The power of recombinant DNA technology stems from the ability it

Transcription

1 1966 Transposition of the Lac Region of Escherichia coli. I. Inversion of the Lac Operon and Transduction of Lac by φ80 J. R. BECKWITH AND E. R. SIGNER The power of recombinant DNA technology stems from the ability it provides to create novel DNA joints; this method uses enzymes to join unrelated DNA fragments in vitro. For example, two novel DNA joints are created when a gene is cloned in vitro onto a small plasmid vector. Transposition and illegitimate recombination also create novel DNA joints. Accordingly, when harnessed properly by genetic selection, these reactions provide an equally powerful experimental tool for the clever bacterial geneticist. Beckwith and Signer first demonstrated this with the isolation of φ80lac. In effect, they cloned the lacz gene with toothpicks. In 1966 Beckwith and Signer were postdoctoral students in the lab of François Jacob. At that time there were two genetic methods for cloning genes from Escherichia coli, and both were severely limited. Certain genes could be incorporated into specialized transducing phage; for example, the gal and bio genes could be incorporated into phage λ and the trp and supf (Su3) genes could be incorporated into φ80. However, this method was limited to genes that were adjacent to phage attachment sites in the bacterial chromosome. A more general method involved the isolation of F' factors, but these episomes were extremely large and practically impossible to manipulate biochemically. Beckwith and Signer combined these methods in ingenious fashion. Using an F lac strain, which carried a temperature-sensitive mutation that prevented episome replication, and a strain in which the chromosomal lac region had been deleted, François Cuzin and François Jacob had shown that Hfr strains could be isolated by selecting for Lac + at 42 C. Under these conditions the episome is lost (Lac ) unless it integrates into the chromosome, thus forming an Hfr derivative. Influenced by the work on λ integration by Allan Campbell (Adv. Genet. 11: , 1962), Beckwith and Signer reasoned correctly that F lac integration, if it occurred within a gene, would split that gene and thus destroy its function. This impressive insight occurred years before transposons and insertion elements were discovered in bacteria, at a time when spontaneous insertion mutations were incomprehensible. As it turns out, the T1rec gene, now called tonb, is located adjacent to the attachment site for φ80 (att 80 ). Mutations in tonb confer resistance to several different phages and colicins. Accordingly, directed transposition of lac to a chromosomal location near att 80 could be accomplished by using the Cuzin and Jacob strain by simultaneous selection for Lac + and phage and colicin resistance at 42 C. By using a strain that answered this selection (Hfr EC15), φ80lac could be isolated in the same manner as φ80trp from wild-type φ80 lysogens. The implications of this work were profound. Chromosomes could be redesigned at will, and genes could be moved from one replicon to another. With specialized transducing phage, genetically well-characterized genes such as lac could be studied by biochemical and biophysical methods. In many ways this represents the dawn of genetic engineering. Indeed, the search for new and better ways to make transducing phage led ultimately to modern recombinant DNA technology. THOMAS J. SILHAVY Reprinted from Journal of Molecular Biology 19: Copyright 1966, by permission of the publisher, Academic Press. 414 Microbiology: A Centenary Perspective

2 Molecular Biology and Physiology 415

3 416 Microbiology: A Centenary Perspective

4 Molecular Biology and Physiology 417

5 418 Microbiology: A Centenary Perspective

6 Molecular Biology and Physiology 419

7 420 Microbiology: A Centenary Perspective

8 Molecular Biology and Physiology 421

9 422 Microbiology: A Centenary Perspective

10 Molecular Biology and Physiology 423

11 424 Microbiology: A Centenary Perspective

12 Molecular Biology and Physiology 425

13 426 Microbiology: A Centenary Perspective