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Nucleic Acids Research, 1992, Vol. 20, No. 20 5311-5320
© 1992

MOLECULAR BIOLOGY

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 Erfle1, Martin Sumner-Smith1, Richard W. Bamett1 and Tak W. Mak

The Ontario Cancer Institute and Departments of Immunology and Medical Biophysics, University of Toronto 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada 1Allelix Biopharmaceuticals Inc. 6850 Goreway Drive, Mississauga, Ontario L4V 1P1, Canada

Received July 23, 1992. Accepted September 15, 1992.

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.


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