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Perspectives Copyright Ó 2009 by the Genetics Society of America DOI: 10.1534/genetics.109.110007 Perspectives Anecdotal, Historical and Critical Commentaries on Genetics Letting Escherichia coli Teach Me About Genome Engineering James A. Shapiro1 Department of Biochemistry and Molecular Biology, University of Chicago, Gordon Center for Integrative Science, Chicago, Illinois 60637 ABSTRACT A career of following unplanned observations has serendipitously led to a deep appreciation of the capacity that bacterial cells have for restructuring their genomes in a biologically responsive manner. Routine characterization of spontaneous mutations in the gal operon guided the discovery that bacteria transpose DNA segments into new genome sites. A failed project to fuse l sequences to a lacZ reporter ultimately made it possible to demonstrate how readily Escherichia coli generated rearrangements necessary for in vivo cloning of chromosomal fragments into phage genomes. Thinking about the molecular mechanism of IS1 and phage Mu transposition unexpectedly clarified how transposable elements mediate large-scale rearrangements of the bacterial genome. Following up on lab lore about long delays needed to obtain Mu-mediated lacZ protein fusions revealed a striking connection between physiological stress and activation of DNA rearrangement functions. Examining the fate of Mudlac DNA in sectored colonies showed that these same functions are subject to developmental control, like controlling elements in maize. All these experiences confirmed Barbara McClintock’s view that cells frequently respond to stimuli by restructuring their genomes and provided novel insights into the natural genetic engineering processes involved in evolution. HIS article is the reminiscence of a bacterial genet- The worlds of transcriptional regulation beyond simple T icist studying the processes of mutation and DNA repressor–operator models, signal transduction, chro- rearrangements. I want to emphasize how my experience matin formatting, transcript processing, protein mod- was full of surprises and unplanned discoveries that took ifications, and regulatory RNAs were all in the future. me ever deeper into the mechanisms and regulation of In my particular field, the molecular basis of genetic natural genetic engineering by Escherichia coli cells. change, discoveries about mobile genetic elements, For the benefit of younger molecular geneticists, there reverse transcription, programmed genome rearrange- are at least three points to be made. First, you can find ments, and other aspects of what I call ‘‘natural genetic something truly novel only when you do not know ex- engineering’’ were yet to be made. actly what you are looking for. If the experiment comes The following account relates my own experimental out just as you planned, you have not really learned journey into a new way of thinking about the molec- anything you did not already know or suspect. ular and cellular basis of genetic change. After detail- Second, routine characterization of your experimen- ing the journey, I will explain why and how I believe tal material is critical because it will tell you where your that this new mode of thought is likely to influence our understanding is incomplete—but only when the char- ideas about evolution, the most basic of biological acterizations do not come out as you expect. In other subjects. words, it can be a good thing if an experimental result I would be remiss if I did not acknowledge the power- does not fit your expectations. ful influence of Barbara McClintock on my thinking. Third, science will inevitably lead us in the future to After meeting her in 1976, I realized that she possessed think about the subjects that we are studying in ways that an unmatched depth of experience about all aspects we cannot currently predict. When I began my research, of biology, from natural history to the current status of we thought we understood the basics of genome ex- molecular genetics. We engaged in a 16-year dialogue up pression and mutation because we knew about DNA, to her death in 1992 (Shapiro 1992c). Only after many RNA polymerase, and the triplet code for amino acids. years did I finally come to appreciate the wisdom of her insistence that the ability of cells to sense and respond to 1Address for correspondence: Department of Biochemistry and Molecular Biology, University of Chicago, Gordon Center for Integrative Science, ‘‘genome shock’’ was just as important in determining 979 E. 57th St., Chicago, IL 60637. E-mail: [email protected] what happens to their genomes as are the biochemical Genetics 183: 1205–1214 (December 2009) 1206 J. A. Shapiro Figure 1.—The gal and trp regions of the E. coli chromo- some. The dashed line indicates .400 kb between the two op- erons. This distance makes galU mutations easy to separate from gal operon mutations that inactivate galK expression. The positions of four strongly polar insertion mutations are indicated by the vertical arrows. The figure is based on the de- letion mapping of Shapiro and Adhya (1969). mechanisms that they use in repairing and restructuring DNA molecules (McClintock 1984). DISCOVERING DNA INSERTIONS When I was a student at the University of Cambridge in the mid-1960s, I planned to study the effect of transcription on mutagenesis. Sydney Brenner at the MRC Laboratory for Molecular Biology suggested that a positive selection system for loss-of-function mutations would be the most sensitive experimental arrangement. The E. coli gal operon offered such a system in which the resulting mutations could easily be analyzed. A defect in the galU locus (Figure 1) made the bacteria sensitive to galactose (Gals phenotype). These galU mutants accumulated Gal-1-P, and the phosphorylated sugar inhibited growth. However, when the bacteria lost the galK-encoded galactokinase activity, they no longer accumulated Gal-1-P and shifted from a Gals to a Galr phenotype. Therefore, GalKÀ mutants could be selected Figure 2.—The results of CsCl equilibrium density-gradi- as colonies on medium containing galactose and glyc- ent centrifugation of a mixture of l-phages (l 857, plaque- forming phage; l dg1, phage carrying the wild-type gal erol. Since galU was not linked to the galETK operon À operon; l dgS114 , phage carrying an insertion mutation). (Shapiro 1966), it was easy to separate the GalK-inactivating The fractions on the left contain the densest particles. From mutations from the original galU mutation for reversion Shapiro (1969) with permission. studies and mapping. Following this plan, I isolated a series of spontaneous Galr mutants and proceeded to characterize the un- As I wrote my thesis in 1967, it occurred to me that derlying gal operon mutations (Adhya and Shapiro the mysterious pleiotropic gal mutations might result 1969). Of almost 200 spontaneous GalK-inactivating from insertion of extra DNA into the operon (Shapiro mutations, 14 proved to be pleiotropic or polar and 1967). For my postdoc, I went to the Institut Pasteur, inactivated at least two of the gal operon’s three where I learned to do CsCl density-gradient centrifu- cistrons. Of these, 10 failed to revert, and most of gation so that I could confirm the insertion hypothesis those were easy to interpret as deletions, which was using ldgal transducing phages: the ldgal particles later confirmed by mapping studies (Shapiro and carrying the mutations had extra DNA and were Adhya 1969). But the pleiotropic gal mutations that denser than the parental phage (Figure 2; Shapiro reverted proved to be hard to understand using 1969; Cohen and Shapiro 1980). The Starlinger existing ideas about the mechanisms of genetic group quickly repeated these results (Jordan et al. change. They reverted spontaneously and could be 1968), and electron microscope heteroduplex studies mapped to upstream regions of the galETK operon subsequently showed that the same DNA segments (Figure 1), but they were not traditional point muta- inserted into various positions in the gal and lac tions because reversion was not increased by operons (Fiandt et al. 1972; Hirsch et al. 1972). Thus, either base substitution or frameshift mutagens. we learned that bacteria have the capacity to move Other pleiotropic gal mutations isolated by the Leder- specific segments of DNA through the genome, and berg and Starlinger groups behaved similarly (Morse thesesegmentscametobecalled‘‘insertionse- 1967; Saedler and Starlinger 1967). quences’’ or IS elements (Starlinger and Saedler Perspectives 1207 1972). The world of transposable elements had moved been accomplished by E. coli, not by humans. We from Barbara McClintock’s maize cytogenetics simply selected the right products. (McClintock, 1950, 1953, 1987) to the molecular biology of E. coli (Cohen and Shapiro 1980). PLASMIDS, TRANSPOSONS, PHAGE MU, AND THE MECHANISM OF TRANSPOSITIONAL CLONING lac INTO l AND PURIFYING lac DNA DNA EXCHANGE WITHOUT RESTRICTION ENZYMES In 1975, my unrelated work on Pseudomonas hydro- After Paris, I moved to Jon Beckwith’s laboratory at carbon oxidation led me to the seminal ICN-UCLA Harvard Medical School. Together with Ethan Signer, Squaw Valley Symposium on Bacterial Plasmids. In a way Jon had pioneered in vivo genetic manipulation of the that periodically happens in all fields of science, re- lac operon, targeting insertions of Ftslac plasmids into search from many labs converged at a key meeting to the chromosome and using these insertions to isolate illuminate a general process. In this case, the topic was F80lac transducing phages (Beckwith et al. 1966). transposable elements, and the general process was These in vivo genetic engineering methods comple- DNA restructuring in bacteria. Great interest had been mented my own experience with spontaneous muta- generated by the ability of plasmids to accumulate genesis and specialized transducing phages. One of my multiple antibiotic resistances. Realizing that DNA seg- early projects, together with Karen Ippen, was to try ments could insert at multiple genomic locations pro- making lN-lacZ transcriptional fusions by adapting a vided a critical part of the explanation. Naomi Datta and double selection method from my thesis research to Fred Heffron spoke on the mobile b-lactamase element obtain deletions.
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