A 9.9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the beta-subunit of the succinyl-CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII

Azevedo, Dulce

Barreiros, Tânia

Guerreiro, Paulo

Rodrigues-Pousada, Claudina

Yeast

Repositório Científico do Instituto Politécnico de Lisboa

yeast vol. 13: 275–280 (1997)Sequencing of a 9·9 kb Segment on the Right Arm ofYeast Chromosome VII Reveals Four Open ReadingFrames, including PFK1, the Gene Coding forSuccinyl-CoA Synthetase (â-chain) and Two ORFsSharing Homology with ORFs of the YeastChromosome VIIIPAULO GUERREIRO1, DULCE AZEVEDO1, TANIA BARREIROS1 ANDCLAUDINA RODRIGUES-POUSADA1*1Laborato´rio de Gene´tica Molecular, Instituto Gulbenkian de Cieˆncia, Apartado 14, P-2781 Oeiras Codex, PortugalReceived 23 July 1996; accepted 26 August 1996A 9·9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced andanalysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1,has already been cloned and sequenced and the other one is the probable yeast gene coding for the â-subunit of thesuccinyl-CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (andtheir physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII. Thesequence is in the EMBL data library under Accession Numbers Z73024, Z73025, Z73026, Z73028 and Z73029.? 1997 by John Wiley & Sons, Ltd.Yeast 13: 275–280, 1997.No. of Figures: 4. No. of Tables: 1. No. of Refs: 9.key words— Saccharomyces cerevisiae; chromosome VII; chromosome VIII; PFK1; O44 protein; â-SCSINTRODUCTIONAs a contribution to the European UnionBIOTECH programme to sequence the Saccharo-myces cerevisiae genome, we have determined thesequence of a 9898 bp DNA fragment of the rightarm of chromosome VII. The sequenced region ispart of a 44 kb yeast segment inserted into thepWE15 cosmid that partially overlaps, at its leftside, the insert of cosmid pEGH484 sequenced byH. Steensma’s group and, at its right, the insert ofcosmid pUGH1273 sequenced by M. L. Carbone’sgroup, part of which was also sequenced byourselves.The fragment includes the previously sequencedPFK1 gene, the putative succinyl-CoA synthetaseâ-subunit encoding gene and two open readingframes (ORFs) sharing homology with other genesfrom yeast and rat. The latter presents a stronghomology with ORFs from chromosome VIII andthey both have the same physical arrangement asin this chromosome.*Correspondence to: Claudina Rodrigues-Pousada.Contract grant sponsor: Commission of the EuropeanCommunity under the BIOTECH programme.Contract grant sponsor: Calouste Gulbenkian Foundation.CCC 0749–503X/97/030275–06 $17.50? 1997 by John Wiley & Sons, LtdMATERIALS AND METHODSStrains and plasmidsThe pEGH301 cosmid was provided by H.Tettelin and A. GoVeau (DNA coordinators ofchromosome VII) and consists of a 44 kb DNAfragment of the chromosome VII of S. cerevisiaestrain S288C inserted in the pWE15 cosmid.Plasmid pUC19 was used for subcloning exper-iments. Escherichia coli strain XL1-Blue was thehost used for transformation and amplification ofplasmids. Standard techniques (Sambrook et al.,1989) were used for transformation of E. coli,restriction mapping, plasmid preparation, DNAligation and other manipulations.Sequencing strategyRandom libraries were generated frompEGH301 by digestion with the BglII, ClaI,EcoRI, KpnI and SfuI restriction enzymes. Over-lapping clones that cover the entire DNA frag-ment to be sequenced were chosen to generatesubclones suitable to sequencing. The sequence ofthe 9898 bp of cosmid pEGH301 reported hereincontains: the EcoRI-EcoRI, E18 (3·2 kb) subfrag-ment, the KpnI-KpnI, K2 (2·0 kb) subfragment,the BglII-BglII, Bg7 (6·2 kb) subfragment, theSfuI-SfuI, Sf2 (1·7 kb), Sf12 (0·4 kb) andSf11 (2·3 kb) subfragments, and the ClaI-ClaI,C2 (1·9 kb) subfragment. Clones suitable forsequencing were generated in two ways.Nested deletions were made using the PharmaciaExonuclease III nested deletion kit in order tosequence one of the strands of the E18, Bg7, Sf11and C2 subfragments. Direct deletions in all sub-fragments using suitable restriction enzymes wereused for the remaining sequence. DNA sequenc-ing was carried out by the dideoxy chain-termination method using T7 DNA polymerase(Pharmacia) and the ‘forward’ and ‘reverse’primers. Synthetic oligonucleotides were alsoused to fill a few gaps. DNA strands were se-quenced in both directions and gel compressionswere solved by sequencing the fragment withDeaza G/A T7 sequencing mixtures (Pharmacia).Sequence analysis softwareSequences from the individual reactions wereassembled into longer contigs with the aid ofDNASIS 5.0 analysis software (LKB-Hitachi).Comparison with the DNA sequences and de-duced amino acid sequences to the GenBankand Swiss-Prot databases was done using MIPSfacilities and the GCG package.RESULTS AND DISCUSSIONThe cosmid pEGH301 contains a fragment ofabout 44 kb located on the right arm of S. cere-visiae chromosome VII. Figure 1 presents theEcoRI restriction map of the fragment, the restric-tion map of the sequenced region and the locationof the ORFs herein. The left extremity of the insertFigure 1. Restriction map of the cosmid pEGH301. The upper part presents the EcoRI restriction map ofthe insert of the cosmid. The left part of the insert overlaps the pEGH484 cosmid and the right part overlapsthe pUGH1273 cosmid. The lower part shows the restriction map of the sequenced fragment here presented.The location and direction of the four ORFs are indicated by arrows, and above each arrow is the provisionalname given by MIPS. The filled arrow represents the previously sequenced gene PFK1. Spotted arrowsrepresent ORFs that share homologies with other genes. Bg, BglII; C, ClaI: E, EcoRI; K, KpnI; Sf, SfuI.276 p. guerreiro et al.overlaps H. Steensma’s cosmid pEGH484 and theright extremity overlaps M. L. Carbone’s cosmidpUGH1273.The DNA sequence of the 9898 bp fragment wasdetermined and is deposited in the EMBL datalibrary under accession numbers Z73024, Z73025,Z73026, Z73028 and Z73029. This region com-prises a total of four complete ORFs longer than100 amino acids, which are listed in Table 1. Othergenetic elements, such as tRNAs, Ty elements orintrons, were not found.G8599The G8599 ORF has already been sequencedand identified as the PFK1 gene, which encodes theá-subunit of the enzyme phosphofructokinase(Heinisch et al., 1989). Phosphofructokinase is anoctameric enzyme composed of four á-subunitsand four â-subunits that catalyses the conversionof fructose-6-phosphate and ATP to fructose-1,6-biphosphate and ADP in glycolysis. Seven diVer-ences have been found between the two sequences,Table 1. Characteristics of the ORFs.Name Position StrandaNo. ofamino acids CAIbFASTAopt. scorec Best homology with NotesdG8599 4049–1089 C 987 0·47 100% identical PFK1, phosphofructokinase 1 —G8610 6896–5193 C 568 0·16 1137 YHR161c 1 TM spanG8620 7651–8088 W 146 0·10 509 YHR162w —269 O44 protein (rat) —G8625 9634–8354 C 427 0·19 1080e Succinyl-CoA synthetase â-subunit(N. frontalis)5 ESTsaW, Watson strand; C, Crick strand.bCAI, codon adaptation index following Sharp and Li (1987).cFASTA optimal scores, database search following Pearson and Lipman (1988).dTM, transmembrane span, search following Klein et al. (1985); EST, cDNA tags.eThe best score obtained with succinyl-CoA synthetases from eight diVerent organisms.Figure 2. Alignment of the predicted amino acid sequence encoded by theORF G8610 with the yeast YHR161c protein. Only the amino acids diVeringfrom the first sequence are shown; dots indicate identical amino acids andbars indicate gaps in the sequence that were introduced to improve alignment.277sequencing 9·9 kb on the right arm of chromosome viiprobably due to the use of diVerent strains. One isa deletion located in the 3* non-coding region (oneC is missing at 932/933 position) and the remain-der are located near the C-terminal end of thecoding region. These diVerences do not cause anyamino acid substitution (G to A at positions 1620and 1716, G to T at position 1692, G to C atposition 1782, T to C at position 1794, and A to Gat position 1875).G8610The G8610 ORF encodes a putative protein of568 amino acids that shows 51·4% identity withYHR161c (Figure 2), a putative protein of 637amino acids from chromosome VIII (Johnstonet al., 1994). Indeed, the comparison between thesequence of chromosome VIII and the presentDNA sequence reveals significant similarities.Interestingly enough, both the deduced amino acidsequence and the physical arrangement of G8610and G8620 ORFs on chromosome VII are similarto those of YHR161c and YHR162w on chro-mosome VIII, indicating that duplications/transpositions had occurred during chromosomesegregation. However, the low homology of lessthan 50% observed between the intergenic regionsof G8610/G8620 (755 bp) and of YHR161c/YHR162w (981 bp), suggests that this may repre-sent an old event. Studies regarding the expressionof these ORFs have shown that their transcriptsare not detected by Northern analysis in ex-ponentially growing cells, even when amountscorresponding to 20 ìg of total RNA were used.G8620The G8620 ORF encodes a protein that shows73·6% identity with YHR162w (Johnston et al.,1994) and 45·9% identity with the rat O44 protein(Figure 3; Tsou et al., 1986), whose functions areunknown. However, the rat O44 gene was found tobe transcribed into nearly 100 diVerent mRNAsdue to alternative transcription initiation sitesand polyadenylation sites. A putative alternativesplicing site was also detected that producesmRNAs coding for proteins with diVerentC-termini. The O44 gene is expressed in all tissuestested but in brain it transcribes longer mRNAsthan those transcribed in liver or kidney (Tsouet al., 1986).G8625The G8625 ORF encodes a protein of 427 aminoacids that is strongly homologous to the â-subunitof the succinyl-CoA synthetase from eight organ-isms (Figure 4), showing a maximum of 55·7%identity with the rumen fungus Neocallimastixfrontalis enzyme. This suggests that G7742 definesthe yeast SCS-â gene. SCS is a mitochondrialheterodimeric enzyme of the citric acid cycle thatcatalyses the conversion of succinyl-CoA to succi-nate and CoA together with the formation of GTP(Bridger, 1974). Assuming that G8625 is the yeastsuccinyl-CoA counterpart, it will be the longestSCS so far detected, with an extended N-terminusand three unique residues at position 245–247.The first 30 residues of the protein share typi-cal characteristics of presequences of importedFigure 3. Alignment of the amino acid sequence encoded by ORF G8620 withthe yeast YHR162w protein and the rat O44 protein. Only the amino acidsdiVering from the first sequence are shown; dots indicate identical amino acidsand bars indicate gaps in the sequence that were introduced to improvealignment.Figure 4. Alignment of the amino acid sequence encoded byORF G8625 with the â-subunit of the succinyl-CoA synthetasefrom the rumen fungus Neocallimastix frontalis (PIR2: S52384),the protozoa Trichonomas vaginalis (PIR2: A45242), pig (PIR2:A44529) and the bacteria Coxiella burnetii (EMBL: X77919),Escherichia coli (PIR1: SYECSB), Haemophilus influenzae(PIR2: C64189), Methylobacterium extorquens (PIR2: B55230)and Thermus aquaticus (PIR2: S15950). Only the amino acidsdiVering from the first sequence are shown; dots indicateidentical amino acids and bars indicate gaps in the sequencethat were introduced to improve alignment.278 p. guerreiro et al.Figure 4.279sequencing 9·9 kb on the right arm of chromosome viimitochondrial proteins, namely seven basic resi-dues (M1, R4, K5, K12, R18, R28 and R29), fiveserines (S3, S6, S8, S11 and S17) and no acidicresidues (Hurt and van Loon, 1986).ACKNOWLEDGEMENTSWe thank H. Tettelin, coordinator of sequencingchromosome VII, for providing us with the recom-binant cosmid. We wish to acknowledge K. Kleineat MIPS for his help in computer analysis. Thiswork was supported, in part, by the Commissionof the European Community under the BIOTECHprogramme for sequencing the yeast genome andby grants of the Calouste Gulbenkian Foundationgiven to C.R.-P.REFERENCESBridger, W. A. (1974). Succinyl CoA synthetase. InBoyer, P. D. (Ed.), The Enzymes. Academic Press,Inc., New York, pp. 581–606.Heinisch, J., Ritzel, R. G., von Borstel, R. C., Aguilera,A., Rodicio, R. and Zimmermann, F. K. (1989). Thephosphofructokinase genes of yeast evolved from twoduplication events. Gene 78, 309–321.Hurt, E. C. and van Loon, A. P. G. M. (1986). Howproteins find mitochondria and intramitochondrialcompartments. Trends Biochem. Sci. 11, 204–207.Johnston, M., Andrews, S., Brinkman, R., et al. (1994).Complete nucleotide sequence of Saccharomycescerevisiae chromosome VIII. Science 265, 2077–2082.Klein, P., Kanehisa, M. and Delisi, C. (1985). Thedetection and classification of membrane-spanningproteins. Biochem. Biophys. Acta 815, 468–476.Pearson, W. R. and Lipman, D. J. (1988). Improvedtools for biological sequence comparison. Proc. Natl.Acad. Sci. USA 85, 2444–2448.Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989).Molecular Cloning, 2nd edn. Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York.Sharp, P. M. and Li, W.-H. (1987). The codon adapta-tion index—a measure of directional synonymouscodon usage bias, and its potential applications. Nucl.Acids Res. 15, 1281–1295.Tsou, A. P., Lai, C., Danielson, P., Noonan, D. J. andSutcliVe, J. G. (1986). Structural characterization of aheterogeneous family of rat brain mRNAs.Mol. Cell.Biol. 6, 768–778.280 p. guerreiro et al.

Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII

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Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII

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