TY - JOUR
T1 - Random mutagenesis of the human immunodeficiency virus type-1 frans-activator of transcription (HIV-1 Tat)
AU - Siderovski, David P.
AU - Matsuyama, Toshifumi
AU - Frigerio, Elena
AU - Chui, Stephen
AU - Min, Xia
AU - Erfle, Heather
AU - Sumner-smith, Martin
AU - Bamett, Richard W.
AU - Mak, Tak W.
N1 - Funding Information:
We thank Raya Kuperman for the oligonucleotide syntheses, Neil Parkin and Nahum Sonenberg for the gift of HeLa TARCAT cells and pSVTat, Wai-Ping Fung-Leung for pSVE183, and Alexandra Ho and Lorenz Trumper for technical assistance. D.P.S. was supported by a Ph.D. Studentship from the Medical Research Council of Canada. This work was supported in part by a grant from the National Health Research and Development Program of Canada (T.W.M), and a National Cooperative Drug Discovery grant (M.S.-S. and T.W.M.).
PY - 1992/10/25
Y1 - 1992/10/25
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0026641650&partnerID=8YFLogxK
U2 - 10.1093/nar/20.20.5311
DO - 10.1093/nar/20.20.5311
M3 - Article
C2 - 1437550
AN - SCOPUS:0026641650
SN - 0305-1048
VL - 20
SP - 5311
EP - 5320
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 20
ER -