The Development of Site-directed Mutagenesis by Michael Smith
Year: 2006
Type: article
Abstract: Mutagenesis at a specific position in a DNA sequence.Journal of Biological ChemistryVol. 253Issue 18PreviewPredefined changes in a known DNA sequence were introduced by a general method. Oligodeoxyribonucleotides complementary to positions 582 to 593 of the viral DNA strand of the bacteriophage phiX174 am3 mutant (pGTATCCTACAAA), and to the wild type sequence in this region (pGTATCCTACAAA), were synthesized and used as specific mutagens. Each of these oligonucleotides was incorporated into a complete circular complementary strand when used as primer on a genetically heterologous viral strand template, by the combined action of subtilisin-treated Escherichia coli DNA polymerase I and T4 DNA ligase. Full-Text PDF Open Access Mutagenesis at a Specific Position in a DNA Sequence (Hutchison, C. A., III, Phillips, S., Edgell, M. H., Gillam, S., Jahnke, P., and Smith, M. (1978) J. Biol. Chem. 253, 6551–6560) Michael Smith (1932–2000) was born in Blackpool, England, a working class town populated largely by coal miners and factory workers. By passing the “Elevenplus” examination in elementary school, Smith was able to obtain a scholarship to the local private school, Arnold School, which would prepare him for university. It was at Arnold School that Smith became interested in science, particularly chemistry. His ability in the sciences qualified him for admission to Cambridge University, but he did not have the required Latin credit. So, in 1950 he entered the chemistry honors program at the nearby University of Manchester and graduated 3 years later. He remained at Manchester for graduate studies and worked on cyclohexane diols with H. B. Henbest. Smith finished his Ph.D. degree in 1956. During his last year of graduate school, Smith wrote to various American professors seeking a postdoctoral fellowship. He had no luck in obtaining his desired fellowship on the west coast of the United States, but he did hear of a young scientist in Vancouver, Canada, named Gobind Khorana, who might have a fellowship to work on the synthesis of biologically important organophosphates. Smith wrote to the future Nobel Laureate and was awarded a fellowship after an interview in London with the British Columbia Research Council. Smith arrived in Vancouver in September 1956. His first project was to develop a general, efficient procedure for the chemical synthesis of nucleoside 5′-triphosphates based on the synthesis of ATP by Khorana in 1954. This study led to more extensive investigations of the reactions of carbodiimides with acids, including phosphoric acid esters, and to a general procedure for the preparation of nucleoside 3′,5′-cyclic phosphates. In 1960, the Khorana group, along with Smith, moved to the Institute for Enzyme Research at the University of Wisconsin. There, Smith worked on the synthesis of ribo-oligonucleotides. However, in 1961, he realized that it was time to move on, and he accepted a position with the Fisheries Research Board of Canada Laboratory in Vancouver. During this time Smith devised a new synthetic method for nucleoside 3′,5′-cyclic phosphates. In 1966, Smith became a faculty member in the Department of Biochemistry at the University of British Columbia, where he remained for the rest of his academic career. At the University of British Columbia, Smith continued to study oligonucleotide synthesis and formulated a method that made possible the synthesis of deoxyribo-oligonucleotides up to 12 to 13 nucleotides in length. This proved to be a significant breakthrough for his small group because it allowed them to undertake a number of fairly ambitious molecular biological projects between 1970 and 1980, when oligonucleotides were not generally accessible. In the fall of 1975, Smith started a year-long sabbatical in Fred Sanger's laboratory, helping with the sequencing of the Escherichia coli phage ϕX174. An important component of the sequencing process came from defining the position and reading frame of these genes using nonsense mutants suppressible by amber or ochre suppressors. For Smith, this highlighted the need for a specific mutagenic method that would target a specific base pair in the genome and introduce a predetermined change with sufficiently high efficiency. Given that the DNA of phage ϕX174 is single-stranded, Smith's earlier studies, which demonstrated that small oligonucleotides could form stable duplexes at low temperature even with a mismatch, suggested that oligonucleotide-directed mutagenesis should be possible. Several years earlier, Clyde A. Hutchison and Marshall H. Edgell had achieved mutagenesis with small fragments of ϕX174 and restriction nucleases (1Hutchison C.A. Edgell M.H. Genetic assay for small fragments of bacteriophage ϕX174 deoxyribonucleic acid.J. Virol. 1971; 8: 181-189Crossref PubMed Google Scholar, 2Middleton J.H. Edgell M.H. Hutchison C.A. Specific fragments of ϕX174 deoxyribonucleic acid produced by a restriction enzyme from Haemophilus aegyptius, endonuclease Z..J. Virol. 1972; 10: 42-50Crossref PubMed Google Scholar). They showed that point mutants could be reverted by annealing mutant phage ϕX174 DNA with fragments from the complementary strand of wild-type DNA prior to transfection. Hutchison, who was also spending a year in Sanger's group sequencing ϕX174, teamed up with Smith, and the pair realized that an obvious route to a mutagenic method was to use a mutant oligonucleotide primer for E. coli DNA polymerase I on a circular single strand template, which would produce a product that could be converted to a closed circular duplex by enzymatic ligation. Their resulting technique for site-directed mutagenesis is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here. Smith and Hutchison decided to use a 12-nucleotide oligomer with a centrally positioned single nucleotide mismatch as primer, ϕX174 DNA as template, and E. coli DNA polymerase I in which the 5′-exonuclease had been inactivated by subtilisin to construct a closed circular double-stranded DNA with the oligonucleotide in one strand. The specific mutations chosen for the experiment were the production and reversion of a known nonsense mutation, am3, since convenient phenotypic screens were available. The mutations involved the interconversion of a Trp codon, TGG, and an amber codon, TAG, by G-T and A-C mismatches. In the initial experiments, mutation was achieved but at low efficiency. This was increased after removal of incompletely closed duplexes by adsorption to nitrocellulose or treatment with a single-strand-specific nuclease under conditions where a single base pair mismatch was not degraded. Further studies on the mutagenesis of phage ϕX174 demonstrated that it was possible to produce transversion mutations and single nucleotide deletions also using very short oligonucleotides. Smith later commented, “We could not have anticipated the explosion of gene isolations, the improvements in DNA sequence determination methodology and the advances in the chemistry of nucleic acids synthesis that have occurred since 1978. This has resulted in an amazing increase in the use of site-directed mutagenesis as an analytical tool in biochemistry and biology. And it has been accompanied by continual improvements in the basic methodologies and versatility of site-directed mutagenesis and the initiation of new scientific publications such as Protein Engineering and Protein Science.” In recognition of his research, Smith was awarded one-half of the 1993 Nobel Prize in Chemistry “for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies.” Kary B. Mullis received the other half of the prize for developing polymerase chain reaction. Smith gave half of his Nobel Prize money to support postdoctoral fellowships in schizophrenia research, and the other half went to the Vancouver Foundation to fund public science education through Science World and the Society for Canadian Women in Science and Technology. In 1981, Smith, along with Ben Hall and Earl Davie, founded the biotechnology company, Zymos. The company, in collaboration with the Danish pharmaceutical company Novo, developed a process for producing human insulin in yeast. In 1986, Smith established a new interdisciplinary institute, the Biotechnology Laboratory, at the University of British Columbia. He also became Acting Director of the Biomedical Research Centre, a privately funded research institute on the Campus of the University of British Columbia, in 1991. In addition to the Nobel Prize, Smith received numerous awards and honors. These include the Jacob Biely Faculty Research Prize from the University of British Columbia (1977), the Boehringer Mannheim Prize of the Canadian Biochemical Society (1981), the Gold Medal from the Science Council of British Columbia (1984), the Gairdner Foundation International Award (1986), the Killam Research Prize from the University of British Columbia (1986), the Award of Excellence from the Genetics Society of Canada (1988), the G. Malcolm Brown Award from the Canadian Federation of Biological Societies (1989), and the Flavelle Medal from the Royal Society of Canada (1992). Smith was also a Fellow of the Royal Society of Canada and the Royal Society (London) and a Laureate of the Canadian Medical Hall of Fame. 1Biographical information for Michael Smith was taken from Refs. 3Smith M. Synthetic DNA and biology.in: Malmström B.G. Nobel Lectures, Chemistry 1991–1995. World Scientific Publishing Co., Singapore1997Google Scholar and 4Smith M. Michael Smith—Autobiography.in: Frängsmyr T. The Nobel Prizes 1993. Nobel Foundation, Stockholm1994Google Scholar. 1Biographical information for Michael Smith was taken from Refs. 3Smith M. Synthetic DNA and biology.in: Malmström B.G. Nobel Lectures, Chemistry 1991–1995. World Scientific Publishing Co., Singapore1997Google Scholar and 4Smith M. Michael Smith—Autobiography.in: Frängsmyr T. The Nobel Prizes 1993. Nobel Foundation, Stockholm1994Google Scholar. Smith's collaborators on the JBC paper, Clyde A. Hutchison III and Marshall H. Edgell, met in Robert L. Sinsheimer's laboratory at Cal Tech where Hutchison was a graduate student and Edgell was a postdoctoral fellow. In 1968 Hutchison and Edgell moved to the University of North Carolina at Chapel Hill, where they joined the faculty of the Department of Bacteriology and Immunology. There they dissected the genome of phage ϕX174 with restriction enzymes. During this period Hutchison and Edgell also applied restriction enzymes to the analysis of mammalian mitochondrial DNA, identifying restriction fragment length polymorphisms and demonstrating maternal inheritance of mitochondrial DNA in mammals. After the JBC Classic paper was published, the Hutchison laboratory developed methods for “complete mutagenesis,” in which each residue in a protein is individually altered. Hutchison and Edgell continued their collaboration in Chapel Hill, using the new sequencing and cloning technologies to sequence the β-globin gene cluster in the mouse. They also discovered and characterized the L1 retroposon, the most common long repetitive element in the mammalian genome. In 1990 Hutchison began work with Mycoplasma genitalium, which resulted in a collaboration with The Institute for Genomic Research to sequence the entire genome of the organism. He also began to work with Hamilton Smith and others at the Institute for Biological Energy Alternatives in 2003, which soon resulted in assembly of the genome of bacteriophage ϕX174 from a single pool of chemically synthesized oligonucleotides. Hutchison is now Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill, where he continues to do research and train graduate students and postdocs. He is also Distinguished Investigator at the J. Craig Venter Institute in Rockville, Maryland, and a member of the National Academy of Sciences and a fellow of the American Academy of Arts and Sciences. Edgell is a Distinguished Professor and Joint Appointee in the Department of Microbiology and Immunology and the Department of Biochemistry and Biophysics at Chapel Hill.
Source: Journal of Biological Chemistry
Language: English
Subfield: History and Philosophy of Science
Field: Arts and Humanities
Domain: Social Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1888429472