Random mutagenesis of the human immunodeficiency virus type-1 frans-activator of transcription (HIV-1 Tat)

David P. Siderovski, Toshifumi Matsuyama, Elena Frigerio, Stephen Chui, Xia Min, Heather Erfle, Martin Sumner-smith, Richard W. Bamett, Tak W. Mak

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11 Scopus citations

Abstract

A new method is described for the direct construction of randomly mutagenized genes by applying the polymerase chain reaction (PCR) to an oligonucleotlde synthesized using doped nucleotide reservoirs. We have demonstrated the utility of this method by generating a library of mutant HIV-1 tat genes. Several arbitrarily selected, inactive tat clones were sequenced to evaluate the extent of the mutagenesis. Moreover, fourteen recombinants encoding varying levels of transcriptional frans-actlvator activity were isolated by transient transfection of sub-library pools into a HeLa cell line bearing an HIV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene. Sequence data revealed a spectrum of alterations including nucleotide substitutions, Insertions, and deletions, suggesting that mutations arose from both the doped DNA synthesis and the subsequent PCR 'rescue' of full-length product. Sequence comparison between inactive and active Tat clones revealed a selection pressure against amino-acid substitutions within the N-tenminal domains of Tat, indicating the importance of this region to trans-activation competence. In addition, single and double missense mutations within the basic-rich, TAR RNA-binding domain were seen to be tolerated within active Tat clones.

Original languageEnglish
Pages (from-to)5311-5320
Number of pages10
JournalNucleic Acids Research
Volume20
Issue number20
DOIs
StatePublished - 25 Oct 1992

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    Siderovski, D. P., Matsuyama, T., Frigerio, E., Chui, S., Min, X., Erfle, H., Sumner-smith, M., Bamett, R. W., & Mak, T. W. (1992). Random mutagenesis of the human immunodeficiency virus type-1 frans-activator of transcription (HIV-1 Tat). Nucleic Acids Research, 20(20), 5311-5320. https://doi.org/10.1093/nar/20.20.5311