Premium
An efficient system for site‐directed mutagenesis to make various mutants of the env gene of human immunodeficiency virus type 1
Author(s) -
Shimizu Nobuaki,
Hoshino Hiroo
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00687-5
Subject(s) - plasmid , mutagenesis , gene , biology , site directed mutagenesis , genetics , restriction site , mutant , insertional mutagenesis , oligonucleotide , directed mutagenesis , dna , mutation , microbiology and biotechnology , restriction enzyme
We developed an efficient system of site‐directed mutagenesis for the envelope ( env ) gene of human immunodeficiency virus type 1 (HIV‐1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, Mlu I, and an in‐frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV‐1 strain, NL4‐3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR‐amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with Mlu I and competent env genes were formed by suppression of the in‐frame termination. Various site‐directed mutants of the env gene of HIV‐1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site‐directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV‐1 precisely and rapidly.