Expression of enzymatically active reverse transcriptase of simian immunodeficiency virus in bacteria: sensitivity to nucleotide analogue inhibitors.
A fragment of the SIVmac251 pol gene was expressed in Escherichia coli as a trpE fusion protein. Analysis of extracts from bacteria containing this expression plasmid revealed the presence of a reverse transcriptase activity dependent on Mg2+ as divalent cation and active on both poly(rA).oligo(dT) and poly(rC.oligo(dG) templates. In comparative studies, the SIV and HIV-1 reverse transcriptases expressed in bacteria displayed very similar high sensitivities to the chain terminator inhibitors AZTTP and ddTTP. The reverse transcriptase of Moloney murine leukemia virus and the DNA polymerase of E. coli were both more resistant to ddTTP, and the E. coli enzyme was significantly more resistant to AZTTP.
Department of Biochemistry, Columbia University, College of Physicians and Surgeons, New York, New York.
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Prasad
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Myrick
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Haseltine
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Goff
D054306:Dideoxynucleotides; D004926:Escherichia coli; D015870:Gene Expression; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D015302:Simian Immunodeficiency Virus / Q000201:enzymology* / Q000235:genetics; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000494:pharmacology
D054306:Dideoxynucleotides; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D015215:Zidovudine; D012194:RNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate