In vitro replication of recombinant plasmids carrying chromosomal segments of Xenopus laevis.

Abstract

Recombinant plasmids carrying a segment of Xenopus laevis chromosomal DNA were constructed with plasmid pBR322 as the vector. A recombinant plasmid pXY65 carrying a 3.2-kilobase BamHI segment of the chromosome of X. laevis has been found to contain a repetitive sequence dispersed throughout the X. laevis chromosomes. This plasmid initiated replication in vitro when the supercoiled circular molecules were incubated in a replication system. The other recombinant plasmids tested and the pBR322 vector were not replicated. Electron microscopic analysis of the replicative intermediates showed that the replication was initiated at a specific site in the 3.2-kilobase BamHI segment of pXY65 and that the replication usually proceeded bidirectionally. Analysis of the reaction products by centrifugation in alkaline sucrose gradients indicated that short pieces were synthesized in the in vitro replication system. DNA synthesis was inhibited in vitro by the addition of aphidicolin and by omission of dNTPs. These results indicate that the X. laevis segment cloned in pXY65 contains a site capable of initiating replication in vitro.The replication of eukaryotic DNA occurs by initiation at many chromosomal sites which are usually clustered in specific regions characteristic of cell type and the stage of the cell cycle (1-9). Recently, several investigators have tried to isolate replication origins by using in vitro recombination techniques (10-13). Many eukaryotic "autonomously replicating sequences" ofyeast as well as other higher organisms have been described. These sequences from higher organisms may act as initiators of replication in yeast cells (9,10). However, there is no evidence that these cloned eukaryotic DNA segments can also act as replicators in cells of the original higher organism.Watanabe and Taylor (13) have described the isolation of a recombinant plasmid which had a 500-base-pair segment of Xenopus laevis DNA and was replicated semiconservatively after microinjection into unfertilized Xenopus eggs. Harland and Laskey (14) also reported that circular double-stranded DNA molecules of simian virus 40, polyoma virus, bacteriophages, and plasmids were replicated in unfertilized eggs ofX. laevis. However, they concluded that a specialized DNA sequence was not essential for initiation of DNA replication in the Xenopus egg.To study DNA replication in vitro, Benbow et al. (15) prepared an extract from unfertilized Xenopus eggs and fractionated it by DEAE-cellulose chromatography. The active fraction contained several activities including DNA polymerases a, P, and y, RNA polymerases, DNA ligase, a DNA-binding protein, and RNase H (16). Using this system, Benbow et al. were successful in obtaining replication of a recombinant plasmid that

An inhibitor of DNA topoisomerase I from Xenopus laevis ovaries

“…DNA. Plasmids pUC19 (Yanisch-Perron et al, 1985) and pXY65 (Hiraga et al, 1982) were isolated from Escherichia coli HB101 by alkaline lysis and purified by CsCl gradient centrifugation (Sambrooke/al., 1989). [3H]-labeledpXY65 was prepared as described by Hines and Benbow (1982).…”