cM) (Mortimer et ul., 1989) does not reflect the physical distance, which can be estimated to be less than 20 kb from data from Isono or the European Yeast Sequencing project. Hot spots of recombination have been described in yeast. They result in higher recombination rates and thus in larger genetic distances. It seems very likely, from these data. that some DNA element(s) favouring DNA recombination is(are) present between MAT and T H R 3 . The comparison between physical and genetic distance along chromosomes should lead to the identification of regions containing important elements for recombination. ACKNOWLEDGEMENTS

Genetic, utid physicul 1oculi:ation The DNA fragment in hPM5240 was thought to be more than 50 cM apart from the M A T locus. This localization was based upon the construction of contigs labelled by the SUP61 gene and using an averaged

We thank A. Goffeau for general organization of the project, S. G. Oliver for the supply of hPM5240 and P4 clones and Dr Mewes for the MIPS facilities. This work has been supported by the EEC BAP program (to J. M. B. and M. J . )

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REFERENCES Adams, A. E. M., Johnson, D. I,, Longnecker, R. M., Sloat, Hughes, D. A., Fukui, Y. and Yamamoto, M. (1990). Homologous activators of ras in fission and budding B. F. and Pringle, J. R. (1990). “CDC42 and CDC43, yeast. Nature. 344,355-357. two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharo- Johnson, D. I. and Pringle, J. R. (1990). Molecular characterization of CDC42, a Saccharomyces cerevisiae gene myces cerevisiae. J . Cell Biol. 111, 131-142. involved in the development of cell polarity. J . Cell Astell, C. R., Ahlstrom-Jonasson,L. and Smith, M. (1981). Biol. 111,143-152. The sequence of the DNAs coding for the mating-type Marck, C. (1988). “DNA strider”: a “C’ program for the fast loci of Saccharomyces cerevisiae. Cell 27, 15-23. analysis of DNA and protein sequences on the Apple Bender, A. and Pringle, J. R. (1989). Multicopy suppression Macintosh family of computers. Nuc. Acids. Res. 16, of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related 1829-1836. Messing, J. (1983). New M13 vectors for cloning. Methods gene RSRI. Proc. Natl. Acad. Sci. USA 86,9976-9980. Enzymol. 101,20-78. Boy-Marcotte,E., Damak, F., Camonis, J., Garreau, H. and Jacquet,M. (1989).The C-terminal part of a gene partially Mortimer, R. K., Schild, D., Contopoulou, C. R. and Kans, J. A. (1989). Genetic maps of Saccharomyces homologous to CDC25 gene suppresses the cdc25-5 cerevisiae. Yeast 5,321403. mutation in Saccharomyces cerevisiae. Gene 77,21-30. Camonis, J. H., KalBkine, M., GondrB, B., Garreau, H., Nasmyth, K. (1982). The regulation of yeast mating-type chromatin structure by SIR: an action at distance Boy-Marcotte,E.andJacquet,M.(1986).Characterization, affecting both transcription and transposition. Cell 30, cloning and sequence analysis of the CDC25 gene 567-578. which controls the cyclic AMP level of Saccharomyces cerevisiae. EMBO J. 5, 375-380. Newlon, C. S . (1988). Yeast chromosome replication and segregation.Microbiol. Rev. 52,568-601. Chant, J., Corrado, K., Pringle, J. R. and Herskowitz, I. (1991). The yeast BUD5 gene, which encodes a putative Sanger, F., Nicklen, S . and Coulson, A. R. (1977). DNA GDP-GTP exchange factor, is necessary for bud-site sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. selection and interacts with bud-formation gene BEMl. Thieny, A., Fairhead, C. and Dujon, B. (1990).The complete Cell 65,1213-1224. sequence of the 8.2 kb segment left of MAT on Crechet, J. B., Poullet, P., Mistou, M. Y., Parmeggiani, A., chromosome I11 reveals five ORFs, including a gene for Camonis, J., Boy-Marcotte, E., Damak, F. and Jacquet, a yeast ribokinase. Yeast. 6,521-534. M. (1990). Enhancement of the GDP to GTP exchange of ras proteins by the carboxyl-terminal domain of Yoshikawa, A. and Isono, K. (1990). Chromosome I11 of Saccharomyces cerevisiae: an ordered clone bank, a SCD25 gene product. Science 248,8664368. detailed restriction map and analysis of transcripts Damak, F., Boy-Marcotte, E., Le-Roscouet, D., Guilbaud, suggest the presence of 160 genes. Yeast 6,383401. R. and Jacquet, M. (1991). SDC25, a CDC25 like gene, which contains a RAS-activating domain is a dispens- Zaret, S . and Sherman, F. (1986). DNA sequence required for efficient transcription termination in yeast. Cell. 28, able gene of Saccharomyces cerevisiae. Mol. Cell. Biol. 563-573. 11,202-212.