Many of the Reflections articles in this journal have focused on particular biological problems that the authors have devoted their careers to or on particular techniques that have facilitated their biological studies. Although I began my career as a biochemist, one of the most common threads to my research has been the exploitation of a genetic approach, the analysis of suppressor mutations. Particularly in the last 20 years, the success of this approach has led me into biological problems that required a remembering or relearning of my early training in biochemistry. This article will describe the voyage from chemistry and biochemistry to genetics and then to a recovery of remnants of my biochemical memory. As a graduate student working under Lowell Hager at Harvard in the late 1950s, my thesis research was chemical and biochemical, as I performed the chemical synthesis and studied the biosynthesis of the chlorinated mold product caldariomycin (2,2-dichloro-1,3-cyclopentanediol) (1Beckwith J.R. Hager L.P. J. Org. Chem. 1961; 26: 5206-5208Crossref Scopus (6) Google Scholar, 2Beckwith J.R. Clark R. Hager L.P. J. Biol. Chem. 1963; 238: 3086-3090Abstract Full Text PDF PubMed Google Scholar, 3Beckwith J.R. Hager L.P. J. Biol. Chem. 1963; 238: 3091-3094Abstract Full Text PDF PubMed Google Scholar). With his postdoctoral fellow Paul Shaw, Lowell had discovered an enzyme, chloroperoxidase, that was capable of utilizing chloride ion to chlorinate organic compounds (4Shaw P.D. Hager L.P. J. Biol. Chem. 1959; 234: 2565-2569Abstract Full Text PDF PubMed Google Scholar). Part of my work was to determine how this enzyme might be involved in the biosynthesis of caldariomycin. Apparently, the work became somewhat well known. This I learned when, toward the end of my Ph.D. work, I attended a FASEB meeting in Atlantic City to give a short presentation. As I walked into the conference center on the first day, a scientist passing by me stopped to look at my badge, hesitated a moment, and then commented “Beckwith... hmmm, oh yes, I remember... chlorination!” Despite this really quite limited chemical/biochemical notoriety, I became more and more fascinated by bacterial genetics in graduate school. I proceeded to do postdoctoral work in several bacterial genetics laboratories. I had not studied any genetics (not even high school biology) until I audited an introductory genetics course late in my graduate career. I am not sure I had even heard of the word “suppressor” when, unwittingly, I happened to work with strains that contained suppressor mutations in the course of a postdoctoral research project. In 1961, Arthur Pardee, my first postdoctoral mentor, gave me a project he had started that was to lead me to an appreciation of the power of suppressor analysis. Art had recently returned from a sabbatical at the Institut Pasteur in Paris, where along with François Jacob and Jacques Monod, he provided the crucial argument for repressor control of gene expression (5Pardee A.B. Jacob F. Monod J. J. Mol. Biol. 1959; 1: 165-178Crossref Scopus (523) Google Scholar). After Art's lab moved from Berkeley to Princeton University, he came up with an idea that was inspired by the Pasteur group's characterization of the lac operator. As initially conceived by Jacob and Monod, the operator, located at the beginning of the operon, served both as the site where transcription of the lac operon began and the site where the repressor acted to block this initiation (6Jacob F. Perrin D. Sánchez C. Monod J. C. R. Acad. Sci. (Paris). 1960; 250: 1727-1729Google Scholar). This model was based on two classes of mutations: operator-constitutive mutations (Oc) that were resistant to repression and Oo mutations that mapped early in the operon and eliminated the activity of both genes of the operon, lacZ and lacY. Because the two classes of mutations appeared to map in the same general region, the Pasteur group proposed that both types of mutations were in the same site, the lac operator. The operator was both the start site for transcription of the operon and the binding site for the repressor. This explanation presented a mechanism for gene regulation that was quite satisfying, probably because of its parsimony, as repression could be readily explained by direct competition for access to the operator between the repressor and the transcription machinery (RNA polymerase). Although these studies obviously included the concept that there was a start site for gene transcription, the term “promoter” had not even been coined at this point. Art, assuming that this characterization of the lac operator was correct (as did I), initiated a project that was, to my mind, ahead of its time in thinking about an issue related to gene expression. Proteins are found in cells in widely different amounts, some as high as 100,000 molecules/cell or as low as only a few molecules/cell. Art asked what determines each gene's level of expression, particularly constitutive genes, for which there is no regulation. He postulated that the DNA sequence of the “operators” for different constitutive genes could vary, leading to different potentials for expression for each gene (still accepting the notion that operators included transcription initiation sites). Art thought to obtain evidence for his hypothesis by selecting for revertants of the lacOo mutants that restored lac operon expression and determining whether they exhibited different levels of expression. He had already begun the project when he invited me to help him out with it. He found exactly the mutants he predicted; a set of lacOo revertants selected for growth on lactose each expressed different levels of β-galactosidase. We imagined that we had generated an array of mutant operators (think promoters) with different potentials for expression (7Pardee A.B. Beckwith J.R. Biochim. Biophys. Acta. 1962; 60: 452-454Crossref PubMed Scopus (5) Google Scholar). Art gave me the project, and I followed it up by setting out to define the locations of the mutations that restored the varying expression levels. I very quickly discovered to my surprise that the mutations that caused the restoration of lac operon function did not map to the operator site (8Beckwith J.R. Biochim. Biophys. Acta. 1963; 76: 162-164Crossref PubMed Google Scholar). They did not even map to the lac region but instead were located at different loci around the Escherichia coli chromosome. They were extragenic suppressor mutations. We had not even thought of that possibility because we had not envisioned that any kind of trans-effect could restore gene transcription. Today, the idea that promoters could vary in their activity (the concept behind this project) may seem self-evident and the error we had made a result of lack of rigor, but in those early days of molecular biology, molecular biologists had little sense of the kinds of complexity that we would meet as we began to explore issues of gene regulation more deeply. I continued this project in a second postdoctoral position in London in the laboratory of Bill Hayes at Hammersmith Hospital. While there, I was contacted by Sydney Brenner, who had followed my work. He suspected that the suppressors of the lacOo mutation I had isolated might define a new class of protein chain-terminating mutations. He invited me to spend some time in his laboratory in Cambridge, and our results led to a paper showing that the lacOo suppressors restored translation to what Sydney named the ochre chain-terminating codon (9Brenner S. Beckwith J.R. J. Mol. Biol. 1965; 13: 629-637Crossref Scopus (110) Google Scholar), later shown to be UAA (10Brenner S. Stretton A.O. Kaplan S. Nature. 1965; 206: 994-998Crossref PubMed Scopus (117) Google Scholar). This finding, along with other work with Oo mutations I had started in Pardee's lab and continued as a postdoctoral student in Hayes' lab, strongly suggested that these mutations were not in the operator (or promoter, if you will) (11Beckwith J.R. J. Mol. Biol. 1964; 8: 427-430Crossref PubMed Scopus (35) Google Scholar). Instead, they were chain-terminating or frameshift mutants early in the lacZ gene with strong polar effects on the downstream lacY gene. While still in Pardee's lab and at the suggestion of a student, John Gerhart, I had begun to look for other kinds of suppressor mutations of the Oo mutations. Because it was possible to select for restoration of the lacY gene by asking for growth on melibiose as carbon source, I could seek revertants that might eliminate the polarity effects of the mutation but not restore β-galactosidase. Among these I found one set of mutations that mapped to from the lac region (8Beckwith J.R. Biochim. Biophys. Acta. 1963; 76: 162-164Crossref PubMed Google Scholar). these were shown to be suppressor mutations in the gene for the transcription of coli C. C. Acad. Sci. S. PubMed Scopus Google Scholar). Art and I had started with about the Oo our of suppressors of the mutations had led to the of a second chain-terminating codon and new the of the protein required for transcription and results that the evidence to for a site both the repressor and transcription initiation site on the result of this project was that François Jacob me a postdoctoral position which I I had started my laboratory at Harvard in the I had from the work on Oo mutants led me to on the site of the transcription initiation region, the With John who came from Hayes' lab to do postdoctoral work with we were to obtain mutations of the lac operon Beckwith J.R. Biol. PubMed Scopus Google Scholar). We later that these mutations were in the binding site for the protein J. R. J. Mol. Biol. PubMed Scopus Google Scholar). by the of the characterization of Oo we to obtain suppressors of our new mutations. these out to be quite suppressors and to In the that the of that we were did not seem to for the of was not until I began to my research in the early from gene regulation to protein that suppressor analysis was to a in my of the we to how the of was the of gene idea was to in which the enzyme was at its to an of an We asked how of the protein was to of to define the region of the protein that was to that This work was begun the of We initiated this gene work with involved in the of and into the coli a that had been by at the Institut to my lab as a postdoctoral had time in laboratory and of the to my We several of this the protein and the protein Beckwith J. Acad. Sci. S. PubMed Scopus Google Scholar, Beckwith J. J. PubMed Google Scholar). served both as a for and as an for our most of the of to and in of β-galactosidase. 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Full Text PDF PubMed Scopus Google Scholar). that restored of the mutant in its the growth mapped to a gene they The mutations a later to be which is the of the machinery in the bacterial and is a of the protein in our two for suppressor mutations most of the of the protein machinery C. Beckwith J. J. Scholar, Beckwith J. PubMed Google Scholar, J. PubMed Scopus Google Scholar, C. Beckwith J. J. PubMed Scopus Google Scholar). We had initiated the genetic and analysis of the bacterial protein machinery that would be by in by Bill group R. D. J. 8: PubMed Scopus Google Scholar). of mutations in a particular gene are to to the function of that gene's product or of the in which that product is but this is not the result of suppressor and the in the of suppressor We learned this when we began to our analysis of protein by for suppressors of mutations in the gene. with a we selected for growth at high to mutations in genes that with The first mutation we was in the gene for a This led to that the protein with the protein of the an that the between protein synthesis and protein in cells S. Beckwith J. Full Text PDF PubMed Scopus Google Scholar). of the for suppressors in my and other that mutations in different genes involved in protein synthesis and even the of low levels of an of protein restored growth at high to mutants C. Beckwith J. J. PubMed Google Scholar, J. 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The in my lab of the for was not We had been how were into for some time as a result of our studies on the of the enzyme This enzyme two for its and student had mutants in the sequence of that its to the and caused it to in the In this was of activity S. J. Beckwith J. J. PubMed Google Scholar). We then that this lack of activity was to the of its Beckwith J. J. 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PubMed Scopus Google Scholar). the if one were to there were no in the would have been of the of the first our that the of caused to in appeared to be with that We in with their were any that the mutations eliminated the activity that in the and could in my lab that the was not In our studies that the to in other in the mutant strains not on the of but on the of The in the in the F. Beckwith J. J. PubMed Scopus Google Scholar). As this work one of the it the of a in the lab from the of a had to studies on in particular a protein with He suggested that we the sequence of it would be and whether the enzyme would into its in the mutant We followed this suggestion and found that of could in the of the mutant but not in a D. Beckwith J. PubMed Scopus Google Scholar). of a with an a of because there were to to in the for both research and by the of the we asked whether we could coli to even levels of first to the for in coli was to the mutation with a mutation the (or the of the even the mutant did not as we had eliminated the two required to the enzyme, in the for these a student, found that of to the their we the of they made of in the those in the mutant F. Beckwith J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). While working with the we that when the mutant was on growth not mutations that restored growth at high mutations mapped to the they were extragenic suppressor mutations located in the gene which a bacterial gene is based on the for this enzyme, named because it as well as of the suppressor mutations with our that they had the that it could to cells D. J. Beckwith J. PubMed Scopus Google Scholar, D. D. Beckwith J. Mol. Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In we had suppressor mutations that generated a new for to the function of could be to that of a of was not as the activity of its to we found with our that a which exhibited growth was quite for high levels of in the coli F. Beckwith J. Acad. Sci. S. PubMed Scopus Google Scholar). We later suppressor mutations in that could even the growth of a mutant both and the S. Beckwith J. Acad. Sci. S. PubMed Scopus Google Scholar). In these strains the suppressor mutations appeared to the that it could or The appeared to have to its by We had first suppressor mutations by the that the mutant on where it could not at high that could I had how suppressor analysis could it to me that we might be to suppressor analysis to other for could be of in of biological and for We imagined that these might be generated in several of of which have been on the coli by a approach PubMed Scopus Google of new of to that did not of one of the other or of enzyme to for a different of or other of into the or seek different types of suppressors from those with the we strains that were the any one of which could to and The mutant could not but could be for by a a postdoctoral and isolated and suppressor mutations. our the mutations mapped not to genes involved in but instead to genes involved in the initiation of DNA and we quickly that a of the suppressors in genes for DNA and the we were was that we were selecting for the activity of which is for for DNA studies with the suppressor mutations in and that they the restoration of growth by of R. S. Beckwith J. Acad. Sci. S. PubMed Scopus Google Scholar). Apparently, the levels of by its by which was to have very activity for these studies led to the of a mechanism that the synthesis of with the initiation of DNA S. J. Beckwith J. J. PubMed Scopus Google Scholar). We our for suppressors of the mutant by selecting for mutations that would restore growth to a We did this to the very suppressor mutations that were in a of other mutations. With the we suppressor mutations that restored of which mapped to the gene for the enzyme is the of the that is to of protein as and the activity of its two in to these two to their the to the activity of these enzyme is to be capable of in J.R. C. Biophys. PubMed Scopus Google Scholar). in genetic evidence in this very project that the in results in the of the of (or which then to S. J. and J. one of the mutations a with a the that of the of the protein in evidence that this both as an and a the enzyme and a in a R. C. PubMed Scopus Google Scholar). We have not at the end of this but it that our suppressor mutations have or the activity of that the cells or Because we found that the suppressors restore growth to a that is the to be to in the that to of have been a for genetic to biological mutations that restore a selected to cells in a particular gene may give on the function of that gene or of the mutation the ochre the power of bacterial one strains for of suppressor mutations that of the and that are to restore a may an protein to an not only on how of new but the function of J. PubMed Google Scholar). some of the most results of suppressor are the In our work, more suppressors that were ochre codon suppressors of lacOo and mutations in a to obtain mutations in protein and mutations DNA or in a gene for a of to be but it that if you select for suppressors of mutations an there is a you will even it may not be what you In some these kinds of thinking that is that the is a more of and we had one or more we and it by we might not have The suppressor approach and the of but results one of the for and In years, it has been a to my lab suppressor mutations that in well biochemical of these or new as protein The biochemistry and the that we had to in graduate school had from the I had my to but as we up these suppressor of the began to I to up the of to in my the biochemical and that I had and to to my what the that few are up I of the of who had been Lowell Ph.D. and I to my Ph.D. at the of I Lowell to him I had an for and I had not that had discovered a we were working Lowell quickly me out as a student with he had been the one who had discovered the enzyme L.P. J. Chem.
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