Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

Abstract

We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BgllI-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Ak-cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences.The replication of eucaryotic DNA occurs by initiation at many chromosomal sites which are usually clustered in a specific region characteristic of the cell type and the stage of the cell cycle (18). Recently, several investigators have tried to isolate replication origins by using in vitro recombination techniques (6). Another approach to the isolation of replication origin is to search autonomous replicating sequences (ARSs). Many ARSs of Saccharomyces cerevisiae, as well as those of other eucaryotic organisms, have been described (7, 9, 13-15, 17, 20, 22-24). These sequences from higher organisms may act as initiators of replication in S. cerevisiae cells. However, there is no evidence that these cloned eucaryotic DNA segments can also act as replicators in the cells of their original higher organisms. We have already reported that the cloned mouse DNA fragment pMU65 can replicate in a simian virus 40 (SV40) T antigen-dependent system in vivo and in vitro (5). The 2.5-kilobase (kb) mouse DNA fragment of pMU65 hybridized to half of a 27-nucleotide perfect palindrome present in the origin of SV40 DNA replication, and the initiation of DNA replication started from this fragment in an SV40 DNA replication system in vitro (5).We constructed the chimeric plasmid (p65-tk) containing 2.5 kb of mouse DNA, the thymidine kinase (tk) gene of herpes simplex virus type 1, and plasmid pKB111. The p65-tk plasmid persisted episomally in transfected mouse FM3A cells and had a good template activity in an in vitro DNA replication system that we have developed from FM3A cells. MATERIALS AND METHODSPlasmid and recombinant DNA constructions. Plasmid pMU65 harboring the mouse genomic DNA was reported previously (5) and is shown in Fig. 1. The 2.5-kb EcoRI-BglIll fragment of pMU65 was inserted into the EcoRI-BglII region of pKSV10, and the tk gene of herpes simplex virus type 1 (10) was further inserted into the BamHI site of the above plasmid. This was named p65-tk (Fig. 1) (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.5), 5 mM KCl, 0.5 mM MgCl2, 0....

Temporal order of gene replication in Chinese hamster ovary cells.

“…We selected a wide variety of DNA sequences for analysis: single-copy genes (e.g., CAD and HPRT); multifamily genes (e.g., (x-globin); oncogenes, because of their close relationship to cell growth (12) and involvement in DNA synthesis (30,43,62); and repetitive sequences, some of which may act as mammalian replicons (3,46). This is the first time that the replication order of various genes or sequences spanning the whole S phase of a single cell line has been quantitatively documented in terms of C values.…”

Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines.

“…In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.The replication of eucaryotic chromosomes is controlled through the selection of sites for the initiation of DNA synthesis (2,6,7,10,12,14,19,21,24,32,39) and through the temporal programming of the activity at these sites (10,12,19,23). Combined with evidence for the existence of proteins which recognize the replication origins of viral minichromosomes (11,17,28,44), these results suggest that chromosome replication can be regulated in trans by DNA sequence-specific protein binding.…”

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

“…Within the limits of resolution of these studies, forks appear to originate from preferred zones or sites. There have also been a few recent reports of small cloned fragments from mammalian genomes that are capable of sustaining limited autonomous replication when introduced by transfection into a suitable mammalian host cell line, suggesting the presence of fixed origins of replication (3,19,33,61). However, none of the latter sequences has yet been shown to actually serve as an initiation site while resident at its natural location in the chromosome (but see reference 14).…”