Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: Positions of viable insertion mutations

L. I. Lobel, S. P. Goff

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

A highly efficient method for the generation of insertion mutations is described. The procedure involves the use of a 220-base-pair (bp) EcoRI fragment bearing the SuIII+ suppressor tRNA gene as an insertional mutagen. The plasmid DNA to be mutagenized is linearized by a variety of means, and the suppressor fragment is ligated into the site of cleavage. Successful insertion mutants can be readily detected in Escherichia coli carrying lac- amber mutations on MacConkey lactose plates; virtually 100% of the red colonies contain insertions of the fragment. Subsequent removal of the SuIII+ gene and recyclization leaves a 12-bp insertion if the original cleavage was blunt-ended and a 9-bp insertion if the original cleavage generated 3-bp cohesive termini. This technique, as well as conventional linker mutagenesis with decamer and dodecamer linkers, was used to generate a large library of insertion mutations in cloned DNA copies of the genome of Moloney murine leukemia virus. A number of viable mutants were isolated bearing 9-, 10-, and 12-bp insertions in various domains of the genome. The map positions of the viable mutations suggest that the viral long terminal repeats and portions of the gag and env genes are quite insensitive to alteration. Although most of the mutations were stable for many passages, some of the mutants lost the inserted DNA; we presume that the insertion was somewhat deleterious in these mutants and that continued passage of the virus selected for overgrowth by a revertant.

Original languageEnglish
Pages (from-to)4149-4153
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number13 I
DOIs
StatePublished - 1 Jan 1984
Externally publishedYes

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: Positions of viable insertion mutations'. Together they form a unique fingerprint.

Cite this