XIV. Yeast sequencing reports. Organization of the centromeric region of chromosome XIV in Saccharomyces cerevisiae

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<ul><li><p>YEAST VOL. 10: 523-533 (1994) </p><p>oo O OO 0' 0 xrv % 0 Yeast Sequencing Reports 0 oooo </p><p>Organization of the Centromeric Region of Chromosome XIV in Saccharomyces cerevisiae DOMINIQUE LALO, SOPHIE STETTLER, SYLVIE MARIOTTE, EMMANUEL GENDREAU AND PIERRE THURIAUX* </p><p>Service de Biochimie et Ginitique Moliculaire, Dipartement der Biologie Cellulaire et Moliculaire, Commissariat a I'Energie Atomique, Centre dEtudes de Saclay, Brit. 142, F 91 191 Gif sur Yvette, France </p><p>Received 20 July 1993; accepted 20 October 1993 </p><p>A 15.1 kb fragment of the yeast genome was allocated to the centromeric region of chromosome XIV by genetic mapping. It contained six bonajde genes, RPC34, FUN34, CITI (Suissa et al., 1984), RLP7, PET8 and MRP7 (Fearon and Mason, 1988) and two large open reading frames, DOM34 and TOM34. RPC34 and RLP7 define strictly essential functions, whereas CITI, PET8 and MRP7 encode mitochondrial proteins. The PET8 product belongs to a family of mitochondrial carrier proteins. FUN34 encodes a putative transmembraneous protein that is non-essential as judged from the normal growth of the fun34-::LUK18( URA3) allele, even on respirable substrates. TOM34 codes for a putative RNA binding protein, and DOM34 defines a hypothetical polypeptide of 35 kDa, with no significant homology to known proteins. The region under study also contains two divergently transcribed tDNAs, separated only by a chimeric transposable element. This tight tDNA linkage pattern is commonly encountered in yeast, and a general hypothesis is proposed for its emergence on the Saccharomyces cerevisiae genome. RPC34, RLP7, PET8 and MRP7 are unique on the yeast genome, but the remaining genes belong to an extant centromeric duplication between chromosome I11 and XIV. The sequences have been deposited in the EMBL/GenBank data libraries under Accession Numbers L11277, L19167, M11344, M22116, V02536, X00782 and X63746. </p><p>KEY WORDS - Mitochondria1 carriers; duplication; citrate synthase; RNA binding; ribosomes. </p><p>INTRODUCTION We have cloned a 15.1 kb genomic fragment of Saccharomyces cerevisiae that was assigned to the centromeric region of chromosome XIV. This region has a marked similarity to the centromeric region of chromosome 111, indicating an extant centromeric duplication of these chromosomes (Lalo et al., 1993a). We report here its complete DNA sequence, which contained two tDNAs, six genes, two open reading frames (ORFs) and one chimeric transposable element. Four of the genes (RPC34, CIT1, RLP7 and MRP7) were described in previous publications (Stettler et al., 1992; </p><p>*Addressee for correspondence. </p><p>Suissa et al., 1984; Lalo et al., 1993b; Fearon and Mason, 1988) and will not be specifically dealt with in this paper. </p><p>MATERIALS AND METHODS Plasmids and sequencing strategy </p><p>YCp34-1 and YCp34-2 were isolated from a genomic library of strain GRF88 (Rose et al., 1987) by colony hybridization with a 1.1 kb RPC34 NcoI-NdeI probe (see Figure 1 and Stettler et al., 1992). Restriction fragments subcloned from these vectors by standard ligation techniques generated the following plasmids: pc34-4 (a 2-6 kb BamHI-EcoRI RPC34 fragment cloned in </p><p>CCC 0749-503W94/040523-11 0 1994 by John Wiley &amp; Sons Ltd </p></li><li><p>524 D. LALO ET AL. </p><p>28 11 39 18 </p><p>A ,.*' @a 1 YCp34-2 1 </p><p>I YCp34-1 1 1 Kb </p><p>I I I I pPET8-5 I pPET8-12 pc34-4 pBS34R I </p><p>1 pPET8-10 I I pPET8-16 I </p><p>I pRL7-2 I </p><p>Figure 1. General organization of the centromeric region of chromosome XIV. (A) Subcloning. Horizontal lines symbolize the inserts of the principal plasmids used in this study. YCp34-1 and YCp34-2 are YCp50 derivates isolated from strain GRF88 (Rose et al., 1987) by colony hybridization in E. coli, using a 1.1 kb NcoI-NdeI probe denoted by the double arrow. The relative positions of the ClaI and SaA sites of YCp50 are indicated in brackets, to give the orientation of the inserts relative to that vector. The inserts of pC34-4, pBS34R, pPET8-5, pPET8-10, pPET8-12, pPET8-16 and pRL7-2 were subcloned from YCp34-1 and YCp34-2 as described in Materials and Methods. (B) Mutational map. Triangles above the restriction map indicate the approximate positions of the rpc34-:: LUK28 (URA3), rpc34-:: LUKll (URA3), rpc34-::LUK39 (URA3) and fun34:--LUK18 (URA3) insertions. Boxes below the restriction map correspond to the rpc34-A::HIS3 (Stettler et al., 1992), rlp7-A::HIS3 (Lalo et al., 1993b), citl-A::LEUZ (Suissa et al., 1984) and mrp7-A::URA3 (Fearon and Mason, 1988) deletions. Black boxes or triangles denote lethal alleles, whilst the dotted triangle defines the viable lac2 fusion allele </p><p>fun34::LUKI8. The suf10 (Cummins et al., 1981) and pet8 (Mortimer et al., 1992) mutations are indicated for the sake of completion, but were not precisely located on the physical map. Only relevant restriction sites are indicated. The map is oriented according to the conventional orientation of the genetic map of chromosome XIV (Mortimer et al., 1992). (C) Functional map. Shaded boxes correspond to genes, open boxes to ORFs, filled boxes to tDNAs and striped boxes to transposable elements. The oval box denotes the centromere (Neitz and Carbon, 1985). Arrows indicate the transcriptional orientations. Two potential origins of replication (1 111 1 match with a DTTWATVTTTH consensus, see Williamson, 1985) are indicated by a star (W=A or T; D=non-C; V=non-T and H=non-G). The corresponding sequences are (from left to right): X63746, X00782, L11277, L19167, V02536 and M22116. </p><p>pUC19); pPET8-10 (a 5.6 kb SaZI-Clal FUN34- CITI fragment cloned in the centromeric vector pUN75 of Elledge and Davis (1988), where the Sari border originates from the YCp34-2 vector); pBS34R (a 3.4 kb EcoRI fragment overlapping FUN34 and CITl, cloned in the pBS + vector from Strategene); pPET8-5 (a 2-9 kb CZaI-SaZI CENl4- DOM34 fragment cloned in pUN75); pRL7-2 (a 4.8 kb Hind111 RPL7-PET8 fragment cloned in </p><p>pUC19); pPET8-12 (a 6.4kb SaZI-CZaI PET8- TOM34-MRP7 fragment cloned in pUN75, where the CZaI border originates from the YCp34-2 vec- tor) and pPET8-16 (a 3.6 kb XhoI PET8-TOM34 fragment in pUN75). Note that YCp34-1, YCp34-2 and pPET8-5 are dicentric and therefore not suitable for amplification in S. cerevisiae. These overlapping inserts were shortened by further subcloning, or by nested deletion using </p></li><li><p>CENTROMERIC REGION OF S. CEREVISIAE CHROMOSOME XIV 525 </p><p>Table 1. Yeast strains </p><p>YNN28 1 * YNN282* </p><p>CD34-6a* 801 ade2-I01 This work </p><p>CD34-6a* 801 ade2-I01 This work </p><p>CD34..5a* rpc34::LUK39( URA3) This work </p><p>CD34.8a* rpc34-::LUK28( URA3) This work </p><p>CD34.7A* 801 RPC34 +lrpc34::LUK44( URA3) This work </p><p>CD34.1 A* uyra3-52 RPC34lrpc34: : HIS3 Stettler et al. (1992) GRF88 MATa gal2 his4-18 Rose et al. (1987) K393-27~ Klapholz and Easton-Esposito (1 982b) DJY36 Lee et al. (1 984) D8 1 - 5 ~ Offspring of DJY36 x CD34-6a </p><p>MATa ura3-52 his34200 trpl-A1 lys2-801 ade2-I01 MATa ura3-52 his3-A200 trpl-A1 lys2-801 ade2-I01 </p><p>MATa fun34::LUKl8 ura3-52 his3-A200 t r p l d l lys2- </p><p>MATa fun34::LUK18 ura3-52 his3-A200 trpl-A1 lys2- </p><p>MATa ade2-101 lys2-801 his3-A200 trpl-A1 ura3-52 </p><p>MATa ura3-52 his34200 trpl-A1 lysa2-801 ade2-101 </p><p>MATaIMATa trpl-A1 his3-A200 ura3-52 ade2-101 lys2- </p><p>MATaIMATa ade2-101 lys2-801 trpl-A his3-A200 </p><p>Hieter et al. (1985) Hieter et al. (1985) </p><p>MATa ura3 his2 leu1 lysl met4 pet8 MATa prp2-1 adel ade2 ura3 </p><p>MATa prp2-1 fun34::LUK18( URA3) pet8 adel ura3 </p><p>*Isogenic strains. </p><p>exonuclease I11 digestion (Henikoff, 1984). The corresponding plasmids, amplified in the Escherichia coli strain DH5a (endAl, hsdRl7, supE44, thi-1, recA1, gyrA96, relA1, AlacU169 ((p8OlacAM1.5)) were sequenced on both strands of alkali-denaturated DNA, using the dideoxy nucle- otide chain-termination method (Sanger et al., 1977) with a modified T7 DNA polymerase (Sequenase 11, USB). This sequence partly over- laps the X00782 (Suissa et al., 1984), M11344 (Neitz and Carbon, 1985) and M22116 (Fearon and Mason, 1988) sequences. X00782 derives from strain FLlOO (Suissa et al., 1984; F. Lacroute, personal communication), M 1 1344 from strain AB230, and M22116 from strain X2180 (Snyder and Davis, 1985). The sequences were analysed with the Fasta (Pearson, 1990) and DNA Strider (Marck, 1988) softwares. </p><p>Yeast strains and genetic techniques Strains are listed in Table 1. The rpc34- </p><p>::LUk28( URA3), rpc34-::LUKll (URA3), rpc34- ::LUK39( URA3) and fun34-::LUK18( URA3) alleles were generated on pC34-4 by in vivo mu- tagenesis with the URA3 TnlOLUK chimeric transposon of Huisman et al. (1987). Rpc34- ::LUK28( URA3) and rpc34-::LUK39( URA3) are viable alleles corresponding respectively to a TnlOLUK insertion immediately upstream and downstream of the RPC34 coding sequence. Rpc34-::LUKll, an insertion in the middle of the </p><p>coding sequence, has a lethal phenotype. Rpc34- A::HIS3 and rlp7-A:: HIS3 alleles were constructed by inserting a 1.7 kb BamHI HIS3 cassette within partial deletions of the RPC34 or RLP7 (Stettler et al., 1992; Lalo et al., 1993b). These constructions were transferred as heterozygous alleles to YNN281 x YNN282 diploid cells as described by Rothstein (1983). Microdissection was performed with a de Fonbrunne micromanipulator. Growth media were previously described (Stettler et al., 1992). P-galactosidase assays were done on perme- abilized cells (Huisman et al., 1987) grown on YPD or YPG plates with 40mg/l of 5-bromo-4- chloro-3-indolyl p-D-galactoside. </p><p>RESULTS AND DISCUSSION Physical and genetic map </p><p>YCp34-1 and YCp34-2 delineate an insert of 15.1 kb centred on CENZ4 (Figure 1). The 295 bp CENl4 fragment (M 1 1344), previously sequenced by Neitz and Carbon (1985) from strain AB320, differed by a 3 bp change (AAAITTT) in the present work, presumably reflecting strain poly- morphism. The region also included two hitherto unmapped genes, MRP7 (M22116, Fearon and Mason, 1988), encoding a protein of the large mitochondria1 ribosomal subunit, and CITl (X00782, Suissa et al., 1984), encoding the mito- chondrial citrate synthase (E.C.4.1.3.7). Two puta- tive ARS elements were identified, one very close </p></li><li><p>526 D. LALO ET AL. </p><p>Figure 2. Similarity of the centromeric regions of chromosome I11 and XIV. Filled boxes correspond to homologous genes, ORFs or pseudogenes (XYZ3 on chromosome 111). Dotted boxes correspond to genes or ORFs present on one chromosome only. Arrows give transcriptional orientations (virtual in the case of the truncated delta34 element). The nomenclature of chromosome I11 ORFs was taken from Oliver et al. (1992). In contrast to Figure 1, chromosome XIV was inverted relative to the orientation of the genetic map (Mortimer et al., 1992), in order to align it directly with the chromosome 111 sequence. </p><p>to the centromere and one upstream of RPC34 (Figure 1). The latter is probably functional, since it is included in a 2.6 kb EcoRI-BarnHI fragment which, when cloned into an integrative plasmid, converted it into a replicative one (data not shown). DNA sequence and mutational analysis revealed a total of six genes (RPC34, FUN34, CITl, RLP7, PET8, MRP7), two ORFs (DOM34 and TOM34), two tDNAs and one chimeric trans- posable element (tau34ldelta34). The coden adap- tation index (Sharp and Cowe, 1991) of the eight genes and ORFs ranged between 0-1 1 (PET8) and 0.25 (CITl), suggesting that the corresponding proteins were expressed at a low or moderate level. Finally, a genomic insert containing the SISl gene (homologous to dnaJ in E. coli, Luke et al., 1991) overlapped the region analysed in the present work (K. Arndt, personal communication), putting SISl approximately 1.5 kb downstream of MRP7 (data not shown). </p><p>We have previously noted a strong homology in the centromeric regions of chromosomes I11 and XIV, implying that these regions are related by an extant duplication (Lalo et al., 1993a). The organization of the two regions is summarized in Figure 2. The presence of unique genes on chro- mosome I11 (e.g. CDClO and RVS161) suggests </p><p>that homologous genes were removed from chro- mosome XIV by deletions, or that insertional events led to a local gene transfer on chromosome 111. A pericentric inversion is also required to explain the opposite transcriptional orientation of TOM34 and YCLll C. </p><p>To map fun34-::LUK18, strain CD34-6a (MATa ura3 trpl fun34-::LUK18) was crossed to the MATa strains K393-27c (ura3 pet8) and DJY36 (ura3 prp2-1, see Table 1 for the complete genotype of these strains), and tetrads were analysed after sporulation of the corresponding diploids. The segregation of fun34::L UK18 (moni- tored by its uracil prototrophy in a ura3 back- ground) was assessed relative to TRPl (a tight centromeric marker, Mortimer et al., 1992) and to the PET8 and PRP2 genes of chromosome XIV. The high rate of prereductional segregation of fun34-::LUK18 relative to TRPl (33 parental ditypes, 34 recombinant ditypes and only one tetratype) indicated a strong centromeric linkage, in keeping with the physical map of Figure 1. Fun34-::L UK18 almost invariably cosegregated with pet8-1 (68 parental ditypes and one recombi- nant tetrad due to a 3:1 gene conversion offun34- ::L UK18), which corresponded to a genetic distance of 0.3 centimorgans. Furthermore, its </p><p>Figure 3. (A) Hydrophobicity plots of the FUN34 and YCRlOC coding sequences. The distribution of hydrophobic (positive values) and hydrophilic (negative values) residues was determined using the Strider software (Marck, 1988). It is consistent with a transmembrane organization, where the two hydrophilic regions (A and C) are on opposite sides of the hypothetical membrane. The two genes are characterized by N-terminal and C-terminal hydrophilic ends (A and E), with a small internal hydrophilic region (C) between two large hydrophobic domains (B and D). The latter two domains contain stretches of amino acids that are compatible with transmembrane a helices. (B) Alignment of the predicted amino acid sequence of FUN34 and YCRIOC. The alignment was generated using the Fasta software (Pearson, 1990). Identities between residues are shown by vertical lines and conservative changes by two dots. Note the less extensive similarity of the three hydrophilic domains A, C and E. </p></li><li><p>CENTROMERIC REGION OF S. CEREVISIAE CHROMOSOME XIV 527 </p><p>A 3 2 1 0 -1 -2 -3 -4 </p><p>4 3 2 1 0 </p><p>-1 -2 -3 </p><p>B FUN3 4 </p><p>YCRlOC </p><p>FUN3 4 </p><p>YCRlOC </p><p>FUN3 4 </p><p>YCRlOC </p><p>FUN3 4 </p><p>YCRlOC </p><p>FUN3 4 </p><p>YCRlOC </p><p>3 2 1 0 -1 -2 -3 -4 </p><p>4 3 2 1 0 -1 -2 -3 </p><p>4 </p><p>l l l : l l : l l l l : : l l I : 1 1 : : : ::: : : : : I : I : : I : I I I I : I : 1 1 1 1 1 1 1 1 1 1 </p><p>A M S D R E Q S S G N T A F E N - P K A L D S S E G E F I S E N N D Q S R H S Q E E Y I Y I G R Q 59 </p><p>MSDKEQTSGNTDLENAPAGYYSSHDNDVNGVAEDERPSHDSLGKIYTGGDNNEYIYIGRQ 60 </p><p>d I I I : : I l : : 1 1 1 1 1 1 l I I I l I I I I I I l 1 1 1 l I l I l I : l 1 1 1 l I I I I I I I I I I I I l I : l I l K F L R D D L F E A F G G T L N P G L A P A P V H K F A N P A P L G L S G F A L 119 </p><p>K F L K S D L Y Q A F G G T L N P G L A P A P V H K F A N P A P L G L S A F A L 120 </p><p>c WGCAMFYGGLVQLIAGIWEIALENTFGGTALCSFGGFWLSFGAIYIPWFGILDAYKDKE 1 7 9 </p><p>WGCAMFYGGLVQLIAGIWEIALENTFGGT...</p></li></ul>

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