YEAST

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7: 761-772 (1991)

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111

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Yeast Sequencing Reports

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The Complete Sequence of a 7.5 kb Region of Chromosome I11 from Saccharomyces cerevisiae that lies between CRYI and MAT BARTON L. WICKSTEED, A. BRE?T ROBERTS*, FRANCIS A. SAGLIOCCO AND ALISTAIR J. P. BROWN Molecular & Cell Biology, University of Aberdeen, Marischal College, Aberdeen AB9 IAS, U.K. *Department of Genetics, University of Glasgow, Church Street, Glasgow G I 1 SJS, U.K. Received 26 February 1991; revised 18 April 1991

We report the sequence of a 7.5 kb region lying between the CRY1 and MAT loci of chromosome 111 from Saccharomyces cerevisiae. This region lies in the overlap between two major contigs used for the generation of the complete nucleotide sequence of this chromosome. Comparison of this sequencewith those reported previously for this overlap [Thierry et al. (1 990) Yeast 6,521; Jia et al. (1991) Yeast 7,4131 reveals 38 nucleotide differences, 45% of which generate changes in the amino acid sequences of the four genes in this region (YCR591, YCR592, YCR.521 and YCR522). These differences appear to reflect true sequence polymorphisms between the two yeast strains used to generate the clones used in the sequencing project. Three of the four genes in this region display weak homologies to proteins in the PIR database. Some properties of YCR521 are analogous to those of ribosomal protein genes. However, the functions of all four genes remain obscure. KEY WORDS -Chromosome

111; strain polymorphisms; DNA replication; mRNA stability; ribosomal proteins.

INTRODUCTION We have sequenced a 7.5 kb region of chromosome I11 from Saccharomyces cerevisiae as part of the European project to sequence the entire chromosome. The 7.5 kb region lies towards the left end of a large contig (Map Unit Merge 0277) which was generated during the physical mapping of the Saccharomyces genome by Olson and co-workers (1986) (Figure 1). This mapping was performed using DNA from the strain AB972 (MATalpha, rho', trpl) which was cloned into the vectors LambdaMG3 and LambdaMG14; the 7.5 kb sequence described here represents the left half of LambdaPM7 121. Map Unit Merge 0277 was originally thought to lie distal to THR4 on the right arm of the chromosome 111, and to be separate from a second major contig used in the European sequencing project. This second contig, which was 0749-503X/91/070761-12 $06.00 0 1991 by John Wiley & Sons Ltd

CRY 1

TEb

MAT

'

YIP

Lambda I Figure 1. Overlap between two contigs used to sequence chromosome 111. The approximate posifion of the region sequenced in this study (black box) is shown relative to the overlap between the YIP and Lambda contigs used in the EEC Yeast Genome Sequencing Project. The YIP contig extends towards the centromere (Newlon et al., 1986) whereas the Lambda contig extends towards the telomere on the right arm of the chromosome (Olson et al., 1986).

generated by Newlon and co-workers using YIp5based clones of chromosome I11 DNA from the strain XJ24-24a (MATa, ade6, arg4, trpl, tyr7), extends to the MAT locus on the right arm of the chromosome (Newlon et al., 1986). During the

762 European sequencing project, comparison of the sequences generated towards the left of Map Unit Merge 0277 with those at the right of the YIp5 contig showed that these two contigs overlap (Figure I ) . An important consequence of this is that there is a large discrepancy between the genetic and physical distance between TSMl and THR41SEC55 (Mortimer et al., 1989), and so the region of chromosome I11 present in LambdaPM7121 is completely contained within the YIp5 clone, E5F. Since E5F has been completely sequenced (Thierry et al., 1990; Jia et al., 1991), the sequence we present here represents a comparison of 7.5 kb of analogous DNA sequence obtained from the two strains used in the European sequencing project. It also provides an indication of the quality of the DNA sequence generated by the project as a whole. MATERIALS AND METHODS Strains The E. coli strains used in this study were NM522 [hsd A5, A(lac-proAB), supE, thi, (F, proAB, 1acIq.Z M15)] for isolation of M13 and plasmid DNA, and LE392 [F-, hsdR514, (rk-, mk-), supE, supF,~(lacIZY), galK, galT, metB, trpR, Lambda-] for preparation of Lambda DNA. The S. cerevisiae strains used for the RNA analysis were RY137 [MATa, ura3, his4, lys21 and RY262 [MATalpha, ura3, his4, rpbl] which were kindly donated by Richard Young (Nonet et al., 1987). DNA isolation and manipulation Standard procedures were used throughout (Sambrook et al., 1989). Sequencing strategy

B. L. WICKSTEED ETAL.

H

E

HH

B

EE

E

Figure 2. Sequencing strategy for the left half of Lambda PM7121. The length of the sequence read from each reaction is shown (arrows). H = HindIII; E = EcoRI; B = BumHI. The scale is in kilobase pairs.

University of Glasgow), were all 17 to 19 nucleotides in length and had a G : C content of between 35 and 59%. Each was designed to generate approximately 50 nucleotides of overlap with the previous sequence. Three further subclones were generated in pTZ18R (Mead et al., 1986) to complete the sequence. The 3.5 kb HindIII-Hind111 fragment was subcloned to sequence the left-hand 0.6 kb HindIIIEcoRI fragment and across the junction with the 2.9 kb EcoRI-Hind111 fragment. The junction between the 2.9 kb EcoRI-Hind111 and 2.0 kb HindIII-EcoRI fragments was sequenced using a subclone of the 3.6 kb EcoRI-BamHI fragment. A 4.1 kb BamHI-SalI subclone was used to sequence across the junction between the 2.1 kb HindIIIEcoRI and 1.6 kb EcoRI-EcoRI fragments, and to sequence into the right-hand half of Lambda PM7121. Dideoxy sequencing (Sanger et al., 1977) was performed upon double-stranded plasmid DNA prepared from each of the pTZ18R subclones according to standard procedures (Sambrook et al., 1989). Once again, each sequence was initiated using the universal MI 3 sequencing primer or the universa1 reverse primer, and extended using synthetic and Eco I oligonucleotides. - ~ ~ ~ Small~ HindIII-Hind111 ) . RI-EcoRI fragments of approximately 0.2 kb and were found at two junctions Oa16kb, (Figure 2). The sequence was anabed using the UWGCG Programs (Devereuxetal., 1984) on the Daresbury Laboratory computer, the DAP program for protein searches (Collins and Coulson, 1987), and the MIPS package.

LamhdaPM7121 DNA was a gift from Steve 0liver (UMIST, ~ ~ ~The 2.9 ~ kb E h~~R ~ HindIII, 2.1 kb HindIII-EcoRI and 1.6 kb EcoRIEcoRI fragments from the left half of the yeast genomic insert from LambdaPM7121 (Figure 2) were gel purified and subcloned separately into M13mp18 and M13mp19 (Sambrook et a/., 1989). Viral-strand DNA was sequenced by the dideoxy chain termination method (Sanger et a/., 1977) using Sequenase (U.S.B.). The sequence of each clone was initiated using the universal M13 sequencing primer, and extended using synthetic RNA analysis oligonucleotides. The oligonucleotides, which Methods for the analysis of mRNA half-lives were provided by Dr Veer Math (Biochemistry, using the conditional-lethal RNA polymerase 11

THE COMPLETE SEQUENCE OF A 7.5 KB REGION OF CHROMOSOME 111

763

groups involved in the project (Thierry et al., 1990; Jia et al., 1991; Figure 4). They assigned the names YCR591, YCR592, YCR521 and YCR522 to the four ORFs. The genomic inserts within LambdaPM7121 and E5F were derived from two different yeast strains; AB972 and XJ24-24a, respectively (Olson et al., 1986; Newlon et al., 1986). A comparison of the LambdaPM7121 and E5F sequences reveals a total of 38 differences out of the 7532 bp. These differences are summarized in Table 1, and are shown in Figure 3. Only five of the 38 sequence differences lie in non-coding regions, but this may reflect the high density of ORFs. Sixteen of the sequence differences do not lead to amino acid changes, and of the remainder, four differences result in non-conservative amino acid changes. One of these non-conservative changes (A to C at position 4246, generating a change from Asp to Ala) leads to a restriction polymorphism at this position through the introduction of a BamHI site (Figure 4). Restriction analysis of the LambdaPM7121 and E5F clones clearly demonstrates that this BamHI polymorphism does exist, and Southern analysis of the AB972 and XJ24-24a genomes using an E5F probe reveals other strain polymorphisms in this region of chromosome I11 (not shown). Careful analysis of the raw sequence data by each of the groups (Herbert, personal communication), and the resequencing of specific regions using new oligonucleotide primers have reduced sequence errors to a minimum. Therefore, an unexpectedly high number of sequence differRESULTS AND DISCUSSION ences (0.500/) appear to exist in this region of the genome for AB972 and XJ24-24a. Sequence analysis Although the YCR591, YCR592, YCR521 and The complete nucleotide sequence of 7532 bp is YCR522 genes all appear to be transcribed presented in Figure 3. The HindIIIIEcoRI map of (Yoshikawa and Isono, 1990), separate gene disrupthis region is consistent with that obtained for tions for each of these ORFs have shown that none LambdaPM712 1 by Olson and co-workers. How- is essential (Thierry et al., 1990; Jia et al., 1991). ever, the sequencing revealed extra HindIII-Hind111 Therefore, it is conceivable that yeast is able to (21 1 bp) and EcoRI-EcoRI (160 bp) fragments that tolerate at least a low frequency of amino acid subwere too short to be resolved during their physical stitutions in the YCR591, YCR592, YCR521 and mapping of the yeast genome (Olson et al., 1986). YCR522 proteins. The sequence, which has a G : C ratio of 38.0°h, contains two complete ORFs and the C-termini of Expression of ORFs two further ORFs (Figure 4). These ORFs account for 85.4% of the sequence, and this dense packing is Of the four ORFs present in the sequence, three consistent with that previously observed in other are transcribed away from the centromere, and one regions of the yeast genome (Barry et al., 1987; ( YCR522) is transcribed towards the centromere Kaback et al., 1989; Yoshikawa and Isono, 1990). (Figure 4). Transcripts of the appropriate length for The genomic fragment within LambdaPM7121 is each ORF have been observed in the appropriate completely contained within the YIPS clone, ESF, position on the transcript map of chromosome 111, which has been completely sequenced by two other and therefore, all of the genes appear to be expressed mutation (rpbl; Nonet et al., 1987) were adapted from those described by Herrick and co-workers (1990). The yeast strains RY 137(RPBI)and RY262 (rpbl) were grown at 26°C in 100 ml of YEPD (2% glucose, 2% bacteriological peptone, 1 YO yeast extract) in a 21 flask with shaking at 200rpm to mid-exponential growth phase ( A m = 0.5) whereupon an equal volume of YEPD at 48°C was added to each to bring the cultures immediately to the restrictive temperature, 37°C. RNA was isolated (Lindquist, 1981) from cells harvested immediately before, and at various times following the temperature upshift. Equal amounts of each RNA preparation (1 5 pg per lane) were denatured by heat-treatment in the presence of formaldehyde, electrophoresed on 1.5% (w/v) agarose gels containing formaldehyde (Lehrach et al., 1977), and transferred to nylon membranes (Thomas, 1980). Filters were fixed, prehybridized, probed, washed and autoradiographed as described previously (Santiago et al., 1986). The probe was a 2.3 kb Bum HIIEcoRV fragment from LambdaPM7 121 which contains sequences from the YCR592 and YCR521 genes. This purified fragment was radiolabelled by random priming (Feinberg and Vogelstein, 1983). RNA loadings were initially measured by the absorbance at 260nm, but these were confirmed more accurately by quantitating the 18s ribosomal RNA in each sample by dot blotting (Santiago et al., 1987).

1

A AAGCTTTAGAAAGTCCTTAT GTATCAGCACATTTACATGA ATGGATTGATTTGATATTTG GTTACAAACAAAAGGGGGAC ATTGCTGTGAAATCTGTTAA A L E S P Y V S A H L H E W I D L I F G Y K Q K G D I A V K S V N

101

T CGTATTCAACAGATTGAGTT ACCCAGGCGCTGTAAATCTA GATAACATTGACGATGAAAA TGAGCGCAGAGCTATCACAG GCATTATTCACAACTTTGGT V F N R L S Y P G A V N L D N I D D E N E R R A I T G I I H N F G

201

301

401

CAAACGCCTTTACAAATATT TCAGGAACCTCATCCGGAAA AAATAGCCTGCAATGTTCAA CAGCTAACAACAGAGGTATG GCGTAAGGTTCCAATGAAGC Q T P L Q I F Q E P H P E X I A C N V Q Q L T T E V W R K V P M K T CAATATTTGAGAAGACAATC TTTAATTTGAATGAAAAGAA CAGGTCCGTCGATTATGTTA TACACGATCCTAGTTACTTC GATTCATTATACTGGAGGGG P I F E X T I F N L N E K N R S V D Y V I H D P S Y F D S L Y W R G

CAACGCTTTCCCAAACTTGT TTTTCAGAACGGAAGAATCG TTAGTGTCATTGAGAATTGT GCATAAAAATTGGTTAAAAA TTGGACTAGATATTTTTAAA N A F P N L F F R T E E S L V S L R I V H K N W L K I G L D I F K

501

T AAGACGCATATGGCTCAGAT TACATCGTTTGCGTACTGGA AGTTGGGTGAATTCATAACT GGCGATAAAAATGGGCTGAT AAAAGTTTGGAAATATCGTA K T H M A Q I T S F A Y W K L G E F I T G D K N G L I K V W K Y R

601

A AAGATAAGCATTCGGTTTCA GGTAACCTTGAGAACRAAAA AACAATGTTTGGGCACCTAT GCGAGCTAAAGGAGATGCGC TGTTATCACGACTACAATAC K D K H S V S G N L E N K X T M F G H L C E L K E M R C Y H D Y N T

701

G A GCTTTTAACCTTAGACATCA GCGGCTTAGTATATGTCTGG GACATGATTAATTTCGAACT AGTAAGGCAAATAACAAATG ATGCGCAAAAGGTCGCAATA L L T L D I S G L V Y V W D M I N F E L V R Q I T N D A Q K V A I

801

TCTCAACATGCAGGGAGCAT TATGGTATTGACTAAGAATA ACGCCATTTCGATCTTCAAT CTAAATGGACAAATATATAC ATCAAAGAAATTCGAACCAG S Q H A G S I M V L T K N N A I S I F N L N G Q I Y T S K K F E P

901

CTAAAATTGTAAGCTCAATT GATTTTTTTGACTTCACTAA GTTAGACGCAGGTTACAGAA AGCATATCTATTGGAAAGAG ATGGAAATACTACTAGTGGG A K I V S S I D F F D F T K L D A G Y R K H I Y W K E M E I L L V G

1001

1101

1201

G A G CTTTGAAGATGGAACTATAG AAATTTACGAGCTCTTTTTG AATTTTCATAATGAATGGGC GATAAAGCTACTGAAGCAGC TCTGTACCGAAAAAGGGAAA F E D G T I E I Y E L F L N F H N E W A I K L L K Q L C T E K G K T R GCCATAACTAGCATTAAGGG ACAGGGGAAGACATACCTGT CCCAGAAAAGACGCAAGGAT ACAGCAGAGCCTCATGAGAT AGAAGTGATTGCGGGAACAT A I T S I K G Q G K T Y L S Q X R R K D T A E P H E I E V I A G T G TAGATGGCAGATTAGCTATT TGGTACTAGGCATCACATCG TAACGCCTTTCTTTAAATGA TTCAATTTTTGTAGTTTATA TCTTTACTTTTGAAACTGAT L D G R L A I W Y *

1301

C TTCTCATCCCATCTAGTATT GTAATTGCGTACGTATCCAA TATCATTACCAACGCCGGGT ATTTTTTTCTAGTATTTCTT CTCCATTTCGCCTATGGAAA

1401

A ACGGCAAAAGGGTAAAAGAA AAAAACAAACGATTAATTCT TCTTTGAATTATGTAAAAAT CAAAACGCAACCGCAGATTT AATAGAGACCAGAAATTCGG

1501 ATTACTATTGACTTTGTGCA CCACCTTCAAATTTACTCAT TGTTTAAGACAGGCAGTGGG AAAGAAGCCGTCATATTGCT CGAATCCTTAACAAGCAAAA 1601

TATACAACCACTAAATTATT

CCGAAAGGGCCTGCTTAATA ATTTGCCTACTAACTTGTGC ATAGAACAGCAAACAGAAAC AAAGCGTAAGAAACATGGGG M G

1701

TATCCGCCACCTACACGAAG GCTTGGAGATAAGAAAAGGT ACCATTATTCCAATAATCCT AACCGAAGGCATCCTTCCGC TGTTTATTCCAAGAATAGCT Y P P P T R R L G D K K R Y H Y S N N P N R R H P S A V Y S K N S

1801

TTCCAAAATCAAGCAATAAT GGATTTGTATCTTCTCCTAC TGCCGATAATTCAACAAATC CGTCTGTAACTCCCAGTACT GCATCTGTACCTCTTCCTAC F P K S S N N G F V S S P T A D N S T N P S V T P S T A S V P L P T

Figure 3.1.

1901

AGCGGCACCTGGAAGCACGT TTGGTATCGAAGCACCCAGG CCATCTCGATATGATCCGAG CTCAGTCAGTAGGCCTTCGT CATCATCTTATTCGTCAACA A A P G S T F G I E A P R P S R Y D P S S V S R P S S S S Y S S T

2001

AGAAAAATTGGAAGCCGTTA TAACCCAGATGTGGAAAGAT CCTCTTCAACCACTAGTTCA ACTCCGGAAAGTATGAATAC GAGCACCATAACACACACCA R K I G S R Y N P D V E R S S S T T S S T P E S M N T S T I T H T

2101

ATACGGATATCGGAAACTCA CGCTATTCTCGAAAAACCAT GAGCAGATATAATCCTCAAT CTACTAGTTCTACAAACGTT ACCCACTTTCCCTCGGCATT N T D I G N S R Y S R K T M S R Y N P Q S T S S T N V T H F P S A L

2201

ATCAAACGCTCCACCGTTTT ATGTTGCCAACGGGAGTTCT CGGAGACCTCGATCAATGGA TGATTATAGTCCTGATGTAA CGAACAAGCTCGAAACAAAT S N A P P F Y V A N G S S R R P R S M D D Y S P D V T N K L E T N

2301

AATGTTTCATCTGTTAATAA TAACAGCCCTCATTCTTATT ACTCTAGGAGCAACAAATGG AGATCCATTGGAACGCCTTC CAGACCACCATTTGATAATC N V S S V N N N S P H S Y Y S R S N K W R S I G T P S R P P F D N

2401

ATGTCGGCAATATGACGACC ACCAGCAATACTAACTCGAT CCATCAAAGGGAACCTTTTT GGAAAGCAAATAGTACTACT ATTTTAAAATCAACTCATTC H V G N M T T T S N T N S I H Q R E P F W K A N S T T I L K S T H S

2501

ACAGTCATCGCCTTCCCTTC ATACTAAAAAATTTCACGAT GCGAATAAATTGGACAAACC AGAGGCTTCAGTTAAAGTTG AAACACCCAGTAAAGATGAG Q S S P S L H T K K F H D A N K L D K P E A S V K V E T P S K D E

2601

2701

2801

2901

3001

A ACAAAAGCCATATCGTACCA TGATAACAATTTTCCACCAA GAAAATCAGTTTCTAAACCT AATGCACCTTTAGAACCCGA TAATATCAAGGTTGGCGAAG T K A I S Y H D N N F P P R K S V S K P N A P L E P D N I K V G E T AAGATGCATTGGGGAAAAAA GAAGTACATAAAAGTGGGCG TGAGATAGCAAAGGAACATC CTACTCCTGTAAAAATGAAA GAGCATGATGAACTAGAAGC E D A L G K K E V H K S G R E I A K E H P T P V K M K E H D E L E A A A TCGCGCTAAAAAAGTAAGTA AAATCAATATTGATGGAAAG CAGGACGAAATTTGGACGAC GGCAAAAACAGTGGCCAGTG CAGTCGAAGTTTCCAAAGAA R A K K V S K I N I D G K Q D E I W T T A K T V A S A V E V S K E N T T C AGTCAAAAGGAACTAACACG CTCTGTTGAAAGGAAGGAAA GCCCAGAAATTAGAGATTAT GAAAGAGCATACGATCCGAA AGCCCTGAAAACAGACGTAA S Q K E L T R S V E R K E S P E I R D Y E R A Y D P K A L K T D V I3 A G CAAAGTTGACAGTAGACAAT GATAATAAAAGTTACGAAGA ACCTCTTGAAAAAGTGGAGG GGTGTATTTTCCCATTACCA AAAGCAGAAACGAGATTATG T K L T V D N D N K S Y E E P L E K V E G C I F P L P K A E T R L W

D 3101

3201

3301

3401

3501

3601

GGAATTGAAAAACCAGAAAA GAAACAAAATAATAAGTGAA CAAAAGTACTTACTGAAAAA GGCAATTAGGAATTTCTCAG AGTATCCTTTTTACGCACAG E L K N Q K R N K I I S E Q K Y L L K K A I R N F S E Y P F Y A Q G AACAAACTTATACATCAGCA GGCTACCGGAGTTATCTTGA CGAAAATTATATCAAAGATA AAAAAGGAAGAACATTTGAA AAAAATAAATTTAAAACATG N K L I H Q Q A T G V I L T K I I S K I K K E E H L K K I N L K H

ATTATTTCGATCTCCAGAAG A A G T A T G W G A A T G C G A AATTTTGACTAAACTGAGTG AAAATTTAAGGAAGGAAGAA ATCGAAAATAAACGTAAAGA D Y F D L Q K K Y E K E C E I L T K L S E N L R K E E I E X K R K E T A GCACGAACTAATGGAGCAGA AAAGACGTGAAGAAGGTATC GAAACAGAAAAGGAAAAAAG CTTACGGCATCCATCCTCGT CTTCCTCATCTCGTCGCAGA H E L M E Q K R R E E G I E T E K E K S L R H P S S S S S S R R R

AATAGGGCTGACTTCGTTGA TGATGCGGAAATGGAAAATG TATTGCTACAAATCGACCCA AATTATAAACATTATCAGGC TGCTGCAACAATTCCTCCGC N R A D F V D D A E M E N V L L P I D P N Y K H Y Q A A A T I P P T TAATTTTAGATCCAATCCGC AAACACTCTTACAAATTCTG TGATGTAAATAACTTGGTTA CAGACAAAAAGCTTTGGGCG TCTAGAATATTGAAAGACGC L I L D P I R K H S Y K F C D V N N L V T D K K L W A S R I L K D A Y c

3701

CTCTGACAACTTTACTGACC ATGAGCACTCTTTATTTTTG GAGGGTTATTTAATTCATCC TAAAAAATTTGGTAAAATTT CTCACTACATGGGCGGCTTA S D N F T D H E H S L F L E G Y L I H P K K F G K I S H Y M G G L

Figure 3.2.

3801

3901

4001

4101

4201

AGAAGTCCTGAAGAGTGTGT CCTACATTATTATAGAACAA AGAAAACTGTGAATTATAAA CAACTTCTTATCGATAAGAA CAAGAAAAGAAAAATGTCAG R S P E E C V L H Y Y R T K K T V N Y K Q L L I D K N K K R K M S G CCGCTGCCAAGCGCCGCAAG AGGAAGGAAAGAAGTAATGA CGAGGAAGTCGAAGTTGATG AGAGTAAAGAAGAGTCAACG AACACGATAGAGAAGGAAGA A A A K R R K R K E R S N D E E V E V D E S K E E S T N T I E K E E T AAAAAGTGAGAACAATGCCG AGGAAAATGTTCAGCCGGTT CTAGTTCAAGGTTCTGAAGT GAAAGGTGATCCATTAGGTA CACCGGAAAAAGTTGAAAAT K S E N N A E E N V Q P V L V Q G S E V K G D P L G T P E K V E N D A T A ATGATTGAACAGAGAGGCGA AGAGTTTGCAGGTGAATTGG AAAATGCTGAGAGGGTAAAC GACTTAAAAAGGGCGCATGA TGAAGTTGGAGAAGAGAGCA M I E Q R G E E F A G E L E N A E R V N D L K R A H D E V G E E S K I A C G G ATAAGTCCAGTGTAATAGAA ACCAACAATGGGGTACAAAT AATGGATCCAAAAGGAGCTG TTCAGAATGGTTATTATCCA GAGGAGACCAAAGAACTTGA N K S S V I E T N N G V Q I M D P K G A V Q N G Y Y P E E T K E L D E A G R

4301

CTTCAGTTTAGAGAATGCGT TACAGAGAAAGAAACACAAA TCTGCACCAGAGCATAAAAC AAGTTATTGGAGTGTTCGTG AATCTCAACTCTTTCCAGAA F S L E N A L Q R K K H K S A P E H K T S Y W S V R E S Q L F P E

4401

TTGTTGAAGGAGTTTGGCTC TCAATGGTCTCTCATATCAG AAAAACTGGGTACCAAATCT ACTACAATGGTAAGGAATTA CTACCAAAGAAATGCAGCTC L L K E F G S Q W S L I S E K L G T K S T T M V R N Y Y Q R N A A

4501

GCAATGGATGGAAATTACTG GTTGATGAAACCGACTTAAA GCGAGATGGGACTAGTTCAG AATCTGTACAACAATCTCAA ATTTTGATACAACCAGAACG R N G W K L L V D E T D L K R D G T S S E S V Q Q S Q I L I Q P E R

4601

ACCAAACATCAATGCCTATA GTAATATTCCTCCTCAACAA AGACCGGCTTTGGGTTATTT TGTTGGACAACCAACTCATG GGCATAATACATCTATTTCA P N I N A Y S N I P P Q Q R P A L G Y F V G Q P T H G H N T S I S

4701

TCTATCGATGGCTCTATAAG ACCATTTGGGCCTGATTTTC ATCGTGATACCTTTTCTAAA ATTAGTGCTCCTTTAACCAC TTTACCACCACCAAGACTAC S I D G S I R P F G P D F H R D T F S K I S A P L T T L P P P R L

4801

CATCTATTCAGTTTCCTCGT TCAGAAATGGCAGAACCTAC AGTGACAGATTTGCGTAACA GGCCCTTAGACCATATTGAC ACGTTGGCTGATGCAGCTTC P S I Q F P R S E M A E P T V T D L R N R P L D H I D T L A D A A S

4901

GTCAGTAACAAATAATCAAA ACTTCAGTAATGAAAGGAAT GCAATTGACATTGGCCGTAA ATCGACGACAATCAGCAATC TATTGAATAATTCGGATCGA S V T N N Q N F S N E R N A I D I G R K S T T I S N L L N N S D R

5001

AGCATGAAATCTTCTTTCCA AAGCGCTTCAAGACACGAAG CACAGCTCGAAGACACTCCC AGCATGAACAATATTGTAGT ACAAGAAATAAAACCGAATA S M K S S F Q S A S R H E A Q L E D T P S M N N I V V Q E I K P N

5101

TTACTACGCCAAGATCGAGT TCTATTTCTGCATTACTAAA TCCTGTAAATGGGAATGGGC AATCAAACCCAGATGGAAGG CCGTTGCTGCCATTTCAGCA I T T P R S S S I S A L L N P V N G N G Q S N P D G R P L L P F Q H

5201

TGCTATTTCTCAAGGCACTC CTACTTTCCCTTTACCGGCC CCTCGCACTAGTCCAATAAG TCGTGCGCCTCCAAAGTTCA ATTTTTCGAATGATCCGTTG A I S Q G T P T F P L P A P R T S P I S R A P P K F N F S N D P L

5301

GCAGCTTTGGCTGCGGTTGC CTCCGCGCCAGATGCAATGA GCAGTTTTTTATCTAAAAAG GAAAATAATAATTGAACAAA CGGCTGAGACGGGCAATACA A A L A A V A S A P D A M S S F L S K K E N " *

5401

TATGCTCTACTTCTTTTCCA TCCAATGGTTGGTGAAACTC TCGAGCATACATTACCTTAC GTGTGTTAGTGTACTATATT A T A T A T A T A T A T A T G e

5501

ATATAMGGGAGGAGTTTTT A A T E T T G T A A T T T C G T ATTTTTTCTGCATTATACAG TTTTTTCCGATTTTAAACGA CTTTATTTAAGTGTCGTGTA

5601

A A AATATGTCACATTTTATTTT E A C G T A T T C A C A T C T C C T GGCGTGCGGCCATTGCTGAA AATCGCAAAACCCACAGAGA AATAAACATCGCGAAAAAGT

5701

CaatgaaaaatTGGAAAATa tttttcattTCACTATTATC CACAAGCAATTTTGTACAAA GTGAAAAGGTTGAACTAATT ATCTTCGTCTAGAAGCCATG

M

Figure 3.3.

5801 AATTCACTCGTTACTCAATA TGCTGCTCCGTTGTTCGAGC GTTATCCCCAACTTCATGAC TATTTACCAACTTTGGAGCG ACCATTTTTTAATATTTCGT N S L V T Q Y A A P L F E R Y P Q L H D Y L P T L E R P F F N I S

5901

TGTGGGAACATTTCGATGAT GTCGTCACTCGTGTAACTAA CGGTAGATTTGTTCCAAGCG AATTCCAATTCATTGCAGGT GAATTACCATTAAGCACTTT L W E H F D D V V T R V T N G R F V P S E F Q F I A G E L P L S T L

6001 GCCCCCTGTGCTATACGCCA TCACTGCCTATTACGTTATT ATTTTTGGTGGCAGGTTTTT GTTAAGTAAGTCGAAACCAT TTAAATTAAATGGCCTTTTC P P V L Y A I T A Y Y V I I F G G R F L L S K S K P F K L N G L F 6101 CAATTGCATAATTTGGTTTT AACTTCACTTTCATTGACGC TTTTATTGCTTATGGTTGAA CAATTAGTGCCAATTATTGT TCAGCACGGGTTATACTTCG Q L H N L V L T S L S L T L L L L M V E Q L V P I I V Q H G L Y F

6201 CTATCTGTAATATTGGTGCT TGGACTCAACCGCTCGTTAC ATTATATTACATGAATTACA TTGTCAAGTTTATTGAATTT ATAGACACCTTTTTCTTGGT A I C N I G A W T Q P L V T L Y Y M N Y I V K F I E F I D T F F L V

6301

GCTddddCATdAAdAATTGA CATTTTTGCATACTTATCAC CATGGCGCTACTGCCTTATT ATGTTACACCCAATTGATGG GCACCACATCTATTTCTTGG L K H K K L T F L H T Y H H G A T A L L C Y T Q L M G T T S I S W

6401 GTCCCTATTTCATTGAACCT TGGTGTTCACGTGGTTATGT ATTGGTACTATTTCTTGGCT GCCAGAGGCATCAGGGTCTG GTGGAAGGAATGGGTTACCA V P I S L N L G V H V V M Y W Y Y F L A A R G I R V W W K E W V T

6501

GATTTCAAATTATCCAATTT GTTTTGGATATCGGTTTCAT ATATTTTGCTGTCTACCAAA AAGCAGTTCACTTGTATTTC CCAATTTTGCCACATTGTGG R F Q I I Q F V L D I G F I Y F A V Y Q K A V H L Y F P I L P H C G

6601 TGACTGTGTGGGTTCAACAA CTGCCACCTTTGCAGGTTGT GCCATTATTTCTTCATATTT GGTACTATTTATTTCATTTT ACATTAACGTTTATAAACGT D C V G S T T A T F A G C A I I S S Y L V L F I S F Y I N V Y K R

6701 AAAGGCACCAAAACCAGTAG AGTGGTAAAGCGTGCCCACG GCGGTGTTGCCGCAAAGGTT AATGAGTATGTTAACGTTGA CTTGAAAAACGTTCCTACTC K G T K T S R V V K R A H G G V A A K V N E Y V N V D L K N V P T

6801

CATCTCCATCACCAAAACCT CAACACAGAAGAAAAAGGTA AGTGTAAAATCTTTGAAAGA ATTTAAGTATTCAACTTTCG TATATTCGTTTTTTCTTAGT P S P S P K P Q H R R K R ’

6901

GGATCTATTGTTACTATTAT CACTATTATTATATTGTAAA AGACCGGATGGTTTTGTTAT ATATTACATACACATGTTAT CGTTGAAAAAAGTTTTCCGT

7001

‘ I N F R T S L D D A R V R S L L L S R TTCCTTTCGACAGTCATCAG ATAATTTTATCCGAGTCTTT TATATGTTAAATCTTGTTGA CAAATCGTCCGCTCTAACCC TAGATAACAACAATGATCTT

M K I M E P T I K A G G G V L T L Q K Y N G S P A A L I S I T S H I S 1101 TTTATCATTTCTGGCGTTAT TTTAGCGCCTCCTCCCACTA ATGTTAATTGCTTATAGTTA CCTGAAGGAGCAGCGAGGAT GGAGATCGTACTGTGTATAC T

7201

T E E A E T D L D A I L V T N L K D M D I D V E E E E S N D S N Q TTGTTTCTTCCGCTTCAGTA TCTAGGTCTGCTATTAGCAC AGTGTTCAATTTATCCATAT CAATGTCCACTTCCTCTTCT TCAGAGTTATCAGAGTTTTG

7301

L Q C E P D L E V I G Y N S A F A I N K A N I M L P V S K T Q D C TAATTGGCATTCGGGGTCTA ACTCCACTATCCCATAATTT GAAGCAAATGCAATATTCTT TGCGTTTATCATTAACGGTA CTGACTTAGTTTGGTCGCAA

I I E Y T E R I T A S R G R T R I T M R L D S A R E D I F A R P L K 7401 ATAATTTCATACGTTTCTCT TATGGTGGCACTTCTTCCTC TTGTTCTTATAGTCATTCTT AAATCGGACGCACGCTCATC TATAAATGCTCTAGGTAACT

7501

V S Q L A Y M L S N TTACGCTCTGTAAAGCGTAC ATCAAAGAATTC

7532

Figure 3.4. Figure 3. The sequence of the 7532 fragment lying between CRY1 and MAT. The sequence is given from the centromere proximal end (5’) towards MAT(3‘). The amino acid sequences of the four major ORFs that lie in this region are shown. Differences with the previously published data (Thierry et a/., 1990; Jia et al., 1991) in the nucleotide sequence and any resultant changes in the presumed amino acid sequences are given above and below the LambdaPM7121 sequence, respectively. Sequences potentially involved in transcriptional regulation are underlined in bold face (ABFI site at 1342; alpha2 site at 5621; possible TATA or A : T-rich sequences at 1480,1601,5475 and 5524). The sequencescapable of forming the stem of a hairpin structure upstream of YCR.521are shown in lower Palzkill and Newlon, case letters [at 57021. Sequences with homology to the ARS core consensus (S-[A/qTTT[ATAG]TTT[ATJ-3’; 1988) are italicized; the perfect match is on the antisense strand and is highlighted by a line over the sequence (at 6304), whereas the imperfect matches are on the sense strand and are underlined (at 6037,6055,6079,61I 1 and 6277).

768

B. L. WICKSTEED E T A L .

vironments ( A A C A m G G G and A G C C m AAT, respectively) compared with the consensus for efficiently expressed genes in yeast ([A/Y]A[A/ B E HHE HH EE E S EB I II I II I1 I 1 ! 1 E5F T I A E T C T ; Cigan and Donahue, 1987). Taken together, these data suggest that the YCR592 and H H B * EE E s YCR521 proteins are synthesized at low levels. Interestingly, the abundance of the YCR521 transcript is adversely affected by mild heat shock 591 592 521 522 (Figure 5). This has been reported previously for --I)= ORFs ribosomal protein mRNAs; both the transcription Figure 4. Summary of the sequence of the left half of Lambda and the stability of these mRNAs are influenced by PM7121. The relative orientations of the genomic inserts within mild heat shock (Herruer et al., 1988). LambdaPM7121 and E5F are shown with the centromere proxiThe 7532bp sequence was scanned for 100% mal side on the left, and the MATproximal side on the right. The homology to the consensus sequences for DNA filled box represents the region sequenced in this study. The arbinding by the proteins ABFI, ARGRII, CCBF, rows show the position and orientation of the YCR genes. H = HindIII; E = EcoRI; B = BamHI; S = Sun; B* =polymorphic CP1, GAL4, GCN4, RAPl/GRFl/TUF, GRM, BamHI site. The scale is in kilobase pairs. HAP2/HAP3, HSTF, MATalphal, MATalpha2, OBFI, PH02, PPR1, PRTF, SPTlS/TFIID, SW14.5.6 and YAP1 (Verdier, 1990). Potential (Yoshikawa and Isono, 1990). The location of RAPl, SPT15/TFIID and YAP1 binding sites are YCR591 (2167 amino acids; Jia et al., 1991) corre- located within the coding regions of the YCR591, lates well with a transcript of 6.5 kb, YCR592 (1226 YCR592 and YCR521 genes, but their significance is amino acids; Figure 3) correlates with a transcript questionable, and hence these are not highlighted in of 4.2 kb, YCR521(347 amino acids; Figure 3) cor- Figure 3. relates with a transcript of 1.3 kb, and YCR522 (394 The YCR592 gene has a perfect match to the core amino acids; Thierry et al., 1990)correlates with one consensus for the ABF 1 binding site (Verdier, 1990) of two transcripts of 1.5 kb and 1.24 kb (Yoshikawa 354 bp upstream of the initiation codon. ABFl has and Isono, 1990). We have confirmed the existence been implicated in both transcriptional repression of the 1.3 kb YCR521 transcript (Figure 5). While and activation at different loci (Verdier, 1990). This YCR591 and YCR522 appear to be expressed at ABFl site is followed by a TATA box at -215 relatively low levels in YPD, the YCR592 and (Figure 3). Thierry and co-workers (1990) noted an YCR521 transcripts are present at relatively high alternative TATA box at -95 upstream from the levels (Yoshikawa and Isono, 1990). However, ATG, but since transcriptional initiation in yeast when Northern filters of RNA from RY 137 [MATa, usually occurs approximately 100 bp downstream ura3, hisl, lys21 and RY262 [MATalpha, ura3, hisl, from the TATA box, the 5'-end of mRNAs tranrpbl] were probed simultaneously for the two tran- scribed using the TATA at - 95 would probably lie scripts, the YCR521 transcript was reasonably downstream from the putative initiation codon abundant (Figure 5), but the YCR592 transcript was shown in Figure 3. absent (not shown). The basis for the differential YCR521 contains an A : T-rich domain of 33 bp located at about -320 upstream from the start expression of YCR592 is not clear. The sequences of YCR592 and YCR521 have codon, which potentially contains multiple TATA been analysed in detail with regard to their expres- boxes, suggesting that transcription might initiate at sion. Neither appears to carry introns. Although a number of sites at about - 180 (Figure 3). Within the YCR521 transcript is reasonably abundant this putative 5'-untranslated region lies a perfect (Yoshikawa and Isono, 1990), it has a short half-life hairpin structure with a 10 bp stem and 8 base loop (about 10 min; Figure 5). Although the YCR592 (predicted AG= -4.0 kcal; Tinoco et al., 1973). transcript was not detected in this study (not Thierry and co-workers (1990) noted the existence shown), Yoshikawa and Isono (1990) previously of an alpha2 binding site in the promoter region of showed that it is relatively abundant in another this gene at - 178, suggesting that YCR521 tranyeast strain. The YCR592 and YCR521 transcripts scription might be repressed in MATalpha cells by have poor codon biases (0.010 and 0.213, respect- the alpha2 protein (Herskowitz, 1989). We tested ively; Bennetzen and Hall, 1982). In addition, their this by comparing the levels of the YCR521 transtart condons lie in relatively poor sequence en- script in MATa and MATalpha cells (Figure 5; zero 0 i

I

6

4

2

.

.

1

.

1

10

8

.

1

.

I

12

.

I

14

.

I

16

.

I

18

.

I

20

.

I

769

THE COMPLETE SEQUENCE OF A 7.5 KB REGION OF CHROMOSOME 111

Table 1. Summary of sequence differences between LumbduPM7121 and E5F DNA Change in amino acid Nonconservative ~

~

Semiconservative

Conservative

No change in amino acid

Total

~~~~

First base of codon Second base of codon Third base of codon Non-coding DNA Total

0

0

5

1

1

3 1

2 0

3 2

0

7 8 18

-

-

15

-

4

7

6

5 10 20 30 40

RY137

RY262

Figure 5. Northern analysis of the YCR52I transcript. RNA from the strains RY 137 ( M A Ta, RPBI) and RY262 ( M A Talpha, rpbl) was isolated at various times (in minutes) following a shift from the permissive (26°C) to the restrictive temperature (37°C). The RNA was probed for the YCR52I transcript.

timepoints). No significant repression is observed in the MATalpha cells, and therefore the putative alpha2 repression site does not appear to be functional. Function of ORFs Thierry and co-workers (1990) and Jia and coworkers (1991) have disrupted the YCR591, YCR592, YCR521 and YCR522 loci and demonstrated that none of these genes are essential.

-

16

5 38

Further analysis revealed no obvious phenotype for any of the mutants, and computer analyses revealed no significant homologies in the protein sequence databases to any of these ORFs (Thierry et al., 1990; Jia et al., 1991). However, we have observed weak homologies to the presumed amino acid sequences of all the ORFs using the DAP program. Some of these homologies repeatedly targeted specific regions of the YCR proteins, indicating that these homologies may have some biological significance. These are summarized in Table 2. No significant homologies were found for the Cterminal 408 amino acids of YCR591, but two short regions of the C-terminal 164 amino acids of YCR522 showed homology to a divergent group of proteins that appear to have a common feature in their ability to bind nucleotides and in most cases, to cleave the phosphodiester bonds of nucleotides or polynucleotides (Table 2). Thierry and co-workers (1990) have reported that YCR522 would encode a soluble protein with a neutral PI. The putative 1226amino acid protein encoded by YCR592 is very hydrophilic and basic [PI = 10.41. It is rich in proline, particularly in the C- and Nterminal domains. Proline accounts for 7.4% of the amino acids compared with an average proline content of 4.0% for known S. cerevisiae protein sequences (Maruyama et al., 1986). The YCR592 protein was divided arbitrarily into four regions of about 300 amino acids for DAP analysis (Table 2). Two regions of the protein show some homology to a diverse group of proteins, many of which are targeted to various cellular or extracellular compartments, yet YCR592 does not appear to have a signal sequence. DAP analysis of the amino acid sequence of YCR521 reveals two short regions with weak

770

B. L. WICKSTEED ET AL.

Table 2. Homologies to the YCR591, YCR592, YCR521 andYCR522 amino acid sequences contained within the left half of LambdaPM7121 YCR Region (amino acids)

Homology

Region (amino acids)

Score

Quality

441-500 2683 13-72 1124-1183 320-378

137 129 128 126 124

16.81 8.17 19.13 12.23 16.83

0.033 0.152 0.181 0.2 14 0.357

Expected number

YCR591

None

YCR592 39-97 39-98 47-106 46-103 38-96

Dictyosteliurn spore coat protein SP96 Mouse cell surface glycoprotein precursor Yeast glucan 1,4-alpha-glucosidase Chicken vitellogenin I1 precursor Dictyostelium spore germination protein

728-778 737-778 728-774 739-797 749-780

Human parathymosin Bovine non-histone protein HMG-1 Rat Zn++-bindingprotein Rat T-cell CD2 precursor Mouse prothymosin alpha chain

18-69 178-215 18.69 229-287 30-61

114 105 104 103 100

28.64 35.59 26.13 21.87 40.98

YCRS21 13-68 21-63 13-59 13-57

Potato alpha-glucan phosphorylase 2-Hydroxyglutaryl-CoA dehydratase, alpha subunit Rat muscle glycogen phosphorylase Rabbit phosphorylase

734793 273-3 13 167-21 6 159-205

85 82 81 80

16.41 20.76 18.16 18.82

2.640 4.757 5.757 6.995

135-149 97-1 20 138-154 97- 120

Mouse mitochondria1 ubiquinone NADH dehydrogenase E. coli p r o w protein [proline and betaine transporter] Chlamydomonas cytochrome c oxidase polypeptide 1 Caldocellum beta-glucosidase Bos taurus adenylyl cyclase

543-599 135-149 4669 129-145 62 1-644

90 88 87 84 83

16.76 64.23 41.83 50.30 39.90

0.997 1.471 1.788 3.208 3.899

YCR522 91-108 82-109 85-101 8CI12 93-1 37

Bacteriophage T4 exonuclease 46 Halococcus RNA polymerase chain A Brine shrimp ribosomal protein eL12’ Mammalian NAD+ ADP-ribosyltransferases Reovirus mRNA guanylyltransferase

169-186 285-3 11 18-34 784-815 1 0 6 w 110

89 82 80 79 73

60.96 36.61 56.74 30.39 20.5 1

0.217 0.983 1.514 I .879 6.861

130-155 134-157 145-159

E. coli glucose- 1-phosphate adenylyltransferase E. coli phosphoenolpyruvate carboxylase Yeast pyruvate carboxylase

332-357 255-278 569-583

86 81 77

39.63 40.10 63.34

0.4 15 1.220 2.894

105-1 62

0.003 0.009 0.010 0.0 14

0.019

All of the genes revealed weak homologies to amino acid sequences in the PIR database. Therefore, only multiple homologies to specific regions of the genes are given here since these might have some significance with respect to the function of each YCR gene. The parameters are described in Collins and Coulson (1987).

homology to a number of proteins. An N-terminal region has homology to phosphorylases from widely divergent sources, and some of the proteins with homology to a second region of YCR.521 inter-

act with nucleotides. The YCR521 protein is very hydrophobic and basic [PI = 9.951. The C-terminus [PTPSPSPKPQHRRKR*] is extremely rich in proline, and is similar to the rough consensus for a

THE COMPLETE SEQUENCE OF A 7.5 KB REGION OF CHROMOSOME I11

nuclear localization signal (Starr and Hanover, 1990). These properties, combined with the observations that the mRNA is abundant, relatively unstable and affected by mild heat shock, might suggest that YCR521 encodes a ribosomal protein (Herruer et al., 1988). However, YCR.521 does not appear to carry a RAPl binding site: these are found in the promoters of most known ribosomal protein genes (Verdier, 1990), although the L3 and S33 ribosomal protein genes lack RAPl binding sites (All-Robyn et al., 1990). Also, the YCR521 gene is not essential (Thierry et al., 1990), and it does not contain an intron. Therefore, the function of YCR.521remains obscure. ARS element An 11/11 match to the ARS core consensus (5’-[A/T]TTTAT[AG]TTTAT)-3‘; Palzkill and Newlon, 1988) is located at 6301 within the YCR521 coding region. In addition, multiple imperfect ARS consensus sequences are located downstream of the perfect match (Figure 3), and this organization is typical of a functional ARS (Palzkill and Newlon, 1988). The functional ARS sequences within a 200 kb ring derivative of chromosome I11 (which contains the sequences carried in LambdaPM7121) have been mapped (Newlon e t al., 1986). The ARS within YCR.521 appears to correspond to the E5F2 ARS within the 1.55 EcoRIlEcoRI fragment described by Newlon and co-workers (1986). ACKNOWLEDGEMENTS We wish to thank Steve Oliver for providing the Lambda DNA and for helpful discussions. Chris Herbert and Bernard Dujon were kind enough to discuss their data with us prior to publication. We are also grateful to John Sgouros (MIPS) for his help, and to John Collins and Andrew Coulsen (Molecular Biology, Edinburgh University) for their advice on the DAP program. The speedy and reliable supply of oligonucleotides was maintained by Veer Math (Biochemistry, Glasgow University). We also thank Richard Young for the rpbl yeast strain. REFERENCES All-Robyn, J. A., Brown, N., Otaka, E. and Liebman, S. W. (1990). Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein. Mol. Cell. Biol. 10,65446553.

77 1

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772 Newlon, C. S., Green, R. P., Hardeman, K. J., Kim, K. E., Lipchitz, L. R., Palzkill, T. G., Synn, S. and Woody, S. T. (1986). Structure and organisation of yeast chromosome 111. UCLA Symp. Molec. Cell Biol. New Series 33, 21 1-223. Nonet, M., Scafe, C., Sexton, J. and Young, R. (1987). Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis. Mol. Cell. Biol. 7, 1602-1 6 11. Olson, M. V., Dutchik, J. E., Graham, M. Y., Brodeur, G. M., Helms, C., Frank, M., MacCollin, M., Scheinman, R. and Frank, T. (1986). Random-clone strategy for genomic restriction mapping in yeast. Proc. Natl. Acad. Sci. USA 8,7826-7830. Palzkill, T. G. and Newlon, C. S. (1988). A yeast replication origin consists of multiple copies of a small conserved sequence. Cell 53,441450. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, USA. Sanger, F., Nicklen, S. and Coulsen, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Santiago, T. C., Bettany, A. J. E., Purvis, I. J. and Brown, A. J. P. (1987). Messenger RNA stability in Saccharo-

B. L. WICKSTEED ETAL.

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