Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis

Hines, Pamela J.; Benbow, Robert M.

doi:10.1016/0092-8674(82)90243-4

Cited by 50 publications

(21 citation statements)

References 44 publications

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“…Analysis by CsCl equilibrium centrifugation has established that most, if not all, of the incorporation is due to semiconservative replication and not repair synthesis (27,44). As reported by others (27,28,44), replicated [32P]DNA was composed of several bands separated by agarose gel electrophoresis, with the supercoiled monomer DNA as the most prominent (Fig. 2, lane 1).…”

Section: Resultsmentioning

confidence: 70%

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Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Zuber¹,

Tan²,

Ryoji³

1989

Mol. Cell. Biol.

107

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Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase 8 but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase 8 is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase a. Anti-DNA polymerase a alone inhibited plasmid replication by 63%. Thus, DNA polymerase a is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase a antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase 8. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase a, that the structure of DNA polymerase a holoenzyme for chromosome replication is significantly different from that for plasmid replication.Proliferating cell nuclear antigen (PCNA) was initially recognized as a nuclear autoantigen which reacts with autoimmune sera from a certain population of patients with systemic lupus erythematosus (47; for recent reviews, see references 60 and 61). This antigen is an acidic nuclear protein with a molecular weight of about 36,000 (13,42,51,58). It is readily extractable from whole-cell or nuclear preparations and is expressed mainly in mitotically active cells of various tissues and cell lines (58). Independently, Celis and his colleagues identified, by two-dimensional gel electrophoresis, a protein called cyclin which is abundant in proliferating cells (for reviews, see references 13 and 16). It was later shown that cyclin and PCNA are identical (42). The complete amino acid sequence of this protein was determined by cDNA cloning in two independent laboratories (1,43). Immunofluorescence studies with human autoantibodies against PCNA have demonstrated that PCNA expression increases during late Gl and early S phase, immediately preceding the onset of DNA synthesis (14,36,48,55,58). During S phase, the distribution of PCNA changes dramatically within the nucleus (5,7,14,36,41,55,58,61), following closely the sites of chromosomes which are pulselabeled with [3H]thymidine or bromodeoxyuridine (8, 41).These observations strongly suggest an association of PCNA with the DNA replication apparatus. It has also been proposed that PCNA might be involved in DNA repair synthesis, because UV damage of DNA caused an increase in the nuclear PCNA staining of non-S-phas...

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Section: Resultsmentioning

confidence: 70%

“…Frog eggs have the ability to replicate injected foreign plasmid DNA (24). Replication does not require specific DNA sequences, and virtually any circular DNA can be replicated to various extents (27,28,44). Incorporation of a radioactive precursor into DNA takes place linearly for the first several hours after DNA injection (44).…”

Section: Resultsmentioning

confidence: 99%

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Zuber¹,

Tan²,

Ryoji³

1989

Mol. Cell. Biol.

107

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“…Furthermore, Harland and Laskey (30) showed that Xenopus laevis oocytes initiate semi-conservative replication on virtually any injected template and possibly at random positions (although see 31). Interestingly, however, replication is limited to one round per cell cycle, indicating that fixed initiation sites are not a prerequisite for all levels of control.…”

Section: Discussionmentioning

confidence: 99%

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Dijkwel¹,

Vaughn

Hamlin

1994

Nucl Acids Res

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In previous studies, we utilized a neutral/neutral twodimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lying between the DHFR and 2BE2121 genes, and subsequently to move outward through the two genes. In the present study, we have analyzed this locus in detail by a complementary, neutral/alkaline 2-D gel technique that determines the direction in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both directions through the intergenic region, but only outward through the DHFR gene. Surprisingly, however, replication forks also move in both directions through the 2BE2121 gene. Furthermore, in early S phase, small numbers of replication bubbles can be detected in the 2BE2121 gene on neutral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in the intergenic region, but also at a lower frequency in the 2BE2121 gene. We further show that only a small fraction of DHFR amplicons sustains an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in light of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic region.

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“…All of the DNA molecules examined, including simian virus 40 (SV40) and polyomavirus (PyV) DNA which normally replicate only in differentiated cells of a specific mammalian host, replicated to the same extent in amphibian eggs. Some indication of sequence-specific replication has been reported (13,39), but the interpretation of these results has been questioned (58).…”

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confidence: 99%

Sequence-dependent DNA replication in preimplantation mouse embryos.

Wirak¹,

Chalifour²,

Wassarman³

et al. 1985

Mol. Cell. Biol.

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Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one-or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development.Although all of the injected DNAs were stable, replication of plasmid pML-l DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-l, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one-or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the a-,8-core or ,-core configuration of the PyV origin of replication. Although the a-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.Although ori sequences have not yet been demonstrated as functional elements of eucaryotic chromosomes, they are implicated strongly by the temporal order of gene replication (11, 36); the selective amplification of specific genes (53,65,74); the existence of autonomously replicating sequences (26,76); and the fact that all bacterial, viral, plasmid, and organelle genomes require one or more unique cis-acting sequences that act as origins of replication (ori) and interact with specific gene products. However, some observations suggest that DNA replication during the early stages of embryonic development may not require sequence-specific origins of replication to carry out DNA replication.Analysis of DNA replication in Drosophila melanogaster (6,56,85) has revealed that the average distance between chromosome replication bubbles in preblastomere embryos is at least five times smaller than that in differentiated cells, suggesting that embryos initiate replication at many more sequences than do differentiated cells of the same species. Furthermore, injection of DNA into Xenopus eggs revealed that semiconservative DNA replication can occur under the apparent control of the cell division cycle but with no apparent requirement for specific DNA sequences (34,57,58). All of the DNA molecules examined, including simian virus 40 (SV40) and polyomavirus (PyV) DNA which normally replicate only in differentiated cells of a specific m...

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Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis

Cited by 50 publications

References 44 publications

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Sequence-dependent DNA replication in preimplantation mouse embryos.

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