PMID: 6170979Aug 1, 1981Paper

Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C

Proceedings of the National Academy of Sciences of the United States of America
I KudoU L RajBhandary

Abstract

We have cloned the Escherichia coli tyrosine-inserting amber suppressor tRNA gene into the recombinant single-strand phage M12mp3. By using the M13mp3SuIII+ recombinant phage DNA as template and an oligonucleotide bearing a mismatch as primer, we have synthesized in vitro an M13mp3SuIII heteroduplex DNA that has a single mismatch at a predetermined site in the tRNA gene. Transformation of E. coli with the heteroduplex DNA yielded M13 recombinant phages carrying a mutant suppressor tRNA gene in which the sequence G-T-T-C, corresponding to the universal G-T-pseudouracil-C sequence in E. coli tRNAs, is changed to G-A-T-C. The mutant DNA has been characterized by restriction mapping and by sequence analysis. In contrast to results with the wild-type suppressor tRNA gene, cells transformed with recombinant plasmids carrying the mutant tRNA gene are phenotypically Su-. Thus, the single nucleotide change introduced has inactivated the function of the tRNA gene. By using E. coli minicells for studying the expression in vivo of cloned tRNA genes, we have found that cells transformed with recombinant plasmids carrying the mutant tRNA gene contain very little, if any, mature mutant suppressor tRNA. In contrast, the predominant low molecul...Continue Reading

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Citations

Dec 1, 1981·Current Genetics·R Grosskopf, H Feldmann
Aug 15, 1982·Journal of Molecular Biology·R L GourseA E Dahlberg
Jan 1, 1987·Gene·K Foss, W H McClain
Oct 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·F A LaskiP A Sharp
Nov 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·G Dalbadie-McFarlandJ H Richards
Dec 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·B L Seong, U L RajBhandary
Sep 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·F A LaskiG M Rubin
Dec 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·C P Lee, U L RajBhandary
Aug 11, 1982·Nucleic Acids Research·F A LaskiP A Sharp
Mar 11, 1983·Nucleic Acids Research·P J FurdonS Altman
Nov 11, 1986·Nucleic Acids Research·C F WrightK McKenney
Jan 1, 1984·Molecular & General Genetics : MGG·A MarmenoutW Fiers
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Dec 1, 2010·Antimicrobial Agents and Chemotherapy·Noboru NakataMasahiko Makino
May 1, 1989·Genes & Development·M C MullinsG M Rubin

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