Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations.
The finding of G-->A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids. Using a RNA/reverse transcriptase ratio of approximately 1:30, comparable to that within the retroviral replication complex, G-->A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations--i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP. As G-->A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U-->C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G-->A and U-->C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.
Martinez
M A
MA
Unité de Rétrovirologie Moléculaire, Institut Pasteur, Paris, France.
Vartanian
J P
JP
Wain-Hobson
S
S
D000225:Adenine; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D003854:Deoxyribonucleotides / Q000378:metabolism*; D015752:Genes, env; D006147:Guanine; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014779:Virus Replication
D017931:DNA Primers; D003854:Deoxyribonucleotides; D012367:RNA, Viral; D011994:Recombinant Proteins; D006147:Guanine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D000225:Adenine