EXPERIMENTAL
CELL
RESEARCH
203,435-442
(1992)
ORSI 2, a Mammalian Autonomously Replicating DNA Sequence, Is Present at the Centromere of CV-1 Cell Chromosomes D. C. W. MAH, McGill
A. SHIHAB-EL-DEEN,
Cancer Centre, Department
of Medicine,
McGill
G. B. PRICE, AND M. ZANNIS-HADJOPOULOS’ Uniuersity,
3655 Drummond
Street, Mont&al,
Qubbec, Canada H3G 1 Y6
within a given species represents a family of closely related sequences which are not identical [l, 4, 7, 81. The a-satellite DNA family of human chromosomes (alphoid DNA) interacts with the centromere-specific protein B (CENP-B) [g-11], which recognizes and binds a 17-bp motif (CENP-B box) present in a subset of alphoid DNA [ll, 121 and mouse minor satellite DNA [13]. a-satellite DNA may also have an important role in phasing the formation of nucleosomes and higher chromatin structure [14, 151, and organization of chromosomal DNA on the nuclear scaffold [3, 14, 16-181. Several studies on cells from a variety of species generally indicate that the majority of a-satellite DNA is replicated in the latter half of S phase during the cell cycle, although there are species-specific variations in the exact position of peak a-satellite DNA replication in S phase [19-231. Replication of a-satellite is not limited exclusively to late S phase. We have previously reported a-satellite sequences in a library of origin-enriched sequences (or-s) of early-replicating monkey DNA [24-261; satellite DNA has also been detected in DNA replicating in early and mid-S phase [20, 27, 281. orsl.2 is an 812-bp-long DNA sequence (GenBank Accession No. M26225; Ref. [25]), which contains an cr-satellite region extending 168 bp from the 5’ end of the sequence and a non-a-satellite region, which extends from nucleotide position 169 to the 3’ terminus (Fig. 1). The a-satellite portion of ors12 is homologous [25] to the 172-bp repeat of a-satellite-containing HirzdIII restriction fragments of CVl DNA [4]. There are no open reading frames on either the + strand or the - strand of ors12. Computer-assisted sequence analysis of the nona-satellite region revealed three inverted repeats (IRS), an AT-rich DNA sequence domain at the 3’ end of the clone, loose homology (lO/ll-bp matches) with the yeast ARS (autonomous replicating sequences) consensus, a SAR-T (scaffold attachment region) consensus of drosophila, and a CACCC consensus sequence characteristic of transcriptional elements upstream of /3-globin genes (Fig. 1 and Ref [25]). Deletion mutagenesis studies in our laboratory indicate that the IR proximal to the CACCC consensus sequence as well as the AT rich region at the 3’ end of the orsl2, containing se-
orsl2, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. ZannisHadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. N&l. and in a cell-free Acad. Sci. USA 84,6668-6672,1987) system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of orsl2 (nucleotides 1-168) consists of the highly reiterated a-satellite sequence (B. S. Rao et al. Gene 87,233-242, 1990). We have estimated the copy number of the non-a-satellite portion of orsl2 in CV- 1 cells to be
CELL
RESEARCH
203,435-442
(1992)
ORSI 2, a Mammalian Autonomously Replicating DNA Sequence, Is Present at the Centromere of CV-1 Cell Chromosomes D. C. W. MAH, McGill
A. SHIHAB-EL-DEEN,
Cancer Centre, Department
of Medicine,
McGill
G. B. PRICE, AND M. ZANNIS-HADJOPOULOS’ Uniuersity,
3655 Drummond
Street, Mont&al,
Qubbec, Canada H3G 1 Y6
within a given species represents a family of closely related sequences which are not identical [l, 4, 7, 81. The a-satellite DNA family of human chromosomes (alphoid DNA) interacts with the centromere-specific protein B (CENP-B) [g-11], which recognizes and binds a 17-bp motif (CENP-B box) present in a subset of alphoid DNA [ll, 121 and mouse minor satellite DNA [13]. a-satellite DNA may also have an important role in phasing the formation of nucleosomes and higher chromatin structure [14, 151, and organization of chromosomal DNA on the nuclear scaffold [3, 14, 16-181. Several studies on cells from a variety of species generally indicate that the majority of a-satellite DNA is replicated in the latter half of S phase during the cell cycle, although there are species-specific variations in the exact position of peak a-satellite DNA replication in S phase [19-231. Replication of a-satellite is not limited exclusively to late S phase. We have previously reported a-satellite sequences in a library of origin-enriched sequences (or-s) of early-replicating monkey DNA [24-261; satellite DNA has also been detected in DNA replicating in early and mid-S phase [20, 27, 281. orsl.2 is an 812-bp-long DNA sequence (GenBank Accession No. M26225; Ref. [25]), which contains an cr-satellite region extending 168 bp from the 5’ end of the sequence and a non-a-satellite region, which extends from nucleotide position 169 to the 3’ terminus (Fig. 1). The a-satellite portion of ors12 is homologous [25] to the 172-bp repeat of a-satellite-containing HirzdIII restriction fragments of CVl DNA [4]. There are no open reading frames on either the + strand or the - strand of ors12. Computer-assisted sequence analysis of the nona-satellite region revealed three inverted repeats (IRS), an AT-rich DNA sequence domain at the 3’ end of the clone, loose homology (lO/ll-bp matches) with the yeast ARS (autonomous replicating sequences) consensus, a SAR-T (scaffold attachment region) consensus of drosophila, and a CACCC consensus sequence characteristic of transcriptional elements upstream of /3-globin genes (Fig. 1 and Ref [25]). Deletion mutagenesis studies in our laboratory indicate that the IR proximal to the CACCC consensus sequence as well as the AT rich region at the 3’ end of the orsl2, containing se-
orsl2, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. ZannisHadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. N&l. and in a cell-free Acad. Sci. USA 84,6668-6672,1987) system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of orsl2 (nucleotides 1-168) consists of the highly reiterated a-satellite sequence (B. S. Rao et al. Gene 87,233-242, 1990). We have estimated the copy number of the non-a-satellite portion of orsl2 in CV- 1 cells to be
