AN EFFICIENT SYSTEM FOR SITE-DIRECTED MUTAGENESIS TO MAKE VARIOUS MUTANTS OF THE ENV GENE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

We developed an efficient system of site-directed mutagenesis for the envelope (mv) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent se lection markers, i,e, a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 do main 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 oligon ucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the pla smids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-d irected mutants of the env gene of HIV-1 were accurately constructed i n 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 th e mr gene of HIV-1 precisely and rapidly. (C) 1998 Federation of Europ ean Biochemical Societies.