YEAST
VOL. 7: 425429 (1991)
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: 00
Yeast Sequencing Reports
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Sequence of the CDClO Region at Chromosome I11 of Saccharomyces cerevisiae H. YDE STEENSMA AND QUIRINA J.M. VAN DER AART Department of Cellbiology and Genetics, Leiden University, Wassenaarseweg 64,2333 AL Leiden, The Netherlands Received 26 November 1990; accepted 4 December 1990
A 4.74 kb DNA fragment from the right arm of chromosome I11 of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDClO gene.
INTRODUCTION
MATERIALS AND METHODS
Sequencing chromosome 111, while still in progress, has already revealed a great number of open reading frames (ORFs). Some of these correspond to previously known, genetically defined genes, others represent new genes, some of which show sequence similarity with interesting genes from higher eukaryotes or other organisms (reviewed in Grivell and Planta, 1990). We have sequenced a chromosome I11 fragment contained in two clones (A2C and part of the overlapping clone D12B). This 4.74 kb fragment is located approximately 600 bp to the right of the centromere and is one of the first yeast sequences cloned, as it is part of a centromere-containing clone isolated by Clarke and Carbon (1980). These authors also reported the presence of the CDClO gene in this area. By deletion analysis, they mapped the gene around a BamHI site, approximately 4 kb from the centromere (Fitzgerald-Hayes et al., 1982). Subsequent, more accurate localization studies of CDClO by electron microscopy of RNA-DNA hybrid molecules (Rloops) and Northern analysis resulted in conflicting results (Feldberg and Kaback, 1985; Yeh et al. 1986). We report the sequence of the 4.74 kb chromosome I11 fragment containing the CDClO gene, and the position of the ORF corresponding to this gene.
Plasmids A2C and D12B (Newton et al., 1986), containing DNA from the CDClO region in the vector YIPS, were distributed by S.G. Oliver at the Manchester Biotechnology Centre. The chromosome 111 DNA in these plasmids originates from strain S288C. E.coli JM 107 (Sambrook et al., 1989) was used for propagation of plasmids and subclones in the vectors pEMBL8 and pEMBL9 (Dente et al., 1983). Standard recombinant DNA methods (Sambrook et al., 1989) were used to generate subclones with inserts between 500 and 1500 bp. Single-stranded DNA was isolated according to Dente et al. (1983). Isolation of double-stranded DNA has been described by Weickert and Chambliss (1989). Sequencing was carried out by the dideoxy method (Sanger et al., 1977) using T7 polymerase (Pharmacia) and [35S]dATPaccording to the manufacturer’s instructions. Oligonucleotides used for primers were purchased from Isogen Bioscience. For all sequences reported both strands were sequenced at least twice. Sequences were analysed using GCG sequence analysis software (Devereux et al., 1984) and compared to the MIPS protein data library by Dr J. Sgouros at the Martinsried Institut fur Proteinsequenzen.
0749-503X/9 1/04042545$05.00 01991 by John Wiley & Sons Ltd
H. YDE STEENSMA A N D QJ.M. V A S DER AART
426
This ORF, the largest in the fragment, was provisionally named YCR022. The ORF is 966 bp long A restriction map of the area sequenced and its corresponding to a peptide of 322 amino acids. The position on the physical map of chromosome Ill are ORF is preceded by potential TATA boxes at positions shown in Figure 1 (Mortimer ef al., 1989; Yoshikawa -13 to -9 (TATAA at 3423 to 3419). -83 to -79 and Isono, 1990). The complete sequence is repro(TAATA at 3493 to 3489) and - 103 to - I00 (TATA at duced in Figure 2A. The sites in the restriction map 3513 to 3510). It is most likely that YCR022 is the were all found at the expected positions of the sequence CDCIU gene, since it is the only ORF in the region and they corresponded to those reported by Clarke and that is sufficiently large. In Figure 2C its position is Carbon (1980) and Feldberg and Kaback (1985). but compared to previous studies. Clarke and Carbon (1980) differed slightly from the maps of Yeh ef a/. (1986) located CDCIO around the BamHl site. The first two and Yoshikawa and Isono (1990). Differences are bases of the YCR022 ORF are indeed part of this site. marked by arrows in Figure 1. The Hind111 site at The R-looping data from Kaback and Feldberg ( 1985) position 1 is at position 2 in the Yoshikawdlsono map. also support our positioning of CDCIU. The localizaThe EcoRI and Hind111 sites (3 and 4) are absent from tion by Yeh ef ul. (1986) differs, but is not reliable the map of Yeh ef al. and the EcoRl site at position 5 since their restriction map differs from all the other is located approximately 300 bp further to the right on maps (cf. Figure I ) . The significance of their 1.3 kb that map. transcript is not clear. This transcript probably originates from a more centromere-distal ORF (M. Aigle and M. Crouzet, personal communication). The mRNA CEY3 from ORF YCR022 would be approximately I kb HYL HI?+ LEY2 C%lO TGKl M+T HYR long. This corresponds with the 0.96-1.2 kb reported for the CDCIU mRNA (Kaback and Feldberg, 1985; . LUA Yoshikawa and Isono, 1990). Thus both the position 50kb and size of YCR022 are in agreement with CDCIU. .* From these results we concluded that YCR022 is Y D indeed the CDCIO gene. During preparation of the manuscript we learned that Pringle and coworkers have recently also sequenced the CDCIO gene (J.R.Pringle, U personal communication). Their sequence was identical I # f! t 3 4 5 1 2 1 kb to the one shown in Figure 2A (position 3410 to Figure.1. Physical map of chromosome 111. 'The small bar under- position 2444). neath the physical map shows the region sequenced. A restriction A small ORF, potentially encoding a peptide of map of this area is shown on a different scale. B = BumHI. H = 67 amino acids, was completely internal to the CDCIU Hindlll. K = K p n l . P = Psrl, R = EcoRI. X = XhoI. Arrows below gene, but was read in the opposite direction. Small the map indicate differences with published restriction maps ORFs are sometimes found in introns (Lazowska et described in the text. al., 1981; de la Salle et al., 1982; Netter et ul., 1982; Open reading frames initiating at an ATG and Magdolen et ul., 1988).There is, however, no indication encoding putative peptides longer than 66 amino acids for intron-specific sequences (Langford et al., 1984) are marked in Figure 2A. The sizes and positions of in CDCIU. This small ORF has not been analysed these four ORFs are summarized in Figure 2B and further. The fourth ORF (YCR041 in Figure 2) encoding a will be discussed below. The most centromere-proximal ORF, named putative peptide of 183 amino acids has previously YCR02 I , corresponds to a hypothetical protein of been sequenced by Aigle and coworkers (M. Aigle 104 amino acids and has no evident homology to any and M. Crouret, personal communication) and will be known gene. It is not clear whether this ORF (YCR021) described elsewhere. From the 4740 bases sequenced, 2 I27 constitute is actually transcribed. Several mRNAs, the smallest of which was 600 nucleotides, were found in this region the three ORFs, giving a potential transcription density by Northern analysis (Yeh et al., 1986), but were not of only 45%. Hence, the transcription density in this included in a recent transcription map of chromosome centromere-adjacent region is low. A similar situation 111 (Yoshikawa and Isono, 1990). There is a potential exists in chromosome I, where no transcripts were TATA box at -136 to -131 (TATATA at 612 to 617 in found in the 3 kb surrounding CENI (Steensma et Figure 2). Splice signals were not found in YCR021 al., 1987; Kaback, personal communication). Given or the region between YCR021 and the next ORE the high transcription density, up to 85%, in other RESULTS AND DISCUSSION
- .
..
.-. ..
... -..-.
Figure.2. Sequences of A2C and part of D12B. (A) Sequence. Position I is the first G of the centromere-proximal BumHl site. Shaded areas represent ORFs beginning with an ATG and encoding peptides larger than 66 amino acids. The middle, largest ORF runs from right to left and hence is represented by its complementary strand. (B) Position of ORFs. The restriction map of the region is shown. Symbols as in Figure I . The positions and sizes of the ORFs are summarized. (C) Localization of CDCIO. Previously published localizations of CDClO are shown. I = Clarke and Carbon, 1980 2 = Kaback and Feldberg, 1985; 3 = Yeh CI a/., 1986.
428 regions investigated thus far (Coleman et al., 1986; Steensma et al., 1987; Yoshikawa and Isono, 1990), it is tempting to speculate that these untranscribed regions are required for some household function of the chromosome. They are obviously not necessary for centromere function per se, since transcripts are found close to several other centromeres (Mann and Davis, 1986; Toh-E and Shimauchi, 1986). The physical distance between CDEIII of CEN3 and the 3’ end of CDClO is approximately 2.9 kb (FitzgeraldHayes et al., 1982), whereas the genetic distance is 0.3 cM (Mortimer and Schild, 1980). The recombination frequency in this region is thus 0.1 cM/kb. This is far below the 04-0-6 cM/kb found for other parts of the yeast genome (Mortimer and Schild, 1985; Strathern et al., 1979; Olson et al., 1986; Kaback et al., 1989). Hence recombination around CEN3, at least on the right side, is decreased. This has also been reported for some other centromere regions (Lambie and Roeder, 1986; Kaback et al., 1989). ACKNOWLEDGEMENT We would like to thank Dr. J. Sgouros for the sequence analyses, Drs M. Aigle and J. Pringle for communicating data prior to publication and our colleagues at the RU Leiden for helpful discussions. We appreciate the technical assistance of B. van der Reyden in part of this work. This study was supported by the EEC in the framework of the BAP project Sequencing of yeast chromosome 111. REFERENCES Clarke, L. and Carbon, J. (1980) Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287,504-509. Coleman, K.G., Steensma, H.Y., Kaback, D.B. and Pringle, J.R. (1986). Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation and characterization of the CDC24 gene and adjacent regions of the chromosome. Mol. Cell. Biol. 6,45 16-4525. Dente, L., Cesareni, G. and Cortese, R. (1983). pEMBL: A new family of single stranded plasmids. Nucleic Acids Res. 11, 1645-1655. Devereux, J., Haeberli, P. and Smithies, 0. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12,387-395. Fitzgerald-Hayes, M., Clarke, L. and Carbon, J. (1982). Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29,235-244. Grivell, L.A. and Planta, R.J. (1990). Yeast: the model eukaryote? TIBS 8,241-243. Kaback, D.B. and Feldberg, L.R. (1 985). Saccharomyces
H. YDE STEENSMA AND
Q J.M. VAN DER AART
cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation. Mol. Cell. Biol. 5, 751-761. Kaback, D.B., Steensma, H.Y. and de Jonge, P. (1989). Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae. Proc. NatLAcad. Sci. USA 86, 3694-3698. Lambie, E.J. and Roeder, S. (1986). Repression of meiotic crossing over by a centromere (CEN3)in Saccharomyces cerevisiae. Genetics 114,769-789. Langford, C.J., Klinz, F.J., Donath, C. and Gallwitz, D. (1984). Point mutations identify the conserved intron contained TACTAAC box as an essential splicing signal sequence in yeast. Cell 36,645-653. Lazowska, J., Jacq, C. and Slonimski, P.P. 1981 Splice points of the third intron in the yeast mitochondria1 cytochrome b gene. Cell 27, 12-14. Magdolen, V., Oechsner, U., Mueller, G. and Bandlow, W. (1988). The intron-containing gene for yeast prolifin (PFY) encodes a vital function. Mol. Cell. Biol. 8, 5 108-5 115. Mann, C. and Davis, R.W. (1986). Structure and sequence of the centrometric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol. Cell. Biol. 6,241-245. Mortimer, R.K. and Schild, D. (1980). Genetic map of Saccharomyces cerevisiae. Microbiol. Rev. 4 4 , 5 19-571. Mortimer, R.K. and Schild, D. (1985). Genetic map of Saccharomyces cerevisiae, Edition 9. Microbiol. Rev. 49, 181-212. Mortimer, R.K., Schild, D., Contopoulos, C.R. and Kans, J.A. (1989). Genetic map of Saccharomyces cerevisiae, Edition 10. Yeast 5, 321-403. Netter, P., Jacq, C., Carignani, G. and Slonimski, P. (1982). Critical sequences within mitochondria introns: Cis dominant mutations of the “cytochrome-b-like” intron of the oxidase gene. Cell 28,733-738. 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 organization of yeast chromosome 111. UCLA Symp. Mol. Cell. Biol. New Series 33, 31 1-223. 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 83,7826-7830. Salle, H. de la, Jacq, C. and Slonimski, P. (1982). Critical sequences within mitochondria1 introns: Pleiotropie mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase. Cell 28, 721-732. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. 3rd edition. CSH Laboratories, Cold Spring Harbor, New York. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Steensma, H.Y., Crowley, J.C. and Kaback, D.B. (1987).
SEQUENCE OF THE CDClO REGION AT CHROMOSOME 111 OF SACCHAROMYCES CEREVISIAE
Molecular cloning of chromosome I of Saccharomyces cerevisiae: Isolation and analysis of the CENl-ADEI CDC1.5 region. Mol. Cell. Biol. 7,410-419. Strathem, J.N., Newlon, C.S., Herskowitz, I. and Hicks, J.B. (1979). Isolation of a circular derivative of yeast chromosome 111: Implications for the mechanism of mating type interconversion. Cell 18,309-3 19. Toh-E, A. and Shimauchi, T. (1986). Cloning and sequencing of the P H 0 8 0 gene and CENl.5 of Saccharomyces cerevisiae. Yeast 2, 129-139.
429
Weickert, M.J. and Chambliss, G.H. (1989). Acid-phenol minipreps make excellent sequencing templates. Editorial Comments USBC 16,5-6. Yeh, E., Carbon, J. and Bloom, K. 1986 Tightly centromerelinked gene (SP01.5) essential for meiosis in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 6, 158-167. Yoshikawa, A. and Isono, K. (1990). Chromosome I11 of Saccharomyces cerevisiae: An ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of 160 genes. Yeast 6,383401.
VOL. 7: 425429 (1991)
0 0 0
: 00
Yeast Sequencing Reports
111 0 0 0 0
Sequence of the CDClO Region at Chromosome I11 of Saccharomyces cerevisiae H. YDE STEENSMA AND QUIRINA J.M. VAN DER AART Department of Cellbiology and Genetics, Leiden University, Wassenaarseweg 64,2333 AL Leiden, The Netherlands Received 26 November 1990; accepted 4 December 1990
A 4.74 kb DNA fragment from the right arm of chromosome I11 of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDClO gene.
INTRODUCTION
MATERIALS AND METHODS
Sequencing chromosome 111, while still in progress, has already revealed a great number of open reading frames (ORFs). Some of these correspond to previously known, genetically defined genes, others represent new genes, some of which show sequence similarity with interesting genes from higher eukaryotes or other organisms (reviewed in Grivell and Planta, 1990). We have sequenced a chromosome I11 fragment contained in two clones (A2C and part of the overlapping clone D12B). This 4.74 kb fragment is located approximately 600 bp to the right of the centromere and is one of the first yeast sequences cloned, as it is part of a centromere-containing clone isolated by Clarke and Carbon (1980). These authors also reported the presence of the CDClO gene in this area. By deletion analysis, they mapped the gene around a BamHI site, approximately 4 kb from the centromere (Fitzgerald-Hayes et al., 1982). Subsequent, more accurate localization studies of CDClO by electron microscopy of RNA-DNA hybrid molecules (Rloops) and Northern analysis resulted in conflicting results (Feldberg and Kaback, 1985; Yeh et al. 1986). We report the sequence of the 4.74 kb chromosome I11 fragment containing the CDClO gene, and the position of the ORF corresponding to this gene.
Plasmids A2C and D12B (Newton et al., 1986), containing DNA from the CDClO region in the vector YIPS, were distributed by S.G. Oliver at the Manchester Biotechnology Centre. The chromosome 111 DNA in these plasmids originates from strain S288C. E.coli JM 107 (Sambrook et al., 1989) was used for propagation of plasmids and subclones in the vectors pEMBL8 and pEMBL9 (Dente et al., 1983). Standard recombinant DNA methods (Sambrook et al., 1989) were used to generate subclones with inserts between 500 and 1500 bp. Single-stranded DNA was isolated according to Dente et al. (1983). Isolation of double-stranded DNA has been described by Weickert and Chambliss (1989). Sequencing was carried out by the dideoxy method (Sanger et al., 1977) using T7 polymerase (Pharmacia) and [35S]dATPaccording to the manufacturer’s instructions. Oligonucleotides used for primers were purchased from Isogen Bioscience. For all sequences reported both strands were sequenced at least twice. Sequences were analysed using GCG sequence analysis software (Devereux et al., 1984) and compared to the MIPS protein data library by Dr J. Sgouros at the Martinsried Institut fur Proteinsequenzen.
0749-503X/9 1/04042545$05.00 01991 by John Wiley & Sons Ltd
H. YDE STEENSMA A N D QJ.M. V A S DER AART
426
This ORF, the largest in the fragment, was provisionally named YCR022. The ORF is 966 bp long A restriction map of the area sequenced and its corresponding to a peptide of 322 amino acids. The position on the physical map of chromosome Ill are ORF is preceded by potential TATA boxes at positions shown in Figure 1 (Mortimer ef al., 1989; Yoshikawa -13 to -9 (TATAA at 3423 to 3419). -83 to -79 and Isono, 1990). The complete sequence is repro(TAATA at 3493 to 3489) and - 103 to - I00 (TATA at duced in Figure 2A. The sites in the restriction map 3513 to 3510). It is most likely that YCR022 is the were all found at the expected positions of the sequence CDCIU gene, since it is the only ORF in the region and they corresponded to those reported by Clarke and that is sufficiently large. In Figure 2C its position is Carbon (1980) and Feldberg and Kaback (1985). but compared to previous studies. Clarke and Carbon (1980) differed slightly from the maps of Yeh ef a/. (1986) located CDCIO around the BamHl site. The first two and Yoshikawa and Isono (1990). Differences are bases of the YCR022 ORF are indeed part of this site. marked by arrows in Figure 1. The Hind111 site at The R-looping data from Kaback and Feldberg ( 1985) position 1 is at position 2 in the Yoshikawdlsono map. also support our positioning of CDCIU. The localizaThe EcoRI and Hind111 sites (3 and 4) are absent from tion by Yeh ef ul. (1986) differs, but is not reliable the map of Yeh ef al. and the EcoRl site at position 5 since their restriction map differs from all the other is located approximately 300 bp further to the right on maps (cf. Figure I ) . The significance of their 1.3 kb that map. transcript is not clear. This transcript probably originates from a more centromere-distal ORF (M. Aigle and M. Crouzet, personal communication). The mRNA CEY3 from ORF YCR022 would be approximately I kb HYL HI?+ LEY2 C%lO TGKl M+T HYR long. This corresponds with the 0.96-1.2 kb reported for the CDCIU mRNA (Kaback and Feldberg, 1985; . LUA Yoshikawa and Isono, 1990). Thus both the position 50kb and size of YCR022 are in agreement with CDCIU. .* From these results we concluded that YCR022 is Y D indeed the CDCIO gene. During preparation of the manuscript we learned that Pringle and coworkers have recently also sequenced the CDCIO gene (J.R.Pringle, U personal communication). Their sequence was identical I # f! t 3 4 5 1 2 1 kb to the one shown in Figure 2A (position 3410 to Figure.1. Physical map of chromosome 111. 'The small bar under- position 2444). neath the physical map shows the region sequenced. A restriction A small ORF, potentially encoding a peptide of map of this area is shown on a different scale. B = BumHI. H = 67 amino acids, was completely internal to the CDCIU Hindlll. K = K p n l . P = Psrl, R = EcoRI. X = XhoI. Arrows below gene, but was read in the opposite direction. Small the map indicate differences with published restriction maps ORFs are sometimes found in introns (Lazowska et described in the text. al., 1981; de la Salle et al., 1982; Netter et ul., 1982; Open reading frames initiating at an ATG and Magdolen et ul., 1988).There is, however, no indication encoding putative peptides longer than 66 amino acids for intron-specific sequences (Langford et al., 1984) are marked in Figure 2A. The sizes and positions of in CDCIU. This small ORF has not been analysed these four ORFs are summarized in Figure 2B and further. The fourth ORF (YCR041 in Figure 2) encoding a will be discussed below. The most centromere-proximal ORF, named putative peptide of 183 amino acids has previously YCR02 I , corresponds to a hypothetical protein of been sequenced by Aigle and coworkers (M. Aigle 104 amino acids and has no evident homology to any and M. Crouret, personal communication) and will be known gene. It is not clear whether this ORF (YCR021) described elsewhere. From the 4740 bases sequenced, 2 I27 constitute is actually transcribed. Several mRNAs, the smallest of which was 600 nucleotides, were found in this region the three ORFs, giving a potential transcription density by Northern analysis (Yeh et al., 1986), but were not of only 45%. Hence, the transcription density in this included in a recent transcription map of chromosome centromere-adjacent region is low. A similar situation 111 (Yoshikawa and Isono, 1990). There is a potential exists in chromosome I, where no transcripts were TATA box at -136 to -131 (TATATA at 612 to 617 in found in the 3 kb surrounding CENI (Steensma et Figure 2). Splice signals were not found in YCR021 al., 1987; Kaback, personal communication). Given or the region between YCR021 and the next ORE the high transcription density, up to 85%, in other RESULTS AND DISCUSSION
- .
..
.-. ..
... -..-.
Figure.2. Sequences of A2C and part of D12B. (A) Sequence. Position I is the first G of the centromere-proximal BumHl site. Shaded areas represent ORFs beginning with an ATG and encoding peptides larger than 66 amino acids. The middle, largest ORF runs from right to left and hence is represented by its complementary strand. (B) Position of ORFs. The restriction map of the region is shown. Symbols as in Figure I . The positions and sizes of the ORFs are summarized. (C) Localization of CDCIO. Previously published localizations of CDClO are shown. I = Clarke and Carbon, 1980 2 = Kaback and Feldberg, 1985; 3 = Yeh CI a/., 1986.
428 regions investigated thus far (Coleman et al., 1986; Steensma et al., 1987; Yoshikawa and Isono, 1990), it is tempting to speculate that these untranscribed regions are required for some household function of the chromosome. They are obviously not necessary for centromere function per se, since transcripts are found close to several other centromeres (Mann and Davis, 1986; Toh-E and Shimauchi, 1986). The physical distance between CDEIII of CEN3 and the 3’ end of CDClO is approximately 2.9 kb (FitzgeraldHayes et al., 1982), whereas the genetic distance is 0.3 cM (Mortimer and Schild, 1980). The recombination frequency in this region is thus 0.1 cM/kb. This is far below the 04-0-6 cM/kb found for other parts of the yeast genome (Mortimer and Schild, 1985; Strathern et al., 1979; Olson et al., 1986; Kaback et al., 1989). Hence recombination around CEN3, at least on the right side, is decreased. This has also been reported for some other centromere regions (Lambie and Roeder, 1986; Kaback et al., 1989). ACKNOWLEDGEMENT We would like to thank Dr. J. Sgouros for the sequence analyses, Drs M. Aigle and J. Pringle for communicating data prior to publication and our colleagues at the RU Leiden for helpful discussions. We appreciate the technical assistance of B. van der Reyden in part of this work. This study was supported by the EEC in the framework of the BAP project Sequencing of yeast chromosome 111. REFERENCES Clarke, L. and Carbon, J. (1980) Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287,504-509. Coleman, K.G., Steensma, H.Y., Kaback, D.B. and Pringle, J.R. (1986). Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation and characterization of the CDC24 gene and adjacent regions of the chromosome. Mol. Cell. Biol. 6,45 16-4525. Dente, L., Cesareni, G. and Cortese, R. (1983). pEMBL: A new family of single stranded plasmids. Nucleic Acids Res. 11, 1645-1655. Devereux, J., Haeberli, P. and Smithies, 0. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12,387-395. Fitzgerald-Hayes, M., Clarke, L. and Carbon, J. (1982). Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29,235-244. Grivell, L.A. and Planta, R.J. (1990). Yeast: the model eukaryote? TIBS 8,241-243. Kaback, D.B. and Feldberg, L.R. (1 985). Saccharomyces
H. YDE STEENSMA AND
Q J.M. VAN DER AART
cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation. Mol. Cell. Biol. 5, 751-761. Kaback, D.B., Steensma, H.Y. and de Jonge, P. (1989). Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae. Proc. NatLAcad. Sci. USA 86, 3694-3698. Lambie, E.J. and Roeder, S. (1986). Repression of meiotic crossing over by a centromere (CEN3)in Saccharomyces cerevisiae. Genetics 114,769-789. Langford, C.J., Klinz, F.J., Donath, C. and Gallwitz, D. (1984). Point mutations identify the conserved intron contained TACTAAC box as an essential splicing signal sequence in yeast. Cell 36,645-653. Lazowska, J., Jacq, C. and Slonimski, P.P. 1981 Splice points of the third intron in the yeast mitochondria1 cytochrome b gene. Cell 27, 12-14. Magdolen, V., Oechsner, U., Mueller, G. and Bandlow, W. (1988). The intron-containing gene for yeast prolifin (PFY) encodes a vital function. Mol. Cell. Biol. 8, 5 108-5 115. Mann, C. and Davis, R.W. (1986). Structure and sequence of the centrometric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol. Cell. Biol. 6,241-245. Mortimer, R.K. and Schild, D. (1980). Genetic map of Saccharomyces cerevisiae. Microbiol. Rev. 4 4 , 5 19-571. Mortimer, R.K. and Schild, D. (1985). Genetic map of Saccharomyces cerevisiae, Edition 9. Microbiol. Rev. 49, 181-212. Mortimer, R.K., Schild, D., Contopoulos, C.R. and Kans, J.A. (1989). Genetic map of Saccharomyces cerevisiae, Edition 10. Yeast 5, 321-403. Netter, P., Jacq, C., Carignani, G. and Slonimski, P. (1982). Critical sequences within mitochondria introns: Cis dominant mutations of the “cytochrome-b-like” intron of the oxidase gene. Cell 28,733-738. 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 organization of yeast chromosome 111. UCLA Symp. Mol. Cell. Biol. New Series 33, 31 1-223. 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 83,7826-7830. Salle, H. de la, Jacq, C. and Slonimski, P. (1982). Critical sequences within mitochondria1 introns: Pleiotropie mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase. Cell 28, 721-732. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. 3rd edition. CSH Laboratories, Cold Spring Harbor, New York. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Steensma, H.Y., Crowley, J.C. and Kaback, D.B. (1987).
SEQUENCE OF THE CDClO REGION AT CHROMOSOME 111 OF SACCHAROMYCES CEREVISIAE
Molecular cloning of chromosome I of Saccharomyces cerevisiae: Isolation and analysis of the CENl-ADEI CDC1.5 region. Mol. Cell. Biol. 7,410-419. Strathem, J.N., Newlon, C.S., Herskowitz, I. and Hicks, J.B. (1979). Isolation of a circular derivative of yeast chromosome 111: Implications for the mechanism of mating type interconversion. Cell 18,309-3 19. Toh-E, A. and Shimauchi, T. (1986). Cloning and sequencing of the P H 0 8 0 gene and CENl.5 of Saccharomyces cerevisiae. Yeast 2, 129-139.
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