Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

Nucleic acids research ·Vol. 5 ·No. 3 ·1978-03-00 ·Pages 905-24

Humphries P, Old R, Coggins LW, McShane T, Watson C, Paul J

Abstract

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.

MeSH Terms

Animals Chromosome Mapping DNA, Recombinant/analysis Escherichia coli/genetics Genes Globins/genetics Nucleic Acid Hybridization Plasmids Protein Biosynthesis RNA, Messenger/genetics Xenopus/genetics

Chemicals

DNA, Recombinant RNA, Messenger Globins

Authors & Affiliations

6 authors, click to expand affiliations / ORCID

Humphries P

Old R

Coggins L W

McShane T

Watson C

Paul J

References (24)

24 references, click to expand

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Article Info

Journal

Nucleic acids research

Abbr.

Nucleic Acids Res

ISSN

0305-1048

DOI

10.1093/nar/5.3.905

Published

1978-03-00

Pages

905-24

Language

English

Region

England

NLM ID

0411011

PMCID

PMC342032

Subset

External Links

PubMed source DOI: 10.1093/nar/5.3.905 PMC Full Text View journal details

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Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

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