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Isolation of Human and Fission Yeast Homologues of the Budding Yeast Origin Recognition Complex Subunit ORC5: Human Homologue (ORC5L) Maps to 7q22
SHORT COMMUNICATION Isolation of Human and Fission Yeast Homologues of the Budding Yeast Origin Recognition Complex Subunit ORC5: Human Homologue (ORC5L) Maps to 7q22 Masamichi Ishiai,*,1 Frank B. Dean,† Katsuzumi Okumura,‡ Makoto Abe,§ Kyeong-Yeop Moon,* Anthony A. Amin,*,2 Kazuhiro Kagotani,‡ Hiroshi Taguchi,‡ Yasufumi Murakami,§ Fumio Hanaoka,§ Mike O’Donnell,† Jerard Hurwitz,* and Toshihiko Eki§,3 *Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 97, New York, New York 10021; †Laboratory of DNA Replication, Rockefeller University, 1230 York Avenue, New York, New York 10021; ‡Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu, Mie 514, Japan; and §Cellular Physiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan Received March 25, 1997; accepted August 19, 1997
Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S. pombe, and S. cerevisiae Orc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified the S. pombe ORC5 gene and located it in a cosmid mapped on chromosome II. q 1997 Academic Press
DNA replication in eukaryotic cells is tightly controlled and coordinated with other events during the cell cycle and cell proliferation. Studies carried out with the budding yeast Saccharomyces cerevisiae led to the identification of an origin recognition complex (ORC; 3). The ORC consists of six polypeptides (Orc1p to Orc6p) and plays an essential role in the initiation of chromosome DNA replication and transcriptional siSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. U92538 and U92539. 1 Present address: Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570, Japan. 2 Present address: Bio-Mega, Boehringer Ingelheim Research Inc., 2100 Cunard Street, Lavel, Quebec H7S 2G5, Canada. 3 To whom correspondence should be addressed. Telephone: 81298-36-9059. Fax: 81-298-36-9137. E-mail: [email protected].
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lencing in the budding yeast (1, 3, 16). This six-protein complex binds in an ATP-dependent manner to several well-defined autonomously replicating sequences that serve as the chromosomal replication origins (3). The genes for all six ORC subunits have been isolated, and deletion of any one of these genes results in lethality (1, 2, 8, 13, 14, 16). The regulation of initiation of metazoan chromosomal DNA replication is poorly understood because chromosomal origins have not been localized to specific DNA sequences. Recently, cDNA clones encoding proteins homologous to the S. cerevisiae ORC have been isolated from Schizosaccharomyces pombe, Xenopus laevis, and human for both Orc1p and Orc2p, from Kluyveromyces lactis for Orc1p, and from Caenorhabditis elegans, Arabidopsis thaliana, and mouse for Orc2p (4, 9, 12, 19, 21, 22). The ORC2 and ORC5 homologues have also been cloned from Drosophila melanogaster (10). A multisubunit protein complex made up of subunits homologous to the S. cerevisiae ORC genes has been purified from Drosophila and Xenopus (10, 21). These results indicate that the architecture of the ORC complex is conserved among eukaryotes and suggest that there may be a common mechanism for the initiation of DNA replication in eukaryotic cells. A homology search in the TIGR database was carried out to isolate other human ORC subunits, and a human cDNA fragment (Accession No. THC83665) with significant homology to Drosophila Orc5p was identified. Two oligonucleotides were used to amplify the homologous region by the polymerase chain reaction (PCR). The primer sequences used were Horc5A, 5*-CTG GAA TTC TGC CCC ACT TGG AAA ACG TGG TGC TTT GTC GCG AGT-3* and Horc5B, 5*-GAT GGA TCC ATT TGT TTA AAA TTT GTT CCA AAA GCA GCC TCA ATG-3*. A PCR product of 249 bp was cloned and used to probe a Lambda ZAPII HeLa cDNA library (Stratagene). From 6 1 105 plaques screened, five positive
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clones were identified. These clones were excised from the Lambda ZAPII vector according to the manufacturer’s protocol and obtained as pBluescript SK(0) phagemids. Restriction enzyme analysis and sequencing demonstrated that all five clones are related to each other but differ in length. The nucleotide sequence of the longest insert, a 1901-bp clone designated pSKHorc5 #2, has been deposited into GenBank (Accession No. U92538). It contains a 5*-untranslated region of 88 bp, a 1308-bp open reading frame encoding a 435amino-acid protein with a predicted molecular mass of 50.2 kDa, and 505 bp at the 3*-untranslated region, including a polyadenylation signal. The S. pombe homologue of ORC5 was identified in cosmid 855 (Accession No. U23729), which was reported to be located on chromosome II (18), by searching the S. pombe genomic DNA sequencing project database at the Sanger Centre, Cambridge, UK (http:// www.sanger.ac.uk/Çyeastpub/svw/pombe.html). This sequence showed significant homology to S. cerevisiae, Drosophila, and human Orc5p. Since we found that this gene had two possible consensus sequences for S. pombe introns from this genomic DNA (15; data not shown), we confirmed this authenticity by direct isola-
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tion of the cDNA sequence. A 1.4-kb PCR product was obtained using two oligonucleotides: F2, 5*-GTC TTT TGC AGG GAA GAT CA-3* and R2, 5*-CCG CCA AAT AAC TAT CGA GA-3*. This fragment was cloned and used as a probe to isolate cDNA clones. Using a Lambda ZAPII S. pombe cDNA library (kindly provided by Dr. D. Young, Calgary University), 6 1 105 plaques were screened, and 32 positive clones were identified in the primary screen. Ten clones were chosen for further characterization. The nucleotide sequence of the longest insert (1604 bp) contained one open reading frame encoding a 455-amino-acid protein with an estimated molecular mass of 51.6 kDa (deposited into the GenBank database, Accession No. U92539). The amino acid sequence of the human Orc5p showed significant identity and percentage similarity to the corresponding genes from Drosophila (35.1, 63.4%), S. pombe (22.3, 47.6%), and S. cerevisiae (19.4, 44.4%). In addition, the human Orc5p showed 19.0% identity and 40.6% similarity to the C. elegans protein, zc168.3, predicted from the Nematoda sequencing project at the Sanger Centre and the Department of Genetics, Washington University (St. Louis, MO). An alignment of the five predicted amino acid sequences is shown in Fig. 1.
FIG. 1. Multiple alignment of the Orc5p homologues. Hs, human (this work); Dm, Drosophila melanogaster (10); Ce, Caenorhabditis elegans ZC168.3 (GenBank Accesstion No. Z70312); Sp, Schizosaccharomyces pombe (this work); and Sc, Saccharomyces cerevisiae (14) were aligned by the clustal method using the Megalign program (DNASTAR Inc,Wisconsin). Identical amino acids are indicated by the shaded regions.
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FIG. 2. Physical mapping of the human ORC5L locus to chromosome 7. FISH analysis was carried out using PAC HOR5L-163O18 DNA as a probe. The corresponding chromosome images stained with propidium iodide are shown. The arrowheads indicate the positions of the fluorescence signals. Twenty-five double dots and 10 single dots on band 7q22 and 5 dots near band 7q22 were detected on the 40 copies of chromosome 7 observed with 20 spreads.
A region that conforms to the P-loop ATP/GTP binding consensus sequence (11) was found in the N-terminal region of human Orc5p (residues 37 to 44), as well as in the other four sequences (10, 14). The human ORC5 gene has been designated ORC5L in accordance with published guidelines for nomenclature. To assign ORC5L to a particular chromosome, we first mapped the gene by Southern analysis using a human/rodent somatic cell hybrid mapping panel (Coriell Cell Repositories, NJ; 6). A 120-bp EcoRI–NruI fragment from pSK- Horc5 #2, corresponding to the 5*untranslated region plus 11 N-terminal amino acids of the human ORC5L cDNA, was used as a probe. A 7.4kb band was specifically detected in lanes containing human genomic DNA and in the lane containing DNA derived from human chromosome 7, but not in lanes with human DNA derived from other chromosomes (data reviewed but not shown). Similar results were obtained when the entire 1.9-kb cDNA fragment was used as a probe (data not shown). PCR analysis of the human/rodent somatic cell hybrid panel was carried out using primers OR5F2 (5*-AAG ACT ATT TAC CAG GGA CCC TGG-3*) and OR5R2 (5*-GGG CTG CTG GCA CGT TCA AAG CGC-3*). These primers amplifed a 188-bp fragment of the 3*-untranslated region of human ORC5L cDNA (nucleotides 1576 to 1763; 657C annealing temperature, 35 cycles). The 188-bp amplified product was detected only in reactions containing human genomic DNA and human chromosome 7 DNA
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(data reviewed but not shown). These results indicate that ORC5L is located on chromosome 7. The chromosomal localization of ORC5L was refined by fluorescence in situ hybridization (FISH) analysis using biotin-labeled human PAC DNAs (clones HOR5L-163O18 and HOR5L-147J1) as probes. The PAC clones were isolated by PCR screening using the above primer set (OR5F2 and OR5R2) from a human genome library (Genome Systems Inc., St. Louis, MO), and the clone name indicates the plate number (e.g., 163), row (e.g., O), and column (e.g., 18) where the PAC clone is located. Chromosome spreads were prepared from lymphocytes of a normal human male by the method of Takahashi et al. (23). PAC DNAs were biotinylated by nick-translation with biotin-16–dUTP and hybridized to R-banded chromosome spreads. The hybridized probe was detected by means of fluorescence isothiocyanate-conjugated avidin (Boehringer Mannheim). Chromosomes were counterstained with 0.2 mg/ ml propidium iodide for R-banding (20). Fluorescence signals were viewed under a Zeiss Axioskop epifluorescence microscope fitted with a CCD camera (Photometrics PXL 1400). Digital images that passed through each fluorescence filter were saved using the software IPLab (Signal Analytics Co.) and were pseudocolored and merged using Adobe Photoshop 2.5J (Adobe Systems Inc.). FITC and propidium iodide fluorescence were stained in green and red, respectively. The merged images were directly printed by Fuji Pictrogra-
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phy. Figure 2 shows the results of a FISH experiment using PAC HOR5L-163O18 DNA as a probe. Twenty metaphase spreads were observed, and the fluorescence signals and the banding patterns of the chromosomes were compared. Double or single fluorescence dots were detected on band 7q22 in 35 of 40 chromosome 7 samples (87.5%) analyzed (Fig. 2). In the case of FISH analyses using HOR5L-147J1 DNA or human ORC5L cDNA as probe, the fluorescence signals were consistently observed on band 7q22 (data not shown). Consistent hybridization signals were not detected on any other chromosomes. YAC clones from the CEPH YAC library were screened using PCR, and only one clone (964H9) was identified as containing ORC5L. This clone contains genetic markers D7S658 and D7S2504, which map within the MIT contig WC7.6 localized on chromosome 7 (YAC data were obtained via the WWW at http:// www-genome.wi.mit.edu/cgi-bin/contig/phys_map). A current YAC contig map indicates that this clone maps to chromosome band 7q22.1 (5), confirming our mapping result by FISH analysis. We have cloned human and fission yeast cDNAs homologous to the budding yeast ORC5 and have mapped the human ORC5 gene (ORC5L) to chromosome 7 band q22. Previously we assigned the genes of two other human ORC subunits, ORC1L and ORC2L, to chromosome bands 1p32 and 2q33, respectively (7). Because the human ORC is likely to be involved in regulating the initiation of DNA replication and gene transcription, mutations in the ORC5 genes might result in deregulation of these processes. Such aberrations could cause various genetic disorders, including cancers. A chromosomal aberration, del(7)(q21– 22q31–32), associates with uterine leiomyoma and maps near the ORC5L locus (17). The relationship between the tumor phenotype and possible alterations in human ORC5L function or expression, however, remains to be determined.
We thank Dr. D. Young, Calgary University, for kindly providing the S. pombe cDNA library, Dr. Z. Kelman for helpful discussions, and H. Wain (University College of London) for ORC5L nomenclature. The cooled CCD camera digital imaging system at the Center for Molecular Biology and Genetics, Mie University, was used in this work. Support was provided by National Institutes of Health Grants GM34559 to J.H. and GM54705 to M.O. and by grants to the Life Science Research Project of RIKEN, the Science and Technology Agency of Japan, in part by a grant from the Ministry of Education, Science, Sports, and Culture of Japan. J.H. is a Professor of the American Cancer Society.
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13. Li, J. J., and Herskowitz, I. (1993). Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262: 1870–1874. 14. Loo, S., Fox, C. A., Rine, J., Kobayashi, R., Stillmam, B., and Bell, S. (1995). The origin recognition complex in silencing, cell cycle progression, and DNA replication. Mol. Biol. Cell 6: 741– 756. 15. Mertins, P., and Gallwitz, D. (1987). Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element. EMBO J. 6: 1757–1763. 16. Micklem, G., Rowley, A., Harwood, J., Nasmyth, K., and Diffley, J. F. X. (1993). Yeast origin recognition complex is involved in DNA replication and transcriptional silencing. Nature 366: 87– 89.
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2. Bell, S. P., Mitchell, J., Leber, J., Kobayashi, R., and Stillman, B. (1995). The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing. Cell 83: 563–568.
12. Leatherwood, J., Lopez-Girona, A., and Russell, P. (1996). Interaction of Cdc2 and Cdc18 with a fission yeast ORC2-like protein. Nature 379: 360–363.
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17. Mitelman, F., Kaneko, Y., and Berger, R. (1994). Report of the committee on chromosome changes in neoplasia. In ‘‘Human Gene Mapping, 1993’’ (A. J. Cuticchia and P. L. Pearson, Eds.),
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pp. 773–812, The Johns Hopkins Univ. Press, Baltimore/ London. 18. Mizukami, T., Chang, W. I., Garkavtsev, I., Kaplan, N., Lombardi, D., Matsumoto, T., Niwa, O., Kounosu, A., Yanagida, M., Marr, T. G., and Beach, D. (1993). A 13 kb resolution cosmid map of the 14 Mb fisson yeast genome by nonrandom sequencetagged site mapping. Cell 73: 121–132. 19. Muzi-Falconi, M., and Kelly, T. J. (1995). Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex. Proc. Natl. Acad. Sci. USA 92: 12475–12479. 20. Okumura, K., Nogami, M., Taguchi, H., Dean, F. B., Chen, M., Pan, Z.-Q., Hurwitz, J., Shiratori, A., Murakami, Y., Ozawa, K., and Eki, T. (1995). Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2–
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q24.3, 3q27, 13q12.3–q13, and 7q11.23. Genomics 25: 274– 278. 21. Rowles, A., Chong, J. P. J., Brown, L., Howell, M., Evan, G. I., and Blow, J. J. (1996). Interaction between the origin recognition complex and the replication licencing system in Xenopus. Cell 87: 287–296. 22. Takahara, K., Bong, M., Brevard, R., Eddy, R. L., Haley, L. L., Sait, S. J., Shows, T. B., Hoffman, G. G., and Greenspan, D. S. (1996). Mouse and human homologues of the yeast origin of replication recognition complex subunit ORC2 and chromosomal localization of the cognate human gene ORC2L. Genomics 31: 119–122. 23. Takahashi, E., Hori, T., O’Connell, P., Leppert, M., and White, R. (1990). R-banding and nonisotopic in situ hybridization: Precise localization of the human type II collagen gene (COL2A1). Hum. Genet. 86: 14–16.
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Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S. pombe, and S. cerevisiae Orc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified the S. pombe ORC5 gene and located it in a cosmid mapped on chromosome II. q 1997 Academic Press
DNA replication in eukaryotic cells is tightly controlled and coordinated with other events during the cell cycle and cell proliferation. Studies carried out with the budding yeast Saccharomyces cerevisiae led to the identification of an origin recognition complex (ORC; 3). The ORC consists of six polypeptides (Orc1p to Orc6p) and plays an essential role in the initiation of chromosome DNA replication and transcriptional siSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. U92538 and U92539. 1 Present address: Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570, Japan. 2 Present address: Bio-Mega, Boehringer Ingelheim Research Inc., 2100 Cunard Street, Lavel, Quebec H7S 2G5, Canada. 3 To whom correspondence should be addressed. Telephone: 81298-36-9059. Fax: 81-298-36-9137. E-mail: [email protected].
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lencing in the budding yeast (1, 3, 16). This six-protein complex binds in an ATP-dependent manner to several well-defined autonomously replicating sequences that serve as the chromosomal replication origins (3). The genes for all six ORC subunits have been isolated, and deletion of any one of these genes results in lethality (1, 2, 8, 13, 14, 16). The regulation of initiation of metazoan chromosomal DNA replication is poorly understood because chromosomal origins have not been localized to specific DNA sequences. Recently, cDNA clones encoding proteins homologous to the S. cerevisiae ORC have been isolated from Schizosaccharomyces pombe, Xenopus laevis, and human for both Orc1p and Orc2p, from Kluyveromyces lactis for Orc1p, and from Caenorhabditis elegans, Arabidopsis thaliana, and mouse for Orc2p (4, 9, 12, 19, 21, 22). The ORC2 and ORC5 homologues have also been cloned from Drosophila melanogaster (10). A multisubunit protein complex made up of subunits homologous to the S. cerevisiae ORC genes has been purified from Drosophila and Xenopus (10, 21). These results indicate that the architecture of the ORC complex is conserved among eukaryotes and suggest that there may be a common mechanism for the initiation of DNA replication in eukaryotic cells. A homology search in the TIGR database was carried out to isolate other human ORC subunits, and a human cDNA fragment (Accession No. THC83665) with significant homology to Drosophila Orc5p was identified. Two oligonucleotides were used to amplify the homologous region by the polymerase chain reaction (PCR). The primer sequences used were Horc5A, 5*-CTG GAA TTC TGC CCC ACT TGG AAA ACG TGG TGC TTT GTC GCG AGT-3* and Horc5B, 5*-GAT GGA TCC ATT TGT TTA AAA TTT GTT CCA AAA GCA GCC TCA ATG-3*. A PCR product of 249 bp was cloned and used to probe a Lambda ZAPII HeLa cDNA library (Stratagene). From 6 1 105 plaques screened, five positive
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clones were identified. These clones were excised from the Lambda ZAPII vector according to the manufacturer’s protocol and obtained as pBluescript SK(0) phagemids. Restriction enzyme analysis and sequencing demonstrated that all five clones are related to each other but differ in length. The nucleotide sequence of the longest insert, a 1901-bp clone designated pSKHorc5 #2, has been deposited into GenBank (Accession No. U92538). It contains a 5*-untranslated region of 88 bp, a 1308-bp open reading frame encoding a 435amino-acid protein with a predicted molecular mass of 50.2 kDa, and 505 bp at the 3*-untranslated region, including a polyadenylation signal. The S. pombe homologue of ORC5 was identified in cosmid 855 (Accession No. U23729), which was reported to be located on chromosome II (18), by searching the S. pombe genomic DNA sequencing project database at the Sanger Centre, Cambridge, UK (http:// www.sanger.ac.uk/Çyeastpub/svw/pombe.html). This sequence showed significant homology to S. cerevisiae, Drosophila, and human Orc5p. Since we found that this gene had two possible consensus sequences for S. pombe introns from this genomic DNA (15; data not shown), we confirmed this authenticity by direct isola-
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tion of the cDNA sequence. A 1.4-kb PCR product was obtained using two oligonucleotides: F2, 5*-GTC TTT TGC AGG GAA GAT CA-3* and R2, 5*-CCG CCA AAT AAC TAT CGA GA-3*. This fragment was cloned and used as a probe to isolate cDNA clones. Using a Lambda ZAPII S. pombe cDNA library (kindly provided by Dr. D. Young, Calgary University), 6 1 105 plaques were screened, and 32 positive clones were identified in the primary screen. Ten clones were chosen for further characterization. The nucleotide sequence of the longest insert (1604 bp) contained one open reading frame encoding a 455-amino-acid protein with an estimated molecular mass of 51.6 kDa (deposited into the GenBank database, Accession No. U92539). The amino acid sequence of the human Orc5p showed significant identity and percentage similarity to the corresponding genes from Drosophila (35.1, 63.4%), S. pombe (22.3, 47.6%), and S. cerevisiae (19.4, 44.4%). In addition, the human Orc5p showed 19.0% identity and 40.6% similarity to the C. elegans protein, zc168.3, predicted from the Nematoda sequencing project at the Sanger Centre and the Department of Genetics, Washington University (St. Louis, MO). An alignment of the five predicted amino acid sequences is shown in Fig. 1.
FIG. 1. Multiple alignment of the Orc5p homologues. Hs, human (this work); Dm, Drosophila melanogaster (10); Ce, Caenorhabditis elegans ZC168.3 (GenBank Accesstion No. Z70312); Sp, Schizosaccharomyces pombe (this work); and Sc, Saccharomyces cerevisiae (14) were aligned by the clustal method using the Megalign program (DNASTAR Inc,Wisconsin). Identical amino acids are indicated by the shaded regions.
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FIG. 2. Physical mapping of the human ORC5L locus to chromosome 7. FISH analysis was carried out using PAC HOR5L-163O18 DNA as a probe. The corresponding chromosome images stained with propidium iodide are shown. The arrowheads indicate the positions of the fluorescence signals. Twenty-five double dots and 10 single dots on band 7q22 and 5 dots near band 7q22 were detected on the 40 copies of chromosome 7 observed with 20 spreads.
A region that conforms to the P-loop ATP/GTP binding consensus sequence (11) was found in the N-terminal region of human Orc5p (residues 37 to 44), as well as in the other four sequences (10, 14). The human ORC5 gene has been designated ORC5L in accordance with published guidelines for nomenclature. To assign ORC5L to a particular chromosome, we first mapped the gene by Southern analysis using a human/rodent somatic cell hybrid mapping panel (Coriell Cell Repositories, NJ; 6). A 120-bp EcoRI–NruI fragment from pSK- Horc5 #2, corresponding to the 5*untranslated region plus 11 N-terminal amino acids of the human ORC5L cDNA, was used as a probe. A 7.4kb band was specifically detected in lanes containing human genomic DNA and in the lane containing DNA derived from human chromosome 7, but not in lanes with human DNA derived from other chromosomes (data reviewed but not shown). Similar results were obtained when the entire 1.9-kb cDNA fragment was used as a probe (data not shown). PCR analysis of the human/rodent somatic cell hybrid panel was carried out using primers OR5F2 (5*-AAG ACT ATT TAC CAG GGA CCC TGG-3*) and OR5R2 (5*-GGG CTG CTG GCA CGT TCA AAG CGC-3*). These primers amplifed a 188-bp fragment of the 3*-untranslated region of human ORC5L cDNA (nucleotides 1576 to 1763; 657C annealing temperature, 35 cycles). The 188-bp amplified product was detected only in reactions containing human genomic DNA and human chromosome 7 DNA
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(data reviewed but not shown). These results indicate that ORC5L is located on chromosome 7. The chromosomal localization of ORC5L was refined by fluorescence in situ hybridization (FISH) analysis using biotin-labeled human PAC DNAs (clones HOR5L-163O18 and HOR5L-147J1) as probes. The PAC clones were isolated by PCR screening using the above primer set (OR5F2 and OR5R2) from a human genome library (Genome Systems Inc., St. Louis, MO), and the clone name indicates the plate number (e.g., 163), row (e.g., O), and column (e.g., 18) where the PAC clone is located. Chromosome spreads were prepared from lymphocytes of a normal human male by the method of Takahashi et al. (23). PAC DNAs were biotinylated by nick-translation with biotin-16–dUTP and hybridized to R-banded chromosome spreads. The hybridized probe was detected by means of fluorescence isothiocyanate-conjugated avidin (Boehringer Mannheim). Chromosomes were counterstained with 0.2 mg/ ml propidium iodide for R-banding (20). Fluorescence signals were viewed under a Zeiss Axioskop epifluorescence microscope fitted with a CCD camera (Photometrics PXL 1400). Digital images that passed through each fluorescence filter were saved using the software IPLab (Signal Analytics Co.) and were pseudocolored and merged using Adobe Photoshop 2.5J (Adobe Systems Inc.). FITC and propidium iodide fluorescence were stained in green and red, respectively. The merged images were directly printed by Fuji Pictrogra-
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phy. Figure 2 shows the results of a FISH experiment using PAC HOR5L-163O18 DNA as a probe. Twenty metaphase spreads were observed, and the fluorescence signals and the banding patterns of the chromosomes were compared. Double or single fluorescence dots were detected on band 7q22 in 35 of 40 chromosome 7 samples (87.5%) analyzed (Fig. 2). In the case of FISH analyses using HOR5L-147J1 DNA or human ORC5L cDNA as probe, the fluorescence signals were consistently observed on band 7q22 (data not shown). Consistent hybridization signals were not detected on any other chromosomes. YAC clones from the CEPH YAC library were screened using PCR, and only one clone (964H9) was identified as containing ORC5L. This clone contains genetic markers D7S658 and D7S2504, which map within the MIT contig WC7.6 localized on chromosome 7 (YAC data were obtained via the WWW at http:// www-genome.wi.mit.edu/cgi-bin/contig/phys_map). A current YAC contig map indicates that this clone maps to chromosome band 7q22.1 (5), confirming our mapping result by FISH analysis. We have cloned human and fission yeast cDNAs homologous to the budding yeast ORC5 and have mapped the human ORC5 gene (ORC5L) to chromosome 7 band q22. Previously we assigned the genes of two other human ORC subunits, ORC1L and ORC2L, to chromosome bands 1p32 and 2q33, respectively (7). Because the human ORC is likely to be involved in regulating the initiation of DNA replication and gene transcription, mutations in the ORC5 genes might result in deregulation of these processes. Such aberrations could cause various genetic disorders, including cancers. A chromosomal aberration, del(7)(q21– 22q31–32), associates with uterine leiomyoma and maps near the ORC5L locus (17). The relationship between the tumor phenotype and possible alterations in human ORC5L function or expression, however, remains to be determined.
We thank Dr. D. Young, Calgary University, for kindly providing the S. pombe cDNA library, Dr. Z. Kelman for helpful discussions, and H. Wain (University College of London) for ORC5L nomenclature. The cooled CCD camera digital imaging system at the Center for Molecular Biology and Genetics, Mie University, was used in this work. Support was provided by National Institutes of Health Grants GM34559 to J.H. and GM54705 to M.O. and by grants to the Life Science Research Project of RIKEN, the Science and Technology Agency of Japan, in part by a grant from the Ministry of Education, Science, Sports, and Culture of Japan. J.H. is a Professor of the American Cancer Society.
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