Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 71 ·No. 9 ·1974-09-00 ·Pages 3455-9

Hershfield V, Boyer HW, Yanofsky C, Lovett MA, Helinski DR

Abstract

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage varphi80pt190 (trp(+)) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp(-) strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the varphi80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes.

MeSH Terms

Chloramphenicol/pharmacology Colicins/metabolism Coliphages/analysis DNA Replication DNA, Viral Drug Resistance, Microbial Endonucleases/metabolism Enzyme Repression Escherichia coli/metabolism Extrachromosomal Inheritance Genes Hydrolysis Kanamycin/pharmacology Operon Polynucleotide Ligases/metabolism RNA, Bacterial/biosynthesis RNA, Messenger/biosynthesis Recombination, Genetic Transformation, Genetic Tryptophan/biosynthesis,metabolism Tryptophan Synthase/analysis

Chemicals

Colicins DNA, Viral RNA, Bacterial RNA, Messenger Kanamycin Chloramphenicol Tryptophan Endonucleases Tryptophan Synthase Polynucleotide Ligases

Authors & Affiliations

5 authors, click to expand affiliations / ORCID

Hershfield V

Boyer H W

Yanofsky C

Lovett M A

Helinski D R

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23 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.71.9.3455

Published

1974-09-00

Pages

3455-9

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC433792

Subset

External Links

PubMed source DOI: 10.1073/pnas.71.9.3455 PMC Full Text View journal details

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