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DNA Cloning: a Personal View After 40 Years Stanley N PERSPECTIVE PERSPECTIVE DNA cloning: A personal view after 40 years Stanley N. Cohen1 Departments of Genetics and Medicine, Stanford University School of Medicine, Stanford, CA 94305 Edited by Joseph L. Goldstein, University of Texas Southwestern Medical Center, Dallas, TX, and approved August 13, 2013 (received for review August 2, 2013) In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer’s laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning. restriction enzyme | pSC101 | EcoRI | genetic engineering | gene cloning In a PNAS paper entitled “Construction of would prevent propagation of genes across a highly public controversy about potential Biologically Functional Bacterial Plasmids different biological domains. Stringent host hazards of “genetic tinkering,” a decision by In Vitro,” mycolleaguesA.C.Y.Chang, range limitations to virus propagation had Stanford University and the University of H. W. Boyer, R. B. Helling, and I reported been observed, and, in some instances, California to seek patents on the technology in November 1973 that individual genes impediments to survival of foreign DNA that Boyer and I had invented, and efforts can be cloned and isolated by enzymatically had been found even among subgroups of by entrepreneurs and industry to implement fragmenting DNA molecules, linking the the same species (9). Supporting the notion DNA cloning methods for commercial pur- pooled fragments to autonomously replicating that DNA was unlikely to survive in cells poses. In the 40 years that have now passed circular bacterial genetic elements known as of an unrelated species was the finding since publication of these PNAS papers, use of plasmids, and introducing the resulting re- that individual biological species maintain DNA cloning methods has produced impor- combinant DNA molecules into bacteria (1). characteristic ratios of A+TtoG+Cbase tant insights about the workings of genes and Boyer and I were young faculty at the Univer- pairs (10, 11). Our discovery that DNA can be cells in health and disease and has profoundly sity of California, San Francisco (UCSF) and transplanted to, and propagated in, a differ- altered the biotechnology and pharmaceutical Stanford, respectively. Annie Chang was a Re- ent species, and even in a different biological industries. I provide here a personal perspec- search Technician in my laboratory and Bob kingdom, by attaching it to a vector indig- tive of these events. Helling was a University of Michigan professor enous to the recipient led to the realization on sabbatical leave in Boyer’s laboratory. A that natural barriers to DNA survival are Plasmids and Antibiotic Resistance few months later, Chang and I reported that not so constraining after all, and that “ge- After the development of antimicrobial agents genes from totally unrelated bacterial species netic engineering”—atleastatthecellular in the 1940s, the notion was prevalent that can be combined and propagated using the level—is possible (8). It also provided a pro- these drugs would end infectious diseases same approach (2) and that interspecies tocol that enabled such engineering to be done caused by bacteria. Of course that did not recombinant DNA molecules can produce by virtually any laboratory having modest happen, and the reason was the occurrence a biologically functional protein in a foreign genetic and biochemical capabilities. of antibiotic resistance. Investigations carried host. Soon afterward, Boyer’s laboratory Our DNA cloning experiments resulted out primarily in laboratories in Japan and the and mine published collaborative experiments from the pursuit of fundamental biological United Kingdom in the early 1960s showed demonstrating that genes from eukaryotic questions rather than goals that most observers that antibiotic resistance in bacteria com- cells can be cloned in bacteria (3). might regard as practical or “translational.” I monly is associated with the acquisition of Bacterial viruses and plasmids had been was investigating mechanisms underlying genes—often multiple genes—capable of de- shown to pick up DNA from the chromo- the ability of plasmids to acquire genes con- stroying antibiotics or otherwise interfering somes of their hosts (4); hybrid viruses from ferring antibiotic resistance and to exist sep- animal cells also had been reported (5, 6). arately from bacterial chromosomes; Herb Author contributions: S.N.C. wrote the paper. However, it had long been known that only Boyer was studying enzymes that restrict The author declares no conflict of interest. closely related species can interbreed and pro- and destroy foreign DNA. The PNAS publica- This article is a PNAS Direct Submission. duce viable offspring, and hybrids displaying tions resulting from these pursuits generated fi — This article was invited in recognition of the 40th anniversary of heritable characteristics of very different spe- considerable scienti c excitement and work the November 1973 PNAS paper by S. N. Cohen, A. C. Y. Chang, cies exist only in mythology; thus, there was aimed at repeating and extending the findings H. W. Boyer, and R. B. Helling reporting a method for constructing uncertainty about whether so-called “nat- was undertaken almost immediately by and cloning biologically functional DNA molecules (1). ural barriers created during evolution” (7, 8) other researchers. The papers also prompted 1E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1313397110 PNAS | September 24, 2013 | vol. 110 | no. 39 | 15521–15529 Downloaded by guest on September 26, 2021 with their actions. The resistance properties Research in the burgeoning field of mo- that are distinguishable visually from thinner, commonly did not map genetically to the lecular biology during the 1960s focused on more kinky regions of single-stranded DNA. bacterial chromosomes, suggesting that the bacteriophages for an important reason: We expected that such experiments would genes encoding resistance were located on a bacterial cell infected by a virus generates provide information about the structural re- separate elements (some had called them thousands of identical copies—clones—of lationships between resistance genes that had episomes) analogous to the fertility factor a single infecting genome during the normal been picked up by plasmids during their (F-factor) discovered earlier (12). Like F- viral life cycle. Thus, phenotypic effects can meandering through bacterial populations. factors, resistance factors (R-factors) were be correlated with the results of biochemical The results of these experiments and also of capable of being transferred between bac- analyses. I realized that elucidation of how separate investigations from Davidson’slab- teria by cell-to-cell contact (13, 14). In 1952, resistance genes function and how R-plas- oratory (27, 28) showed remarkable sequence Joshua Lederberg had given the name “plas- mids evolve required a way to clone in- conservation among large segments of dif- mids” to such extrachromosomal genetic ele- dividual plasmid DNA molecules and to ferent R-plasmids and, importantly, provided ments (15). The antibiotic-inactivating genes isolate the resistance genes. Genetic map- direct physical evidence that plasmid se- carried by resistance plasmids provide a bi- ping of R-plasmid properties had led to the quences associated with interbacterial DNA ological advantage to host bacteria in pop- prediction that bacterial plasmids exist as transfer had become linked covalently to re- ulations exposed to antimicrobial drugs, DNA circles (19, 21, 22), and I proposed to sistance genes to form large circles of ’ and, in barely a decade after the intro- use circularity to isolate intact resistance R-plasmid DNA. Sharp s electron micros- duction of antibiotics to treat human in- plasmid DNA. If I could obtain R-plasmid copy also detected a phenomenon that we — — ’ fi fections, R-plasmid–mediated multidrug circles I reasoned IcouldapplyDNA didn t yet understand the signi cance of: resistance had become a major medical fragmentation approaches I had used to short inverted repeats of DNA sequences λ problem as well as a
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