Open AccessMethod for introducing site-specific mutations into adenovirus 2 genome: construction of a small deletion mutant in VA-RNAI gene.
Author(s) -
Q.S. Kapoor,
G. Chinnadurai
Publication year - 1981
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.78.4.2184
Subject(s) - ecori , biology , microbiology and biotechnology , bamhi , plasmid , mutant , gene , restriction site , dna , pbr322 , in vitro recombination , restriction fragment , genetics , genome , restriction enzyme , molecular cloning , peptide sequence
We have developed a method for introducing mutations into the adenovirus type 2 genome at predetermined sites. Specific mutations are introduced into segments of the viral genome cloned in bacteria by using plasmid vectors. The chimeric DNA is used to construct viral mutants by cotransfection with two viral DNA segments derived from both ends of the viral genome, each of which has overlapping sequence homology with the cloned viral DNA segment. To illustrate this procedure, we cloned a large restriction fragment [EcoRI fragment A, map position (mp) 0--58.5] by using plasmid vector pBR322, and a small deletion mutation was introduced at the BamHI cleavage site located within the VA-RNAI gene (mp 29 at residue +75 on VA-RNAI gene). The mutated DNA was then used to construct viral mutants by cotransfection into human cells with the adenovirus type 2 DNA--protein complex digested with Sal I. In vivo recombination occurred via overlapping sequences between the cloned EcoRI fragment A and Sal I-digested DNA--protein complex at two sites, mp 0--25 (i.e., within Sal I fragment B) and mp 45--58.5 (i.e., overlapping sequences between Sal I fragment A and EcoRI fragment A), generating infectious DNA molecules with intact ends. The viral mutant grows as well as the wild type in KB cells and induces the synthesis of smaller VA-RNAI but normal-size VA-RNAII.