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PMID: 6954512 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 79 ·No. 12 ·1982-06-00 ·Pages 3697-701

Hiraga S, Sudo T, Yoshida M, Kubota H, Ueyama H

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.

MeSH Terms
Animals Cloning, Molecular DNA Replication DNA, Recombinant Plasmids Repetitive Sequences, Nucleic Acid Xenopus laevis/genetics
Chemicals
DNA, Recombinant
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Hiraga S
Sudo T
Yoshida M
Kubota H
Ueyama H
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34 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.79.12.3697
Published
1982-06-00
Pages
3697-701
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC346493
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.79.12.3697 PMC Full Text View journal details
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