Cloning of an origin of DNA replication of Xenopus laevis.

Abstract

ABSTRACr DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli Rlasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3HJ thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (5SO base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. The pNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling. To determine the number of replications of pSW14, a novel mpethod was employed. Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites-two bracketing (4) in which the S phase is 6-7 hr. In some organisms, cells of the early embryo complete the S phase in a few minutes, and many more functional origins have been detected in Drosophila, for example (5). The time interval between cleavages in sea urchin embryos and some amphibian embryos is so short that origins would have to be close together and operating simultaneously to allow completion of replication in the time observed. Callan (6), by the use of DNA fiber autoradiography, has estimated that the interval in Triturus vulgaris (a salamander) might be as small as 10Mim (30,000 bp). However, a study by DNA fiber autoradiography of Chinese hamster (CHO) cells in culture indicated that potentially functional origins might be as close as 4 jm (12,000 bp) in the DNA that replicates early in S phase (7).With these studies in mind we initiated studies on the cloning and characterization of the origins in Xenopus laevts (the African clawed frog), which has been widely used to study the expression (transcription and translation) of foreign genes in oocytes (8-12). It also has been shown that unfertilized X. laevt eggs have the ability to support DNA replication from injected exogenous DNA (13)(14)(15).A number of derivatives of pACYC189 containing segments Escherichia coil SF8 (rjm-lop-1l, rec B-, rec C-) and plasmid pACYC189 were provided by S. N. Cohen through the Plasmid Reference Center (Stanford University). Plasmid pBR322 and E. colh RY13 were obtained from H.

Transformation of frog embryos with a rabbit beta-globin gene.

“…There have been several reports on the replication of foreign DNA injected into unfertilized eggs of X. laevis (7)(8)(9)(10)(11)(12). Recently, Harland and Laskey (12) to unlabeled RNA from injected embryos, treated with S1 nuclease, denatured, fractionated by gel electrophoresis, and autoradiographed.…”

“…More recently, microneedle injection of DNA into Xenopus oocytes has provided a functional test to analyze the biological activity of cloned genes (2-5), a concept named "surrogate genetics" (6). Unfertilized eggs and egg extracts are reported to promote replication of injected DNA (7)(8)(9)(10)(11)(12). Fertilized eggs injected with DNAs (13, 14) or rabbit globin mRNAs (15) are able to continue developing into apparently normal tadpoles.…”

Chromosome replication in cell-free systems from Xenopus eggs

“…Templates that replicate include bacteriophages X, M13, G4 and 4>X174, plasmids Col E l, pBR322, pMB9 and pAT153, together with a wide range of eukaryotic DNA fragments cloned into these vectors (Harland & Laskey 1980;McTiernan & Stambrook 1980;Mechali et al 1983;Mechali & Kearsey 1984). In contrast, there have been claims of sequence-specific initiation of replication in Xenopus eggs (Watanabe & Taylor 1980;Chambers et al 1982;Hines & Benbow 1982). A careful attempt to repeat these experiments (Mechali & Kearsey 1984) contradicted the claims of specific initiation and showed that replication is independent of DNA sequence, but depends on the size of the template DNA molecules.…”

Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

“…Recently, several investigators have tried to isolate replication origins by using in vitro recombination techniques (1, 16,21,41). Another approach to isolation of the replication origin is to get autonomous replicating sequences (ARS).…”