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The Complete Sequence of a 10.8 kb Segment Distal of SUF2 on the Right Arm of Chromosome I11 from Saccharomyces cerevisiae Reveals Seven Open Reading Frames Including the RVS 161, ADPl and PGK Genes JACEK SKALA*, BENEDICTE PURNELLE AND AND& GOFFEAU Uniti de Biochimie Physiologique, Universiti Catholique de Louvain, Place Croix du Sud 2/20, 1348 Louvain-la-Neuve, Belgium

Received 4 February 1992; accepted 10 February 1992

We have entirely sequenced a 10,835 bp segment of the right arm from chromosome 111 contained in the J11D and J11DK3B GF clones. The segment contains seven open reading frames longer then 100 amino acids. Three of them, RVSI61 (Urdaci et al., 1990; Crouzet et al., 1991), ADPl (Purnelle et al., 1991) and PGKl (Hitzeman et al., 1982) have been described previously. YCRIOC encodes a putative membrane protein. YCR8W (encoding a putative protein kinase) and YCRllC extend inside the DlOH (Skala et al., 1991) and 62B5-2D clones respectively. Four ARS elements previously reported by Palzkill et al. (1986) are located between RVSI6I and YCRIOC. KEY WORDS-Yeast;

chromosome 111; RVSI61; ADPI; PGKl; ARS.

INTRODUCTION As a part of the European Community (BRIDGE) project of systematic sequencing the Saccharomyces cerevisiae genome, we have sequenced a 10,835 bp DNA fragment located between the SUF2 and PGKl genes on the right arm of chromosome I11 (Mortimer et al., 1989). MATERIALS AND METHODS The J l l D plasmid is a YIp5 derivative containing 7903 bp from the small ring of S. cerevisiae chromosome I11 (Newlon et al., 1991) of strain XJ24-24 (Strathem et al., 1979). The J11D-K3B GF plasmid contains DNA from strain CN31C (Warmington et al., 1987) inserted in the YCp50 vector (Newlon et al., 1991). *Permanent address: Institute of Microbiology, Wroclaw University, Przybyszewskiego 63,51-I48 Wroclaw, Poland.

0749-503X/92/050409~9$05.00 a1992 by John Wiley & Sons Ltd

Escherichia coli JM 109 (recA 1 supE44 endA 1 hsdR17 gyrA96 relA 1 thi (lac-proAB) F"traD36 proAB' lacP lacZ M15] was used for propagation of plasmids and subcloning. The 3.05 kb BamHI-EcoRI, 2.2 kb and 2-7 kb (the second EcoRI site used for subcloning this fragment was from YIPS) EcoRI fragments from J11D as well as the 4-2 kb and 5.2 kb EcoRI fragments from J11DK3B GF (Figure 1A) were inserted into the pTZ18R or pBluescriptSK(-) vectors in both orientations. Clones suitable for sequencing were generated by exonuclease I11 digestion as described by Barnes et al. (1983). Single-strand DNAs were sequenced by the chain-termination method of Sanger et al. (1977) using the Klenow fragment of DNA polymerase I (Amersham).The sequencing of 148 overlapping clones produced a total of 45,750 nucleotides covering a final sequence of 10,835 bp which was shown to be contiguous to the sequences from the flanking DlOH and 62B5-2D clones. The details of this analysis are deposited in MIPS (Martinsried). Where necessary

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J. SKALA ET AL.

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Figure 1 The restriction, mapping and sequencing scheme. (A) The general restriction map of sequenced plasmids. (B) The sequencing strategy. The arrows indicate the direction and extent of each individual sequencing reaction. (C) The open reading frames. (D) Location of the sequences previously reported in this region by: (a) Aigle et al. (personal communication); (b) Urdaci et al. (1990); (c) Palzkill et al. (1986); (d) Ogden et al. (1986); (e) Hitzeman et al. (1982); (0 Dobson et al. (1982).

oligonucleotide primers were used to verify the junctions between the restriction fragments and to fill in data gaps. The entire sequence was determined on both strands. The open reading frames (ORFs) revealed in the course of sequence analysis were named according to nomenclature proposed by Oliver et al. (1992). Each ORF that was longer then 100 amino acids was assigned a three-letter designation plus a number and strand indication. The meaning of designations is as follows: Y, yeast; C,the third letter of the alphabet for the third chromosome; R,right chromosome arm; the number assigned to each ORF increases progressively from the centromere side to the telomere side; W or C for Watson or Crick strand.

The general restriction map of the sequenced fragment and its orientation on the physical map of chromosome I11 are shown in Figure 1A. The subcloning and sequencing strategy are given in Figure 1A and B. The complete nucleotide sequence is presented in Figure 2. Search for coding regions by Fickett’s (1982) method identifies seven ORFs longer then 100 amino acids (Figure 1C). The ORF YCR8W encoding a putative protein kinase of 603 amino acids overlaps with the adjacent DlOH

The fourARS JllDla, JllDlb, J l l D l c and JllDld, previously reported by Palzkill et al. (1986),are present between the RVS161 and YCRIOC (Figures 1D and 2). The predicted protein corresponding to YCRlOC comprises 283 amino acids (Figure 3) and has a molecular weight of 30,726 Daltons. The value of the codon bias index, calculated according to the method of Bennetzen and Hall (1982), is 0.26, indicating a low level of expression. The search for membraneassociated helices using the methods of Klein et al. (1985), Eisenberg et al. (1984) and Rao and Argos (1986) revealed six putative segments located as shown in Figure 3. In the 5’ non-codingregion of YCRIOC we have found a putative TATA box as well as a GTCATCTGCAGCGA sequence (position 4299 to 43 12 on Figure 2), showing near-perfect match with the ABF-I consensus RTCRYNNNNNACG (Dorsman et al., 1988; Della Seta et al., 1990). The presence of this putative ABF-I factor binding site suggests that the YCRIOC gene is subject to multiple activation of transcriptional regulation. Examination of the 3‘ non-coding region revealed a putative polyadenylation site (AATAAA) as well as the tripartite consensus TAG ...TATGT...TTT (Figure 2) associated with transcriptional termination (Zaret and Sherman, 1982). No significantsimilarity was found between YCRlOC and any known nucleotide or amino acid sequences (MIPS; January 1992).

clone. The detailed analysis of this ORF (previously named YCRZUI)has been published (Skala etal., 1 9 9 1 ) .

The product of the Y C R l l C gene i s a protein of 1049 amino acids with nine putative hydrophobic

RESULTS AND DISCUSSION

The 795 bp long YCR9C corresponds to the R V S Z B Z

gene, which has been sequenced previously (Urdaci e r ai., 1990).

membrane spans. This protein. which presents strong

homologies to several A”-dependent

permeases, has

also been previously described (Purnelle et al.. 1991).

41 1

COMPLETE SEQUENCE OF A 10.8 KB SEGMENT DISTAL OF SUF2 ON THE RIGHT ARM OF CHROMOSOME 111

1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441

GATCCAAAGACGACTTTATCCAACAAGAATCCTCCTACTCT~TTGCAGGCTTCATG A A G C A G G T C G T G G A T A T G G A T G A T A A A T A T C C A G G C G A AATTGTCCTGAAAGGGATATATACAGGTCAGATCAAAAAGAT A A T A T C A C T A C T A C T A A A A A A G A T A G G C A A T G C C G

GAAGTCCTTGGTAAAGGTGCTTTTGGTGTAGTAAGAATATGTCAAAAGAAAAATGTTTCT TCTCAAGATGGTAATAAAAGTGAAAAGCTTTATGCAGTGAAAGAGTTCAAGCGTAGAACA TCCGAATCAGCAGAAAAGTATTCTAAGAGGTTGACTTCTGAATTTTGCATTTCTTCTTCA TTACACCATACAAATATTGTTACTACACTAGATCTTTTCC C TGTGAAGTAATGGAATATTGTGCAGGTGGCGATCTATTCACTTTGGTCG~GCC~CGGAR AAATTAGAATATATGGAAGCAGATTGTTTCTTCAAGCAGCTTATTAGAGGTGTTG~AT 8 ATGCATGAAATGGGTGTTTGTCATAGAGATTTGAAGCCTGAAGCCTGAGAACTTACTGCTTAC~AC GATGGTGTGCTAAAAATTACAGACTTTGGTAACAGCGAATGTTTCAAGATGGCATGGGAA AAAAATATTCACCTTAGTGGAGGCGTTTGCGGTTCATCGC TATATCAAAGAAGAGTTTGATCCAAGACCCGTAGATATAT~CATGTGGTGTCATTTAT ATGGCAATGAGAACTGGTAGACAATTGTGGAG~CTGCTG~GACGATCCATTTTAT ATGAATTATTTAAAAGGACGTAAGGAAAAGGGAGGCTATGAGCCAATCGAAAGTTTAAAA AGAGCCAGGTGTAGGAATGTTATATATTCGATGTTAGATCCCG~CCGTACAGAAGAA~ AACGGGAAACAAATTTTGAACAGTGAATGGGGAAGGGAGATAAAATGCTGCCATAATGGG

CGCGCATTGAAATAAACGAGTACTTCACTTTCAkATATCAAATATCACGATATT~GGAACAATTCG YCRBW s t o p 1 GTATTTTTTACTTAATCTAGTACACTAAGGAATGC~TG~ATCCGGCATTCGTATCTTA TTCCTCGCTTCTATTGTTCTACTTTTATATCCCGTTTGGC ATTGGTAAATCCCATTAATTAAAAAAGAATTGTAACCTTATTT-TAGTAC ATAACAATAAAAAAAAAAAAGATAATAATTTTGAATTTATTGCTAGACATTCTTACGTm ATTGTGTTCATGTTAACACACATGCCCGCGGTTATTTC

TTAAAAAGAGTCGGTTGGTCAGATAGAGCAGATAGTAAATTATAGTGCTATTTAGGTCTG

I

2341

TTAATTTAAATGAAAGAAACATAAATGACCGTAAAAAACT-GGCAAAAGCATTAATTT RVS161 Stop -a ATTTTATCCCGAGCGCACAAATATCTAGGCTTGTCATTTG T TAGTGTCTAATAACCCATTGGCATAGTCGTCTCTTGATTGTTGGTCCAAATATTGTTGAA TCTGCGCTAAACGAGTGTAACCATCAGTACAGAACCTTAGCTGAATC~GATTAAA~TT CAAAACTTGGGTCAAAGTAAGGTACTCTTAATGAAACTAACTGTGGTAGTTCAGTTTTCA R ATTGGTTATTAAGATTTTCGAAAATATCTTTAGCTAAGCTCAA~CTTTTTCA~CC~G V GCAGTTTAGAGGCATCTTTAGCAGGTTTGTCCACTAATCTACGAACT~TGCC~CGCAGS CATCGAAGTCTTGTTTCTTATGGTCTCTCTTTTTTATGGCCTCCTCAA~TCTTTG~T 1 ACGTCGAAAACTTTGTTATTGGATCTAGTACGGTTTCTCTTAAGGGCCCGTCTAATTGCT6 TAACAGTTTCGCTATCAAAATCTTGAACACATTGCAAATAATAGTTACCAACG~GTAAC 1 CACCACCAGCAACATATTTTGAATCGTCATAGAGG~AGAGATGACCTCGGCAATGGTAG TCTGTGATGCTGTCACAGCTCTCAATGAGTCCAAGAAACCTTTGGCTTCCTTTTGTAATG CCTCACCTGCTCTTTGAAGAACTTTATAACGACGTTCTTCCATGTCATACTCT~ATCAA TGGTCTTGTCGACATTCTTAATTATCACACTGTGACCAGCTCTGTTGATAGCTTTCTTAA r JllDla TTTAGCTTTGAAAAACATTTAATTGCGAAGTAACATAATGTTATTAATAGTGTACGACTG

2941

AAACAGCCATTCTATTTAAACATATAAAAGTACGATTAGTTGGTGTGGTTATATAAGTCA

3001

J 1 1 D1 c