YEAST

VOL. 8: 777-785 (1992)

0

0

11

00 0

00

Yeast Sequencing Reports

0 0 0 0

The Sequence of an 8 kb Segment on the Left Arm of Chromosome I1 from Saccharomyces cerevisiae Identifies Five New Open Reading Frames of Unknown Functions, Two tRNA Genes and Two Transposable Elements JACEK SKALA*, LUC VAN DYCK, BENEDICTE PURNELLE AND ANDRE GOFFEAUI Unite de Biochimie Physiologique, Universite' Catholique de Louvain, Place Croix du Sud 2/20, 1348 Louvain-la-Neuve, Belgium Received 21 April 1992; accepted 30 April 1992

The DNA sequence of an 8079 bp ClaI fragment located at 40 kb from the centromere on the left arm of chromosome I1 from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five new open reading frames, tRNAGly and tRNALeUgenes as well as sigma and truncated delta elements. The disruption of the three larger open reading frames shows that they are not essential for mitotic growth. KEY WORDS-Yeast; Saccharomyces cerevisiae; chromosome 11; tRNAGlY; tRNAbU; sigma; delta.

INTRODUCTION

In the framework of the European BRIDGE project for sequencing the Saccharomyces cerevisiae genome, we have determined the sequence of a 8079 bp ClaI fragment of the left arm of chromosome 11. This fragment was subcloned from the alpha1008.5 cosmid and is situated between the 8.5 kb and 12 kb ClaI segments reported by Van Dyck et al. (1992) and Perea and Jacq (in preparation). The sequence revealed four complete open reading frames (ORFs) larger then 300 bp and two tRNA genes (for glycine and isoleucine). The tRNAG*y is affiliated with transposable sigma and truncated delta elements. A fifth ORF YBL03.09, which extends to the neighbour 12 kb fragment (Perea and Jacq, in preparation) is also presented. MATERIALS AND METHODS The alpha1008.5 cosmid was provided by Rolf Stucka and Horst Feldmann (Munchen). It is a pYC3030 der-

ivative containing 32 kb DNA from chromosome I1 of S . cerevisiae alphaS288C. The Escherichia coli JM109 (recAl supE44 endAl hsdRl7 gyrA96 relA 1 thi (lac-proAB) F"traD36 proAB+ laclq lacZ M151) was used for plasmid amplification and subcloning. The S. cerevisiae haploids W303-1B MATa and EVWl-5D MATa (both ade2-1 trpl-1 leu2-3,112 his3-11,lS ura3-I) and diploid (issued from the cross of W303-1B and EVWl-5D) were used in the gene disruption experiments. The alpha1008.5 cosmid DNA was submitted to ClaI digestion, producing four fragments: 22 kb (12 kb of the insert plus 10 kb of the vector), 8.5 kb, 8 kb and 3.4 kb. After electrophoretic separation in 0.9% agarose gel, the 8 kb fragment was subcloned into the pTZl8R vector. The random sequencing strategy of Sambrook et al. (1989) modified by Jauniaux (personal communication) was carried out. About 25 pg of purified plasmid DNA were subjected to 1 min of sonication.

* Permanent address: Institute of Microbiology, Wroclaw University, Przybyszewskiego 63,51-148 Wroclaw, Poland. +

Addressee for correspondents

0749-503x/92/090777~9$08.50 01992 by John Wiley & Sons Ltd

778

J. SKALA E T A L

c

C'C

x

L

II

I

b3.4kbo(c==--

S S C C'BSBB I

I

I

I 1

8 . 5 .k--

-

-Van

I I

12 k b

XS II

I

vector PYC 3 0 3 0

-

1 Perea and Jacq in preparation _I

5 kb

CENTROMERE

Figure 1. Partial restriction map of the cosmid alpha10085 B: BarnHI; C: ClaI; C': ClaI protected by methylation of the DNA; S: SalI: X: XbaI.

A C

5' 3'

C' B

S

B

B

YBL03.13 YBL 03.12

X

s s

C

YBLO3.11 YBL 03.10

5'

-I+

1 0 0 0 bp

Figure 2. (A) General restriction map of the sequenced 8 kb ClaI fragment and sequencing strategy. The arrows indicate the direction and extent of each individual sequencing reaction. The white arrows indicate sequences made with specifically synthesized primers. The restriction sites are designated as described in the legend to Figure 1. (B) Position of the open reading frames and other genetic elements.

MOLECULAR ANALYSIS OF YEAST CHROMOSOME I1 BETWEEN CDMl AND LYS2

DNA was then treated for 30 min (at 37°C) with 20 U of T4 DNA polymerase following by 15 min of incubation (at room temperature) with 10 U of Klenow polymerase I and electrophoresed on a 0.8% agarose gel. Fragments ranging from 500 to 1000 bp were cut out from the gel, purified using Geneclean kit (Bio Rad), ligated with SmaI-digested pSEQl vector DNA and transformed into E. coli JM109 according to the Hanahan (1983) protocol. Transformed cells were spread on LB medium containing 70 pg/ml of ampicillin and 1 pg per plate of X-Gal and IPTG. To ensure the presence of insert, the white clones were tested by colony hybridizations with the coresponding ClaI fragment as a probe. Double-strand DNAs were prepared from positive clones and sequenced using T7 DNA polymerase (Pharmacia) and M13 forward and reverse primers. Sequencing was carried out on both strands by the dideoxy chain termination method (Sanger et al., 1977). Where necessary, oligonucleotide primers were used to fill in data gaps. The entire sequence was determined on both strands. Assembly of the obtained sequences and preliminary data analysis were done using PC/GENE software (IntelliGenetics,Inc.). Gene disruption experiments were performed using the one-step method of Rothstein (1983). The BgnI (from the pFL44 plasmid) and HindIII (from the YEP24) fragments containing the URA3 marker were inserted into the BamHI, BclI and HindIII restriction sites of the YBLO3.12, YBLO3.09 and YBL03.I 1 respectively. Transformations of the yeast diploid and haploid strains were done using the linear DNA fragments and resulting transformants were verified by Southern blotting according to Sambrook et al. (1989). RESULTS AND DISCUSSION Figure 1 shows the restriction map of the alpha1008.5 cosmid and position of the sequenced 8 kb ClaI fragment. The partial restriction map of this fragment, sequencing strategy and location of the ORFs longer than 300 bp and of other genetic elements are shown in Figure 2. The junctions with the adjacent 8.5 kb and 12 kb fragments were sequenced by Van Dyck et al. (1992) and Perea and Jacq (in preparation), showing that those fragments are contiguous. The complete DNA sequence is given in Figure 3. The sequence reveals two genes encoding isoleucine and glycine transfer tRNAs (Figures 2B and 3). The coding parts of both have 100% identity with already published tRNA1le and tRNAGly sequences (Felici and Cesareni, 1987; Gaber and Culbertson, 1982; Drabkin and RajBhandary, 1985), while the 5' and 3'

779

flanking regions are unrelated indicating that they originate from different parts of the yeast genome. At 15 bp from the 5' end of tRNAGIY,a sigma element with the classical 8 bp (TGTTGTAT) inverted repeats (Del Rey et al., 1982) was found (Figure 3). This sigma has 98.5% identity with previously reported sigmas related to tRNAHis (Del Rey et al., 1983) and tRNAA1a(Sandmeyer et al., 1988). At 113 bp from the sigma end we have detected a 83 bp fragment showing 78.3% identity with the delta terminal repeats of the Ty917 transposon (Roeder et al., 1980; Williamson, 1984). The location of this truncated delta is shown in Figures 2B and 3. The conserved sequence TACTAAC (Pikielny et al., 1983), present near the 3' end of all sequenced yeast introns, was found once in the upper strand (from telomere to centromere) and twice in the lower strand at positions 3600-3606, 3979-3973 and 6149-6143 respectively. The GTAYGT 5' splice site consensus sequence was found twice in both the upper and lower strands at positions 1486-1491, 3255-3260, 1212-1207 and 1187-1182. However, the relative position of these sequences suggests that their presence is fortuitous. Search for coding regions by Fickett's (1982) method revealed three large ORFs, YBL03.09, YBLO3.11 and YBLO3-12. The YBLO3.09 extends in the adjacent 12 kb fragment (Perea and Jacq, in preparation) of the alpha1008.5 cosmid. In addition, two small ORFs (YBL03.10 and YBL03.13) were detected (Figures 2B and 3). They are not likely to be expressed because they are included in the complementary strands of much longer genes. The upstream and downstream regions of the three largest ORFs have been analysed for the presence of basic transcription elements. Only a putative TATA box, CAP signal and polyadenylation site were detected. Figure 3 locates these elements. The predicted protein corresponding to the YBL03.09 comprises 759 amino acids and has a molecular weight of 85,693 Daltons. In the carboxy-terminal part of this protein, a long stretch of 18 glycine residues (position 738 to 755) and a PEST region (residue 686 to 712) were found. PEST regions characterize proteins with half-lives shorter than 2 h (Rogers et al., 1986). The search for membrane-associatedhelices using the methods of Klein et al. (1985), Rao and Argos (1986) and Eisenberg et al. (1984) revealed three putative transmembrane segments located as shown in Figure 4A. The putative product of the YBLO3-I1 gene is a protein of 393 amino acids with a molecular weight of 43,544 Daltons (Figure 4B). No functional motif was detected using the PROSITE software.

J. SKALA ET AL.

780

ATCGATAGATGATCGTTGATTATTCCTTCAACAGATTCTGTCTAAAAAATGGTAAAACTG TTGCTACTAATCAATATCATATAATTGATCCTTATTCAACAATATCTACAACGAAAAAGG AGATTAAAAAAATGCATGCTAAGTAAATGAAGGTTTTCATCTGTCCCTGATGTTTTTCTT tRNA1le - - - > CTTCAAGCACGTTAACCAGTAATCAAGGTCTCTTGGCCCAGTTGGTTAAGGCACCGTGCT

-, ~

-

1

AATAACGCGGGGATCAGCGGTTCGATCCCGCTAGAGACCATTTTTTTGCAATTCAAATAA

r

CGTCGTTTATTTTTTATATAAAAATAAAAATCAAAAAGAATTGCGCAAGCCCGGAATCGA L ( - - - tRNAG'Y __ 1

ACCGGGGGCCCAACGATGGCAACGTTGGATTTTACCACTAAACCACTTGCGCTTATTTCT I

m TGGAAGTGTTGTATCTCAAAATGAGATACGTCAGTATGACAATACGTCATCCTGAACGTT

u

CATAAAACACATATGAAACAACCTTATAACAAAACGAACAACATGAGATAAAACCCGGCC

TTCCCTAGCTGAACTACCCAAAAGTATAAATGCCTGAACAATTAGTTTAGATCCGAGATT CCGCGCTTCCACCACTTAGTATGATTCATATTTTATATAATATATAAGATAAGTAACATT CCGTGAATTAATCTGATAAACCGTTTTGACAACTGGTTACTTCCCTAAGACTGTTCATAT m TAGGATTGTCAAGACACTCCGATATTACTCGAGCCCGTAATACAACACTTTTTTTGGTAA I

GTAGTTTATAATAACATGTGTTGAAAGATAACAAATGCCACAAAATATCAATGGCTACTT AAAAGGGTAATTGCAAACCATTGGAATGAAATCCTAATATCATCTTCTTACACCGCACGT ~~

GATAATAAACTAGTAACACGAATACAACTAAACAGATGATATTATAGACTTTCATTCCAA truncated delta CACATCTGTTGACTAGTAAGATGGATATTAGTCAATAGATGATGTTTCTTATTCCAAACT _I

ATCATGGGTTTGTAAGAAAATTTATTTTGAGGGGAATTGATTACATTTCCTTTTTTTTTC AATGTACTTTGAAGTTTTTTCTTGTTTAACAATAAGCTTGCCTAATCATTAAATTAGTGC ATAAAAAAAAAGGTAAGCCGAGATTAAATAAGTAAACAAAGACATACTAAAAAAAATCAT

TCTGGTACATACAGCATTCTAGTTAATGTTAGATTAGCAGTGGAACATTCCATATTTAAT CTTGTATCATTATTAAAATAAGGATAAGTCTGAGAAGTCCATTCAACCGGGACTTAACCG CCACTTTAATTAAATCTATGCAGGATTACCCTTGTGTAGGCCACATAAAATTTACTGGAG ATTTCTGTCCGGAAAGTTATTGAAAGCTTTTAAGTATACTTAAATGATTTCTCTTTTCCA TTTCTACGTTGCCCTTTTATCAATCTTATTGTGGCATCGATCCTTGTACGTCTACTTATT CCATTATTTGATGAAAATTGAATCCCGTCTCTCCGGTCATATTATTTTCTTCACCTTTTA CTGCCGTATACAAATATTCCCTCTCGACGTGTACCATCATTTTGATCCGTGACATAGTAG GAATAGATGGATTATGGTCGATTGAGCTTCCAATACTTTGCGTATTGAGCAAGCTATGCA AATTAAAATATCTATAAACTGATAGGAAATGTATATACTGAAGAAAAT~TTTATTACTTG

1801 1861 1921 1981 2041 21 0 1 21 61 2221 2281 2341

-

AAATAA

denvlation -Ds toolp-v aYBLb3.72

site

~

TTACATTGTTTTGATTACCTGTGCACGCATTATACCTTATCCAAACCCCCGGATCCTTTT AAT

CTTCTTCTTTTTGGCTTTTTGAGAATTACGCGGTTGTGAATCGTTCACAAATGATATCTG CGAGGAAGGGGGCTGCATTAGAGTAAATGCTGGTGTCATTGAATCTGGCAGGACACTTAA GTTTTGAGTGGATTGTGATAACGGAGTAGCTTTTTGTGATTGTGTCTTTAATATTCTTTT CCTCCTTGCCAGTCCGCTACTTTGTGATGATTTGATCGTTGGAATTTGAGATTGCGAATT TAAATTAAATGGAGGCCTGCCATTAAAGGGTGCTGAAAATTGACTATCAAAATTAAATTC ATTAGATTCATCTTCTTCATTGATATCATCCATATCCCAAGAGGAGATAATATCCTGGTA AGGACCACTTAGTGATCGTGAAATTTCGTTTTGAATCGGTTCAAAAAGTGATGGTTTTTC AAGTCTCGCTAAACTCCAGATAATGTCTTTGACTATCTCCTTTGTCAGCAGTTCCGCTTG CGGGGATACCAAAACCCAACATTGCAATAATTTGTTGTAAAAGATATCCAAGCCCGGAAC GTCTTCATGTAAAAACAAATGTAGCAATTTCTCAAAACCAATAAATGTCACATCCTGATC

Figure 3. Complete DNA sequence of the 8 kb CluI fragment from the alpha1008.5 cosmid. One strand in the 5' to 3' orientation from the left arm telomere towards the centromere is entirely shown. The ORFs are boxed from the first possible ATG codon to the first encountered stop codon. The arrows indicate the orientation of the open reading frames (Oms). The tRNA genes and the 8-bp terminal inverted repeats flanking the sigma element are boxed. The truncated delta is underlined. Putative TATA box, CAP signal and polyadenylation sites are indicated. The restriction sites used in the disruption experiments are underlined twice and indicated.

MOLECULAR ANALYSIS OF YEAST CHROMOSOME I1 BETWEEN CDMI AND LYS2

2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181

CTGATAGTACTGGAAAAACTGATCTAGTAATGAAGAAAATTCAGGTATACTCGCGAAGGA ATCTGAATTCTCCAAAAGGTTTTTCAATTTTGAATGGCTCAAAGACCCACTAAGGATGCT TTCATCTTTCGTCTTCTGCCAAGATTCAAGCAATTCGTTTGTAGCTATAGATAAGCGATA TCCCAAATCTTGGAGATATTTATCTTCGTCGTGACTGTTTTCATGCTCAATTAAATTTTG GACTCGAGATATGCGCTCTCTTTCCTTCAACTTAATTTGAGAAACCAGTGCACCGATACT CTCCTTTTCACGTTCATCAGCGTTATTGAAAAGTGACGCCCATTCGGGATGGTCGACAAT Y GATGGGGGTTTCTTGTTTGTTCGGTTCGGAGTTTTGAGTGTTTGATAAAGCATAGTAATA B AACCTCGGATGAGTTCCTTAGTTTGACTAAAAAATCTACAACTAACTCAAAATTTTCATC L TGCATCCTCCTCTCTCCGAGATTCATCGTCTATATGATCCAGTGTTAAGATTGTCTCTAT 0 ACCGGTGGGGGTCCCACCCGGAATTTCAAGAACTGTTGAGCATCCTAAGGATTGAAACAA 3 GTTTGCTTTCCTATGGGAAAATACATGCATATAGATGCGTTTATGCCGCATCGAATATAC AAAGGCCACCAGAAGAATATGGTCAGGCTTTTTAACCTTTTGTACAGTGATTCTGAGCGT ? AGTATCATCTGGATCCAGATCGTGTTTCCAAGATATTCTTCTCACTGGATCATTTGATTC TGATGCCCCTACGATGATTATTTCCCTTGAAGTGAGTAAAATACCATTTTTATCATCTAT s t a r t YBL03.13 - - - >

-

3241

I

3301 3361 3421 3481

GTTCATAAAGTCGATCTCAATCATTTTGGACCTATCAAAGACCAGTATTTTTTGGAAGTG TGAAAACCATTCTATTCTTTTCCATGACGATAATTCTTCTGGATCAAAAATCGTACCGTG GAGGTTATCTATTAACTGTAGTTTCCTTTTGTTATTATTATTGAAATTCTTTGGTATCCT CCCGATACTCCAGTTACCTTTTATATCAATGATTGCGAATTGTTGTAAGTCCCAAGGATT

3541

GAATGCAAAATCTACCACCTGAAGGTCGTCTATTTCAACAAAATACAGTGGTTCTGAACT

3601 3661 3721 3781 3841 3901 3961 4021 4081 41 41 4201 4261 4321 4381 4441 4501 4561 4621 4681

YBL03.13 s t o p ACTAACCATGACATCACATGATCTTGAGTGGACACTTTCAATTCTAAAGATTTGGAAAGA ATTTTCGGTGATTATGCCCACCAGGTTTGATCTCCGCCCGATGGACTCAGAGGCCCCCGG TATCTTAATACTCTTGATAGGTGAATGCAATTCGATACTCGTTACATTGTTATGTCGGTT TAAATGCAATGTATTTTGTCGTGTCAGAACTGCTATATTTAGGACTGAGCCTGTTTTACC CGAGGCATATGCTATGATTTCGGTTCCGTCTCTATAATTTCTTAAATCTGATGCTGTCTG BamHI AATATATTGAGAGTCCAGTCGATTAGCCACAGTAGGATCCCAAAAGAAGGCGTCTTGCAT CGGCCTCGAAGAGTTAGTACTTTCTTGTAATGTATCATCTAAGTTGCGCAGCAAATCTGA AGGAATGTAATCTTGAAATGAAACTGGCGTATCGCAGTTCACACCAAACATAGAATCATT ACCAACGTCAACTTTCTTAAAAAAATGGACATCTTTCGGTACCACGGGTATCACTGGATT GACCACTATAGATGTATCCCCCTGATTGTCCGTATCTTCATCAATATCGCTAGTTATGAG ATCATCGTCGGCATCTGATTCTTGCAAAACCTTGTCGTCGGAAATATATCTTATGGCAGT ATCACAAAGCAGACTTTTGACCACTATATGAAGGTCTAGTGCATCCTCGGCCAATGTATC ATCTACTGGTCTTAGCCATTGTGGGTTTTCTTGCTTCTTCGTAGTGTAATTTTCTTGCGG ACAGTAAAGACTGGCGCCTTGTACACCAACACCCAATTGCGAGCCTAACACATCTGAGCT

-

4741

AGAAGATTGAAAACGGCGTATCAAACAAATGGTTAAAAATGAGAAGAATAACACCTACACG

4801 4861 4921 4981

CCTCCTATTCACATGCAGATATATTTCAAACAATGCTTCTCCACCAGTGCAGCCCTTGAA TGTGCTTTTCTTTGGTAGCGACACTTTCAGTAATTTCTCATTGCAAGCACTCAATGAGTT GCGTCAAAATAATGGAAGCTGTGGTATAGTGGACAATATTCAAGTAGTAACTAGGTCGCC GAAGTGGTGCGGTAGACAGAAGTCTATTTTGAAATACCCGCCGATCTTCGATATGGCAGA Hi n d I I I GAAGCTTCAATTGCCACGCCCAATTACATGCGACACCAAGCAGGAAATGTTGGCGCTAAG CAAACTGACACCCAGTCGCCAAGGAAATCCGGAGAACGACGGCTCCGGTGCTCCGTTCAA CGCGATCATTGCGGTTTCTTTTGGGAAGCTCATTCCGGGTGACTTGATCCGCGCGGTGCC B ATTGGCGCTAAACGTCC9TCCTTCGCTACTTCCCAGACATAAAGGCAGTGCACCTATCCA L GCGAGCTCTGCTCGAGGGTGACACTTACACCGGTGTAACTATACAGACACTGCATCCGGA 0 TCGGTTTGACCATGGTGCAATTGTAGCGCAGACGGAGCCACTGGCGATCGCAACAATGCT 3 GTCAAAAGGGAGAGTCAATGATTCGACGGCAGATTTTAATTCTGAGGGCCTGCCTCGGAG 1 GACTGCCATACTGATGGACCAGTTGGGCGCCCTTGGCGCCCAACTTTTGGGCCAAACGCT 7

5041 5101 5161 5221 5281 5341 5401 5461

Figure 3. (cont’d)

78 1

J. SKALA ET AL.

782

5521 5581 5641 5701 5761 5821 5881 5941 6001 I

6061 61 21 6181

GCCCTACGCGCTATGAAGAAAAAGAAAAGTAGGCCCGCCTTCTTTCCCAGTTGCAGTTTA YBLO3.10 s t o p - m TTTATGATTCGGAAAGATTGATGTTAGTAATGGCTTGTAGTTCATAACAGAGTGAATTAA TATA box GTGTAGGAAGCCCGGAATTAAATATATAGTAAAAAGAGCACAGGGGCGTTTACATCGGGG s t a r t YBLO3.09 - - - >

-

6241

TAAAAAAAAATGCCTGCACCAAAACTCACGGAGAAATTTGCCTCTTCCAAGAGCACACAG

6301 6361 6421

AAAACTACGAATTACAGTTCCATCGAGGCCAAAAGCGTCAAGACGTCGGCTGATCAGGCA TACATCTACCAAGAGCCTAGCGCTACCAAGAAGATACTTTACTCCATCGCCACATGGCTG TTGTACAACATCTTCCACTGCTTCTTTAGAGAAATCAGAGGCCGGGGCAGTTTCAAGGTA r

6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7 1 41 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 81 01 8 1 61 8221 8281 8341 8401 8461 8521

GTCGCCAAGTACCACAACCCGGAAACGAACAGAGATGCAGTGAAAGAATTATTAGATACC ATATCGAAGGGTTTACAATCCGTTACCGTTACATGTTCTGATTATGAAACTTTGATGGTG GTTCAAACGATAAGAAGACTATATATGACACAATTTAGCACCAAGTTACCGTTGCCCTTG ATTGTGGAAATGAACAGAAGAATGGTCAAAGGTTACGAATTCTATAGAAACGATCCTAAA ATAGCGGACTTGACCAAAGATATAATGGCATATAATGCCGCCTTGAGACACTATAATCTT Be1 I CCTGATCACCTTGTGGAGGAGGCAAAGGTAAATTTCGCAAAAAACCTCGGACTTGTTTTT TTTAGATCCATCGGGCTCTGCATCCTCTTTTCGTTAGCCATGCCAGGTATCATTATGTTC TCACCTGTCTTCATATTAGCCAAGAGAATTTCTCAAGAAAAGGCCCGTACCGCTTTGTCC AAGTCTACAGTTAAAATAAAGGCTAACGATGTCATTGCCACGTGGAAAATCTTGATTGGG ATGGGATTTGCGCCCTTGCTTTACATCTTTTGGTCCGTTTTAATCACTTATTACCTCAGA CATAAACCATGGAATAAAATATATGTTTTTTCCGGGTCTTACATCTCGTGTGTTATAGTC ACGTATTCCGCCTTAATCGTGGGTGATATTGGTATGGATGGTTTCAAATCTTTGAGACCA CTGGTTTTATCTCTTACATCTCCAAAGGGCTTGCAAAAGCTACAAAAGGATCGTAGAAAT CTGGCAGAAAGAATAATCGAAGTTGTAAATAACTTTGGAAGCGAATTATTCCCCGATTTC GATAGTGCCGCCCTACGTGAAGAATTCGACGTCATCGATGAAGAGGAAGAAGATCGAAAA ACCTCAGAATTGAATCGCAGGAAAATGCTAAGAAAACAGAAAATAAAAAGACAAGAAAAA

GATTCGTCATCACCTATCATCAGCCAACGTGACAACCACGATGCCTATGAACACCATAAC CAAGATTCCGATGGCGTCTCATTGGTCAATAGTGACAATTCCCTCTCTAACATTCCATTA TTCTCTTCTACTTTTCATCGTAAGTCAGAGTCTTCCTTAGCTTCGACATCCGTTGCACCT TCTTCTTCCTCCGAATTTGAGGTAGAAAACGAAATCTTGGAGGAAAAAAATGGATTAGCA AGTAAAATCGCACAGGCCGTCTTAAACAAGAGAATTGGTGAAAATACTGCCAGGGAAGAG GAAGAGGAAGAAGAAGAGGAAGAAGAAGAAGAGGAAGAAGAAGAAGAAGGGAAAGAAGGA

GATGCGTAG - S t o p YBLO3.09

I

Figure 3. (conr’d)

THE SEQUENCE OF AN 8 KB SEGMENT ON THE LEFT ARM OF CHROMOSOME I1

783

A

1 M P A P K L T E K F A S S K S T Q K T T N Y S S I E A K S V 3 1 K T S A D Q A Y I Y Q E P S A T K K I L Y S I A T W L L Y N 6 1 I F H C F F R E I R G R G S F K V P Q Q G P V I F V A A P H 9 1 A N Q F V D P V I L M G E V K K S V N R R V S F L I A E S S 1 2 1 L K Q P P I G F L A S F F M A I G V V R P Q D N L K P A E G 1 5 1 T I R V D P T D Y K R V I G H D T H F L T D C M P K G L I G 1 8 1 L P K S M G F G E I Q S I E S D T S L T L R K E F K M A K P 2 1 1 E I K T A L L T G T T Y K Y A A K V D Q S C V Y H R V F E H 2 4 1 L A H N N C I G I F P E G G S H D R T N L L P L K A G V A I 2 7 1 M A L G C M D K H P D V N V K I V P C G M N Y F H P H K F R 3 0 1 S R A V V E F G D P I E I P K E L V A K Y H N P E T N R D A 3 3 1 V K E L L D T I S K G L Q S V T V T C S D Y E T L M V V Q T 3 6 1 I R R L Y M T Q F S T K L P L P L I V E M N R R M V K G Y E 3 9 1 F Y R N D P K I A D L T K D I M A Y N A A L R H Y N L P D H 421 L V E E A K V N F A K N L G L V F F R S I G L C I L F S L A 451 M P G I I M F S P V F I L A K R I S Q E K A R T A L S K S T 481 V K I K A N D V I A T W K I L I G M G F A P L L Y I F W S V 51 1 L I T Y Y L R H K P W N K I Y V F S G S Y I S C V I V T Y S 541 A L I V G D I G M D G F K S L R P L V L S L T S P K G L Q K 571 L Q K D R R N L A E R I I E V V N N F G S E L F P D F D S A

6 9 1 A S T S V A P S S S S E F E V E N E I L E

K N G L A S K I

B 1 M V K M R R I T P T R L L F T C R Y I S N N A S P P V Q P L 3 1 N V L F F G S D T F S N F S L Q A L N E L R Q N N G S C G I 6 1 V D N I Q V V T R S P K W C G R Q K S I L K Y P P I F D M A 9 1 E K L Q L P R P I T C D T K Q E M L A L S K L T P S R Q G N 1 2 1 P E N D G S G A P F N A I I A V S F G K L I P G D L I R A V 1 5 1 P L A L N V H P S L L P R H K G S A P I Q R A L L E G D T Y 1 8 1 T G V T I Q T L H P D R F D H G A I V A Q T E P L A I A T M 2 1 1 L S K G R V N D S T A D F N S E G L P R R T A I L M D Q L G 2 4 1 A L G A Q L L G Q T L R E R L Y L P Q N R V Q A P T A Y K P 2 7 1 S Y A H R I T T E D K R I H W A R D S A A E L L N K L E T L 3 0 1 G P L H A F K E A T A A R K D A Q N S V L K R I L F H E C K 3 3 1 V M R D A R L D N G S K P G M F K Y D D I K D C I L V T C R 3 6 1 G N L L L C V S R L Q F E G F A V E R A G Q F M A R C G K D 391 A A P

Figure 4. Putative proteins encoded by: (A) YBL03.09. The double underlined putative transmembrane helices were predicted by the method of Klein ef al. (1985) and simultaneously confirmed by those of Rao and Argos (1986) and Eisenberg er al. (1984). The putative segment not confirmed by the method of Eisenberg et al. (1984) is underlined once. The PEST region is boxed. (B) YBL03.11. (C) YBU3.12. The leucine zipper motif is underlined once. The sugar transport protein signature is underlined twice.

C 1 M S E G Q I P S S D V L G S Q L G V G V Q G A S L Y C P Q E 3 1 N Y T T K K Q E N P Q W L R P V D D T L A E D A L D L H I V 6 1 V K S L L C D T A I R Y I S D D K V L Q E S D A D D D L I T 9 1 S D I D E D T D N Q G D T S I V V N P V I P V V P K D V H F 1 2 1 F K K V D V G N D S M F G V N C D T P V S F Q D Y I P S D L 1 5 1 L R N L D D T L Q E S T N S S R P M Q D A F F W D P T V A N 1 8 1 R L D S Q Y I Q T A S D L R N Y R D G T E I I A Y A S G K T 2 1 1 G S V L N I A V L T R -O N T L H L N R H N N V T S I E L H S 2 4 1 P I K S I K I P G A S E S I G R R S N L V G I I T E N S F Q 2 7 1 I F R I E S V H S R S C D V M V S S S E P L Y F V E I D D L 3 0 1 Q V V D F A F N P W D L Q Q F A I I D I K G N W S I G R I P 3 3 1 K N F N N N N K R K L Q L I D N L H G T I F D P E E L S S W 3 6 1 K R I E W F S H F Q K I L V F D R S K M I E I D F M N N W Q 3 9 1 T E V V Q A K A W S N I R D Y K R I D D K N G I L L T S R E 4 2 1 I I I V G A S E S N D P V R R I S W K H D L D P D D T T L R 4 5 1 I T V Q K V K K P D H I L L V A F V Y S M R H K R I Y M H V 4 8 1 F S H R K A N L F Q S L G C S T V L E I P G G T P T G I E T 5 1 1 I L T L D H I D D E S R R E E D A D E N F E L V V D F L V K 5 4 1 L R N S S E V Y Y Y A L S N T Q N S E P N K Q E T P I I V D 5 7 1 H P E W A S L F N N A D E R E K E S I G A L V S Q I K L K E 6 0 1 R E R I S R V Q N L I E H E N S H D E D K Y L Q D L G Y R L 6 3 1 S I A T N E L L E S W Q K T K D E S I L S G S L S H S K L K 6 6 1 N L L E N S D S F A S I P E F S S L L D Q F F Q Y Y Q D Q D 6 9 1 V T F I G F E K L L H L F L H E D V P G L D I F Y N K L L Q 7 2 1 C W V L V S P Q A E L L T K E I V K D I I W S L A R L E K P 7 5 1 S L F E P I Q N E I S R S L S G P Y Q D I I S S W D M D D I 7 8 1 N E E D E S N E F N F D S Q F S A P F N G R P P F N L N S Q 8 l l S Q I P T I K S S Q S S G L A R R K R I L K T Q S Q K A T P 8 4 1 L S Q S T Q N L S V L P D S M T P A F T L M Q P P S S Q I S 8 7 1 F V N D S Q P R N S Q K A K K K K K R I R G F G

Figure 4. (cont’d)

A H Xh

BK

11

I

+

YBL03.13

Bs BcHh I I j

I

YBL03.11

YBL03.09

L

YBL 03.12

F

YBL03.10

1 4E p

D

0

3

.

1

2

~ D 0 11 3

k

p D 0 3.09

-

1000 bp

H Figure 5 . Strategy of the gene disruption experiments. (A) Map showing the location of the open reading frames (ORFs) and the restriction sites used in the disruption experiments. The white part of the YBLO3.09 arrow shows the part of this O W extending into the neighbour 12 kb fragment (Perea and Jacq, in preparation). (B) Positions and extents of the linear fragments (thick lines) used to transform the yeast strains after insertion of URA3 marker. B: BnrnHI; Bc: Bclk Bs: BstEII; H: HindIII; Hh: HhaI; K: KpnI; Xh: XhoI.

THE SEQUENCE OF AN 8 KB SEGMENT ON THE LEFT ARM OF CHROMOSOME I1

The predicted protein corresponding to the ORF YBLO3.12 comprises 894 amino acids (Figure 4C) and has a molecular weight of 102,033 Daltons. In its amino-terminal part, a leucine zipper pattern (position 43 to 64) and a sugar transport proteins signature (position 251 to 262) were detected (Figure 4C). Despite the presence of the latter signature, no transmembrane spans typical for transport proteins were found. The value of the codon adaptation index calculated according to Sharp and Li (1987) is 0.003 for YBLO3.09, 0.079 for YBLO3.11 and 0.132 for YBLO3-12.No significant similarity between these ORFs and any known nucleotide or amino acid sequences was found (MIPS; March 1992). The ORFs YBLO3.09, YBLO3.11 and YBLO3.12 were disrupted by insertion of the URA3 marker as shown in Figure 5. In all cases, haploid and diploid strains bearing one disrupted ORF were obtained, showing that the genes under study are not essential. ACKNOWLEDGEMENTS We gratefully acknowledge R. Stucka and H. Feldmann for providing alpha1005.5 cosmid, J. Perea and C. Jacq for part of the YBLO3.09 sequence and J. Sgouros for help with computer analysis. This work was supported by the European Communities Commission programme BRIDGE and by matching funds from the Region Wallonne, Belgium. REFERENCES Del Rey, F., Donahue, T.F. and Fink, G.R. (1982). Sigma, a repetitive element found adjacent to tRNA genes of yeast. Proc. Natl. Acad. Sci. USA 79,41384142. Del Rey, F., Donahue, T.F. and Fink, G.R. (1983). The histidine tRNA genes of yeast. J . Biol. Chem. 258, 8 175-8 182. Drabkin, H.J. and RajBhandary, U.L. (1985). Attempted expression of human initiator tRNA gene in Saccharomyces cerevisiae. J . Biol. Chem. 260, 5596-5602. Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R. (1984). Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J . Mol. Biol. 179, 125-142.

785

Felici, F. and Cesareni, G. (1987). Structure of the Saccharomyces cerevisiae gene encoding tRNA1le (IAU). Nucl. Acids Res. 15,364. Fickett, J.W. (1982). Recognition of protein coding regions in DNA sequences. Nucl. Acids Res. 10,5303-5318. Gaber, R.F. and Culbertson, M.R. (1982). The yeast frameshift suppressor gene SUFI6-I encodes an altered glycine tRNA containing the four base anticodon 3'-cccg-5'. Gene 19, 163-172. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 116,557-580. Klein, P., Kanehisa, M. and Delisi, C. (1985). The detection and classification of membrane-spanning proteins. Biochim. Biophys. Acta 815,468476. Pikielny, C.W., Teem, J.L. and Rosbash, M. (1983). Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing. Cell 34,395-403. Rao, J.K.M. and Argos, P. (1986). A conformational preference parameter to predict helices in integral membrane proteins. Biochirn. Biophys. Acta 869, 197-2 14. Roeder, G.S., Farabaugh, P.J., Chaleff, D.T. and Fink, G.R. (1980). The origins of gene instability in yeast. Science 209, 1375-1380. Rogers, S., Wells, R. and Rechsteiner, M. (1986). Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234, 364-368. Rothstein, R.J. (1983). One-step gene disruption in yeast. Methods in Enzymology 101,202-21 1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (Eds) (1989). Molecular Cloning: a Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. Sandmeyer, S.B., Bilanchone, V.W., Clark, D.J., Morcos, P., Carle, G. F. and Brodeur, G. M. (1988). Sigma elements are position specific for many different yeast tRNA genes. Nucl. Acids Res. 16, 1499-1515. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Sharp, P.M. and Li, W.H. (1987). The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications. Nucl. Acids Res. 15, 1281-1295. Van Dyck, L., Purnelle, B., Skala, J. and Goffeau, A. (1992). An 11.4 kb DNA segment on the left arm of yeast chromosome I1 carries the carboxypeptidase Y sorting gene PEPI as well as ACHl, FUS3 and a putative ARS. Yeast. 8,769-776. Williamson, V.M. (1984). Transposable elements in yeast. Int. Rev. Cytol. 83, 1-25.