YEAST

VOL. 8: 4 1 9 4 2 2 (1992)

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Yeast Sequencing Reports

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Sequence of the Novel Essential Gene YJU2 and Two Flanking Reading Frames Located within a 3.2 kb EcoRI Fragment from Chromosome X of Saccharomyces cerevisiae HELENA FORROVA, JORDAN KOLAROV*, MICHEL GHISLAINt AND AND& GOFFEAU' Food Research Institute and *Cancer Research Institute, Slovak Academy of Sciences, 84215 Bratislava, Czechoslovakia 'Unite' de Biochimie Physiologique, Universite' Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium

Received 9 December 1991 ; accepted 28 January 1992

We have isolated and sequenced a novel gene from Saccharomyces cerevisiae coding for an essential protein of unknown function. The gene called YJU2 was borne on a chromosome X fragment as shown by hybridization to intact S. cerevisiae chromosomal DNA fractionated by orthogonal pulsed field electrophoresis. Northern hybridization analysis indicated that the 0.8-kb transcript of YJU2 is expressed in exponential-phase cells grown in rich medium (data not shown). Figure 1 shows the nucleotide and deduced amino-acid sequences of the 834-bp coding region as well as the nucleotide sequences of the 5' upstream region and of the 3' downstream region, together with the flanking neighbouring open reading frames (ORFs), YJUI and YJU3. SEQUENCE ANALYSIS OF THE YJU2 GENE

A search in databases (MIPS, November 1991) did not reveal any significant sequence homology. DISRUPTION OF THE YJU2 GENE The YJU2 coding region was interrupted at the unique ClaI site (nucleotide 117) by insertion of a 3.2-kb BglII fragment carrying the S.cerevisiae LEU2 gene (Figure 2A). The null allele of YJU2 was introduced, by homologous recombination (Rothstein, 1983), into haploid and diploid cells using the isogenic yeast strains W303TM 1A, W303-1B and W303 (respectively MATa or MATa or MATaa leu2-3,112 his3-11 ,I5 trpl -I can1 -100 ade2-I ura3-I). Leu+ prototrophic transformants were recovered in the diploid strain only. After sporulation of these transformants, tetrad analysis showed a 212 viablebethal segregation of the cells. None of the viable spores harbored the LEU2marked YJU2-disruption, indicating that the YJU2 gene is essential for cell survival.

The YJU2 gene encodes a polypeptide of 278 amino acids having a predicted molecular weight of 32,312 Da. This polypeptide is predicted to be hydrophilic using NEIGHBOURING OPEN READING FRAMES the method of Eisenberg et a1.(1984). A putative The YJUI ORF is lacking the initiation AUG codon nuclear targeting signal motif (K,R) (K,R) ( x ) , , , ~(see Figure 1). Comparison with protein and DNA (K,R) (X)"=0-2(K,R) (X),=a-:! (K,R) is localized at the sequences from available databases indicated a 40% carboxy terminus (residues 242-258 and 258-278), identity in a 117 amino-acid span of YJUI and the indicating that the YJU2 gene product could be a product of the yeast SRPI gene belonging to the nuclear protein (Robbins et al., 1991). serine-rich protein family (Marguet et al., 1988).

Address for correspondence: Dr A. Goffeau, Unit6 de Biochimie Physiologique, Place Croix du Sud, 2/20, B-1348 Louvain-la-Neuve, Belgium. Tel. 32/10/473614, FAX 32/10/473872.

0749-503X/92/0504 19-04$05.00 01992 by John Wiley & Sons Ltd

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G C A G C A G A ~ ~ C f f i T T ~ ~ C A T ~ ~ ~ ~ T ~ ~ ~ T ~ T -598 ~ A A D F S P S D S S S S S S A S A S S A S A S S S T K H S S S I E S V E T S T T G T G G A A A C T V E T S S V S S P

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G T C O G C A T ~ T ~ G G T ~ C T ~ T ~ ~ ~ ~ G ~ ~ A A G T ~ ~ T ~ A ~ ~ ~ T A-358 T ~ C T A ~ T ~ V G M G A G A L A V A A A Y L L A G C G T A A T G T A A T A T G A A P ; r G G C C C A T A A T A m A T T C T C T

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YJU2 => G A G T T A m A T T G T A C A T A T T ~ ~ ~ T C A ~ A ~ T G A C C T C A G T A ~ C T A A T f f i T ~ ~ ~ m ~ ~ G A T A G G 3A G A A T A ~ ~ T G M T C T G A A A G A A A A G C T P T T A A ~ G T A C T A T C C A C C G G 1A23 S E R K A I N K Y Y P P D Y N P L E A E K L S R K M A K K L K T M N K S H A S I A G A T T A A T G A ~ m A G T A T G A G G T G m ~ T G T A A C R L M T P F S M R C L E C N E Y I P K S R K F N G K K E L L K E K Y L D S I K I

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T A T A G A C T A A C C A m C A T G T C T T G T G C C A A T T C C A T T 363 Y R L T I S C P R C A N S I A F R T D P G N S D Y V M E V G G V R N Y V P Q K P

A A T G A T G ~ f f i T ~ ~ T ~ G A T ~ T N D D L N A K T A V E S I D E T L Q R L V R E K E M E Q N E K M G I K E Q A D D

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C G C A G C A A A C A C G C A C A A C A A A ~ ~ G A T G A T ~ ~ f f i T ~ T ~ A ~ ~ T ~723 ~ T A ~ T ~ ~ T A R S K H A Q Q E K A V T T D D L D N L V D Q V F D N H R Q R T N K P G N N N D E A A G A G A A C T ~ ~ G ~ A A T C C T ~ T ~ C T ~ ~ T C T A C A T C C A C T A A G843G G A A A A K R T P L F N P T S T K G K I ~ K K S S V R T N P L G I V I K R G K S L K -

A A A G T A A G G A A C G T A C G A T T G G T A T C A T G A T C T C T T G T G A T ~ G T ~ A C T G T G G T T ~ ~ ~ A T ~963 T ~ T ~ A ~ ~ ~ ~ ~ ~ G YJU3 => G T C T G G C C G T T T m C A T G A A m T C T T A T A A A T G A A T A C A T A T 1083 M A P Y

C A T A C A A A G T G C A G A ~ T ~ G A A ~ ~ T ~ ~ G A T G G T ~ f f i T T ~ T ~ T 1203 G ~ ~ ~ G T T ~ T

P Y K V Q T T V P E L Q Y E N F D G A K F G Y M F W P V Q N G T N E V R G R V L T G A T T C A T G G G m G G C G A G T A C A ~ G A ~ ~ T T ~ A T ~ A T C A C T ~ T ~ T T ~ T C A ~ ~ m G A ~ ~ 1~323 T ~ G G T G T T A C

L I H G F G E Y T K I Q F R L M D H L S L N G Y E S F T F D Q R G A G V T S P G G A T C G A A A G G T G T A A C T G A T G A G T A C C A T G T G m A A a ; A T C

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G G G I C L N Y A C Q G K H K N E I S G Y I G S G P L I I L H P H T M Y N K P T Figure 1 The nucleotide and deduced amino acid sequences of the essential YJU2 gene from Saccharomyces cerevisiae (strain S288C). The sequence was determined in both orientations using the progressive deletions method of Barnes et al. (1983) and the dideoxynucleotidechain-terminating method of Sanger et al. (1977). Nucleotides are numbered on the right. Position + I is assigned to the uppermost in-frame initiating codon of YJU2. The two motifs matching the nuclear localization signal at the carboxy-terminus of the polypeptide are underlined. The two neighbouring ORFs (=>), YJUl and YJU3, are marked similarly.

SEQUENCE OF THE NOVEL ESSENTIAL GENE YJU2 AND TWO FLANKING READING FRAMES A

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I M H G Q D D T I N D P K G S E K F I Q D C P S A D K E L K L Y P G A R H S I F ~ ~ ~ T ~ T ~ ~ T G T T ~ T ~ TAMCCATAACCTATAUGGACTrMTCACTTAGACAGTAT A T ~ T f f i T2043 T S L E T D E V F N T V F N D M K Q W L D K H T T T E A K P -

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Figure 1 (cont’d)

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YJU S

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Figure 2 Gene disruption analysis. The S . cerevisiae DNA insert is represented by a full line while the dashed line corresponds to the vector pEMBLl2’ sequence bordering the insert. (A) Disruption of the YJU2 gene. (B) Disruption of the YJU3 gene. Restriction endonucleases are abbreviated as follows: R, EcoRI; S, SphI; C, ClaI; HII, HindII; B, BgnI; H, HindIII.

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422 The YJU3 ORF encodes a polypeptide of 3 13 amino acids with a predicted molecular weight of 35,549 Da. Computer-aided comparisons of the predicted aminoacid sequence of Y J U 3 to sequence databases revealed no sequences with statistically significant similarity to YJU3. A null allele of the YJU3 gene was made by inserting the LEU2 marker gene within the coding region, at the BgZII site (Figure 2B). The disrupted YJU3 gene was then used to transform a haploid Leu- strain. Southern analysis of Leu+ transformants confirmed the predicted structure of the disrupted YJU3 gene by showing the presence of restriction endonuclease sites diagnostic of the transplacement. These Leu+ transformants grew normally and lacked obvious phenotypic abnormalities, suggesting that YJU3 is not essential to mitotic growth. ACKNOWLEDGEMENTS We gratefully acknowledge Dr John Sgouros (MIPS, Munich, Germany) and Dr Elisabetta Balzi (University of Louvain, Louvain-la-Neuve, Belgium) for their help in sequence analysis. This work was supported in part by grants to A. G. from the Services de la Politique Scientifique: Action Sciences de la Vie and

H.FORROVA ETAL,.

by the Fonds National pour la Recherche Scientifique, Belgium. REFERENCES Barnes, W.M., Bevan, M. and Son, P.H. (1983). Kilosequencing: creation of an ordered nest of asymmetric deletions across a large target sequence carried on phage M13. Methods Enzymol. 101,88-122. Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R. (1984). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J . Mol. Biol. 120,97-120. Marguet, G., Guo, X.J. and Lauquin, G.J.-M. (1988). Yeast gene SRPI (serine-rich protein) intragenic repeat structure and identification of a family of SRPI -related DNA sequences. J . Mol. Biol. 202,455470. Robbins, J., Dilworth, S.M., Laskey, R.A. and Dingwall, C. (1991). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615-623. Rothstein, R. J. (1983). One-step gene disruption in yeast. Methods Enzymol. 101,202-21 1. Sanger, F., Nickeln, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467.

Sequence of the novel essential gene YJU2 and two flanking reading frames located within a 3.2 kb EcoRI fragment from chromosome X of Saccharomyces cerevisiae.

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