Rambling and Scrambling in Bacterial Transformation— a Historical and Personal Memoir
Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000*6'(0d;, 百拇医药
In these retrospective comments I hope to accomplish several objectives. I shall briefly review the early history of bacterial transformation, with an emphasis on breakthroughs and personalities. Then, I shall describe my own role in the development of the field with its curious dependence on the interplay of rational experimentation and serendipity. In this context, I shall discuss the importance of mentoring, cooperation, chance, and revelation. My title refers to my good fortune in having support that enabled me to ramble over various aspects of transformation, although from time to time I was obliged to scramble to obtain grant funding, to produce publications, and to generally compete in the field. To achieve within these few pages the multiple objectives mentioned above, I shall be rambling and scrambling once again.*6'(0d;, 百拇医药
EARLY HISTORY*6'(0d;, 百拇医药
Fred Griffith in 1928 reported that heat-killed encapsulated pneumococci could transfer the ability to make a capsule and, hence, to infect mice, when injected together with live, unencapsulated and nonpathogenic pneumococci (18). He termed the phenomenon "transformation." At the time, it was not realized that bacteria contained genes, let alone that DNA was the genetic material. Griffith conjectured that a seed of capsular polysaccharide was perhaps transferred from the heat-killed bacteria, but he went one step further in wondering whether an enzymatically active protein might be the agent transferred. Few people know that Griffith, a medical doctor, was killed, together with a colleague at the Ministry of Public Health, during the German bombing of London in 1941 (5).
Soon after Griffith's discovery, Oswald Avery at the Rockefeller Institute in New York took up the problem, and most of the subsequent developments were connected to his laboratory. In the 1930s Dawson and Sia achieved transformation in vitro as opposed to in mice (8), and Alloway was able to extract the active principle from heat-killed cells (2). However, proof that the transforming principle was DNA awaited the landmark paper of 1944 (3). That work had enormous impact in demonstrating that the genetic material consisted of DNA.?jy, 百拇医药
QUANTITATIVE TRANSFORMATION?jy, 百拇医药
Although capsular transformation was effective in revealing the phenomenon of transformation, it depended on screening for smooth (encapsulated cell) and rough (unencapsulated cell) colonies. For precise quantitation, selectable markers are preferable. Rollin Hotchkiss, a member of Avery's group who carried on the investigation of pneumococcal transformation at Rockefeller, obtained drug resistance markers and devised methods for the quantitative measurement of transformation frequencies. The value of quantitative measurements for understanding molecular mechanisms of transformation, regulation of recipient cell competence, processes of DNA degradation, and genetic analysis by transformation is apparent from the results of Hotchkiss and those influenced by his laboratory. In 1957, using a streptomycin resistance marker, Hotchkiss showed that transformants increased linearly with added marker DNA, until a saturation level was obtained, thereby demonstrating a discrete number of DNA uptake sites on the recipient cells (22). Competence for DNA uptake, measured as transformation frequency, was found to vary systematically during the culture growth cycle (21). Later it was shown that competence depended on accumulation of an extracellular polypeptide (20, 55).
Particularly noteworthy was the demonstration by Marmur and Lane of strand separation on denaturation of the native, duplex DNA and the renaturation of its transforming activity after thermal denaturation by annealing the separated strands at submelting temperatures (44). Although this work was accomplished at Harvard University, Marmur had been a postdoctoral fellow and Lane had been a technician in the Hotchkiss laboratory. Another outstanding contribution from that laboratory was Maury Fox's discovery of the eclipse of donor marker transforming activity immediately after DNA uptake. When DNA is extracted from recipient cells just after uptake, it is unable to transform tester cells, but the ability is fully restored within 10 min at 37°C (14). Several investigators, including Fox (13), Goodgal and Herriott (17), and Lerman and Tolmach (39), pioneered the use of 32P-labeled DNA to demonstrate the physical uptake of DNA in transformation. Sol Goodgal was instrumental in organizing the first Transformation Meeting in 1956. It is to Leonard Lerman that we owe the name of the Wind River Conference; he organized the Transformation Meeting at the Wind River Ranch, Estes Park, Colo., in 1959.
My own work in transformation began in 1955, as the first graduate student of Rollin Hotchkiss. Rollin fostered self-determination in his students, and he agreed to my proposal to study recombination between nutritional mutants—to see if we could analyze genetic fine structure by transformation. For my thesis I isolated and analyzed eight mutants unable to use the sugar maltose (32). Even from this small number of mutants, several significant facts emerged. Three maltose-negative mutations were multisite and ultimately shown to be deletions; one covered the entire locus, and the other two overlapped from opposite ends. Their transformation efficiencies depended inversely on deletion length. (Transformation efficiencies are expressed as the ratio of a maltose marker transformation frequency to that of a standard streptomycin resistance marker to account for variations in culture competence.) Five mutations recombined with each other and appeared to be mutated at single sites; they presumably corresponded to base changes. What was surprising was that these single-site mutations showed transformation efficiencies with wild-type DNA that varied over a 10-fold range. Later, such variation in transformation efficiency was shown to result from differential action of a base mismatch repair system (11, 25). We were able to measure production of the enzyme amylomaltase, product of the malM gene, immediately after DNA uptake by the various mutants. Surprisingly, the amount of enzyme produced was not dependent on the amount of DNA taken up but was proportional, rather, to the transformation efficiency of the marker (33). This, too, was a manifestation of the eclipse; not only was the incoming DNA unable to transform another cell, but it was also unable to direct production of a protein prior to its integration.
MOLECULAR MECHANISM OF DNA UPTAKEmoz-%ew, 百拇医药
In searching for a molecular explanation of the eclipse phenomenon, my initial hypothesis was that DNA was fragmented during entry and that the double-stranded fragments were later connected, presumably while aligned on a recipient chromosomal template, to restore both transforming and transcribing activity. My idea was to label the donor DNA with 32P, break open recipient cells after uptake, and look for 32P-DNA fragments with a methylated-albumin coated kieselguhr (MAK) column. Mandell and Hershey had recently shown that fragments of T-even coliphage DNA were separated on MAK columns in a size-dependent manner (43). However, when I carried out the experiment, ~ 40% of the 32P counts were immediately eluted and only ~ 5% were eluted by the salt gradient, which eluted even the high-molecular-weight recipient DNA; most of the radioactivity was missing, and I was puzzled over the low recovery. That evening I had a moment of "eureka": the remaining counts must have been retained by the column! On returning to the laboratory, I detected, in fact, radioactivity still on the MAK column. When eluted with 0.1 N NaOH, the counts amounted to ~ 50% of the total. Upon more carefully reexamining the paper by Mandell and Hershey, I saw that they had mentioned in passing that denatured DNA sticks to the column. Apparently, the DNA was converted to single strands on uptake. This conclusion was soon confirmed by separation of newly entered and resident DNA in a CsCl density gradient that separated single- from double-stranded DNA.
In reporting my findings in 1962, I proposed that one strand of the duplex DNA was degraded as the other strand entered the cell (23). Such conversion to a single-stranded form explained the eclipse, inasmuch as denatured DNA was known to be poorly taken up by cells and could not serve as a template for transcription. Furthermore, conversion to a single strand would facilitate interaction with the recipient chromosome. In fact, radioactive label passed rapidly from single strands into double stranded, presumably chromosomal DNA (23). Later, it was shown by density labeling that single-stranded segments of donor DNA were indeed incorporated into the transformed chromosome (15).xl}78, http://www.100md.com
I considered the determination of the fate of DNA on entry to be an important new finding for the understanding of transformation. Therefore, I was quite surprised to read, afterward, in a thesis of Pierre Schaeffer published in 1961 the following concluding statement, "Une hypothèse est proposée d'après laquelle la pénétration de la molécule d'ADN dans la bactérie requiert une structure à double chaîne, alors que la synapse, dont dépend l'intégration, requiert au contraire une chaîne polynucléotidique unique" (50a). Translated, this reads "A hypothesis is proposed according to which penetration of the DNA molecule into the bacterium requires a double-stranded structure, while synapsis, on which integration depends, conversely requires a single polynucleotide strand." Schaeffer was a medical doctor, a mature investigator, who had for some years investigated the competition between different DNAs in the transformation of various bacterial species. As was the custom in France, such researchers often took an additional degree of Docteur Sciences later in life. Schaeffer based the uptake part of his hypothesis on his own experimental work, but the conclusion that a single strand was needed for integration was the result of careful thought. He reasoned that only single-stranded DNA, with its bases exposed, could readily interact with recipient DNA. It was sobering for me that these thoughts were presented before publication of my own work. In fact, it is possible that they were expressed during Schaeffer's thesis defense, which I attended in Paris, France, in 1958, when I was working in the laboratory of François Gros, another very fine mentor, at the Pasteur Institute. Despite its proposal on theoretical grounds, I comforted myself in the novelty of my work showing that the strand conversion process occurred during DNA entry. However, I never forgot that scientific understanding is a large edifice to which many contribute.
Because the amount of low-molecular-weight 32P found in the cells approximately equalled the amount in single strands, it appeared that one strand was degraded as the other strand entered (23). However, Morrison and Guild showed that the amount of DNA breakdown inside the cell depends on the size and quality of the donor DNA; more intact DNA gave fewer breakdown products so that a larger proportion of DNA taken up was in the form of single strands (45). In the early 1970s, they and Bill Greenberg and I independently discovered that the breakdown products of the strand complementary to the one that enters appear external to the cells (28, 46). Early on, I hypothesized that a cellular DNase acted on donor DNA to facilitate entry of one strand by degrading the opposite strand (23). Investigation of the pneumococcal nucleases revealed two enzymes active on DNA (27). In vitro, EndA, which was found to be membrane bound (31), degraded DNA to give oligonucleotide products, and ExoA (like ExoIII of Escherichia coli) gave mononucleotides and single strands. To see which, if either, enzyme facilitated entry, I sought mutants lacking them. Useful in this approach was a method for detecting colonies that lacked nuclease activities by growing them in agar containing DNA and methyl green, which stains only intact DNA (26). Colonies around which the DNA was digested showed colorless zones that were absent around mutant colonies (Fig. 1).
fig.ommitted+#k], http://www.100md.com
Nuclease detection in nutrient agar plates containing DNA and methyl green. Cells of S. pneumoniae wild-type strain R6 (endA+ exoA+) and mutant strain 593 (endA1 exoA+) were plated in a ratio of 1:10 and incubated at 37°C for 30 h.+#k], http://www.100md.com
Before discussing another technique helpful in analyzing the DNase mutants, I shall divert briefly to discuss mentoring. I personally was fortunate to have as a mentor Rollin Hotchkiss, who not only advised me wisely as a graduate student but also remained a friend and inspiration throughout my life. Still, I recall that when I first began to look for nutritional mutants of pneumococcus, the medium used in the Hotchkiss laboratory was only semidefined—a good wallop of yeast extract was added to the otherwise defined mix. I set about to find out what was essential in the yeast extract, and after mastering several new techniques, was able to identify the substance as glutamine. "Aha!" said Rollin, "Adams [and Roe (1), who formulated the medium] did add glutamine, but we thought it too unstable to make a difference, and to get good growth we added yeast extract." Earlier, I had a similar experience in my senior research at college. I was to obtain and study petite mutants of yeast, but I could not grow the cells to any significant turbidity in the medium for which I was given the formula. After two weeks of frustration I returned to my mentor, and he discovered an error in his formulation that made the medium a hundredfold too dilute. Next, it was my turn. When a postdoctoral fellow, Allan Rosenthal, who was characterizing DNase mutants, pointed out to me the potential utility of revealing enzyme activity in situ after electrophoresis of extracts in a denaturing gel, I told him to forget about it because it would be impossible to remove sodium dodecyl sulfate (SDS) from the enzyme to renature it. However, when I was away from the laboratory for a week, he tried the experiment, and now we all know how useful such enzyme detection after denaturing electrophoresis can be (50). The importance of mentoring in developing new generations of scientists cannot be overstated; however, skepticism on the part of a student is also warranted.
The in situ detection method helped show conclusively that null mutants of EndA reduced uptake and transformation by a factor of a thousand (35). ExoA mutants had no effect on transformation. Both its membrane location and finding that the external products of in vivo degradation were oligonucleotides (34) supported a role for EndA in bringing one strand into the cell as it degraded the other.3kw'c{^, http://www.100md.com
fig.ommitted3kw'c{^, http://www.100md.com
DNases in wild-type and mutant strains of S. pneumoniae detected after electrophoresis in polyacrylamide gels containing SDS and DNA. Wells contained 25 µg of protein from SDS lysates of S. pneumoniae as follows: wild-type R6 (a), exoA5 (b), endA1 (c), and endA14 (d). (e) This well contained pancreatic DNase I and micrococcal nuclease. Electrophoresis was anodal in the downward direction. After washing to remove SDS, the gel was incubated for 64 h, stained with ethidium bromide, and photographed with UV illumination. Size markers (in thousands) were from a parallel lane of proteins stained with Coomassie blue (not shown). Dark bands correspond to DNases.
DpnI AND DpnII RESTRICTION SYSTEMS8+7:{, 百拇医药
Elimination of the major DNases from the pneumococcal cell still left a weak DNase activity, which gave no acid-soluble products but could be detected by its reduction of DNA viscosity. This enzyme turned out to be DpnI, an unusual restriction endonuclease that cleaves only DNA with adenine methylated at GATC sites (29, 56). Normally, restriction enzymes cleave unmethylated DNA, and DNA is protected from cleavage by methylation. The contrary is true for DpnI. Its discovery was fortuitous, in that E. coli DNA, which happens to be methylated at GATC sites, was used as the substrate for testing DNase activity, and not DNA from the pneumococcal strain, which was unmethylated. Interesting as this enzyme was, it was only the first part of the story.8+7:{, 百拇医药
The concept of serendipity, proposed by Horace Walpole in 1754, derives from an ancient tale entitled The Three Princes of Serendip. In the story, three young men from Serendip, which is an old name for Sri Lanka, set off on a trip abroad and encountered many adventures. By a combination of chance and sagacity, they fared well in their travels. In a personal instance of serendipity, a culture of a strain that should have made DpnI produced instead a different enzyme, DpnII, which is complementary to DpnI and cleaves only at unmethylated GATC. We wondered if by some genetic switch, perhaps related to DNA methylation, the DpnI-producing strain had converted itself into a DpnII-producing strain (30). This turned out not to be the case. This DpnII strain was a contaminant streptococcus that outgrew the original inoculum. However, different isolates of pneumococcus from nature could produce either DpnI or DpnII (47), and the responsible genes were found to constitute mutually replaceable genetic cassettes (38). Similar findings for capsular genes were foreshadowed by this result. An additional unexpected feature of the DpnII system was the presence of two methyltransferases (9), one of which preferentially methylated single-stranded DNA and was able to prevent the restriction of unmethylated plasmid entering a cell by the transformation pathway (6).
CLONING IN STREPTOCOCCUS PNEUMONIAE2o, http://www.100md.com
Among the tools and methods developed in my laboratory, one of the most important was the pneumococcal cloning system using derivatives of the plasmid pMV158 (53). We were forced to turn to this system by our inability to clone many important pneumococcal genes, such as malM, endA, and hexA, in the available E. coli systems, apparently due to the strong promoters of S. pneumoniae and the toxicity of the gene products in E. coli (52). The pneumococcal cloning system was elaborated in a relatively short time by the intense effort of Diane Stassi, Paloma Lopez, and Manolo Espinosa. This followed an initial suggestion by Francis Barany in a telephone conversation with me. Walter Guild provided pMV158; he obtained it from its discoverer, Vickers Burdett. There again, we see the interwoven undergarment of scientific progress.2o, http://www.100md.com
Cloning in pneumococcus not only allowed the analysis of otherwise unclonable genes, but it also provided a facile method for exchanging chromosomal segments present in a recombinant plasmid. Such exchange resulted from chromosomal facilitation of plasmid establishment, in which an entering linear strand from a recombinant plasmid must interact with the chromosome in order to circularize (42). That the DpnI and DpnII system genes resided at the same position in the chromosome was first discovered by the substitution of a DpnI gene segment on transformation of a DpnI cell by a recombinant plasmid containing the DpnII gene segment (38). Cloning in S. pneumoniae of a chromosomal segment containing sul-d, a mutation which conferred sulfonamide resistance, enabled us to carry out the first genetic analysis of folate biosynthesis in any organism (41).
DNA MISMATCH REPAIR%v, 百拇医药
A major thrust of my work has been in DNA mismatch repair. This phenomenon was first discovered in S. pneumoniae, a decade before it was found in E. coli, by examination of transformation frequencies of different mutational markers at the amylomaltase locus. In my doctoral work (circa 1957) I had found different mutations to be transformed at characteristic frequencies or efficiencies (32). Perhaps influenced by both the periodic table, with its categories of elements, and astrophysics, with its grouping of stars by luminosity, I undertook a classificatory approach, grouping mutations of similar transformation frequency. By 1965, I had over 70 single-site mutations in the malM gene, which clustered in four efficiency groups, and I had found that reciprocal transformation of markers gave identical frequencies (24, 25). These findings allowed the attribution of the four efficiency groups, I to IV (ratios of 0.05, 0.2, 0.5, and 1.0), to the six possible classes of mutations giving base mismatches. On the basis of mutagenic origin and changes at the same site, I proposed the following correspondence of efficiency class to mutations giving rise to mismatches in the heteroduplex product of transformation: I, ATGC, GCAT; II, ATTA; III, GCCG; IV, ATCG, GCTA (25). These proposed assignments proved to be generally correct when cloning and sequencing of the malM gene was achieved (37).
I had been impressed by the colligative properties of the efficiency groups with respect to parameters other than transformation efficiency. Marker sensitivity to UV light (24, 40) and speed of recovery from eclipse (16) were both inversely correlated with efficiency. Both effects result from dependence of donor marker excision on single-strand breaks prior to ligation of the donor segment. Repair of UV damage produces subsequent strand breaks that result in additional mismatch repair (36). The apparent earlier recovery of low-efficiency markers reflects their later loss by repair, as was evident from early peaking of their recovery (51). These correlations helped support a model for mismatch repair in which an entire donor strand segment that would normally be integrated in transformation is excluded from integration (7, 25, 36).h^r#5]x, 百拇医药
With important contributions by various investigators, the different transformation efficiencies and other properties were ascribed to a cellular mismatch repair system. A major contributor was Harriett Ephrussi-Taylor. In 1958, she invited me to give a talk at her laboratory in Gif, near Paris. Harriett was very interested in the malM mutant transformation frequencies and recombination results. With Michel Sicard she devised another gene system, based on aminopterin resistance, for analyzing mismatch repair (11). She was the first to propose that the pneumococcal system removed mismatches by an enzymatic mechanism (10). Unfortunately, Harriett died soon afterward; however, Michel and, later, his student Jean-Pierre Claverys, continued to contribute importantly to the field. Our discovery of the hex mutants, in which all markers transformed at high efficiency (26), supported the concept of an enzymatic system for detecting and repairing DNA mismatches, in which large tracts of donor DNA containing the mismatched base were removed. Using such hex mutants, Tiraby and Fox showed that the mismatch repair mechanism also prevented spontaneous mutations (54). T. S. Balganesh in my laboratory (4) was the first to clone a mismatch repair gene, hexA, and that enabled determination of its nucleotide sequence (48). Sequence comparison with mismatch repair genes from other species indicated that the system was universally found in living cells (19, 48, 49). Strikingly, a homologous system in human cells prevents colon cancer (12) (Fig. 3).
fig.ommitted&:$gr, 百拇医药
. From left to right, Bill Greenberg, Sanford Lacks, and Sylvia Springhorn examine autoradiograms that reveal the DNA sequence of genes encoding the restriction enzymes of S. pneumoniae.&:$gr, 百拇医药
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.&:$gr, 百拇医药
ACKNOWLEDGMENTS&:$gr, 百拇医药
I thank the scientists who were my mentors, my collaborators, and those who came to work in my laboratory as visiting scientists, postdoctoral associates, students, and technical associates. Although everyone made important contributions, I regret that I was able to mention only a few by name in this memoir. I am indebted to two associates who worked with me for very long periods, Bill Greenberg and Sylvia Springhorn; they are pictured in&:$gr, 百拇医药
REFERENCES&:$gr, 百拇医药
Adams, M. H., and A. S. Roe. 1945. A partially defined medium for cultivation of pneumococcus. J. Bacteriol. 49:401-409.
Alloway, J. L. 1931. The transformation in vitro of R pneumococci into S forms of different specific types by the use of filtered pneumococcus extracts. J. Exp. Med. 55:91-99.}5e/, http://www.100md.com
Avery, O. T., C. M. MacLeod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J. Exp. Med. 89:137-158.}5e/, http://www.100md.com
Balganesh, T. S., and S. A. Lacks. 1985. Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: Cloning and expression of the hexA gene. J. Bacteriol. 162:979-984.}5e/, http://www.100md.com
British Medical Journal. 1941. Obituary. Br. Med. J. 1941-I:691.}5e/, http://www.100md.com
Cerritelli, S., S. S. Springhorn, and S. A. Lacks. 1989. DpnA, a methylase for single-strand DNA in the DpnII restriction system, and its biological function. Proc. Natl. Acad. Sci. USA 86:9223-9227.}5e/, http://www.100md.com
Claverys, J. P., and S. A. Lacks. 1986. Heteroduplex deoxyribonucleic acid base mismatch repair in bacteria. Microbiol. Rev. 50:133-165.
Dawson, M. H., and R. H. P. Sia. 1931. In vitro transformation of pneumococcal types. I. A technique for inducing transformation of pneumococcal types in vitro. J. Exp. Med. 54:681-699.qtk, 百拇医药
de la Campa, A. G., P. Kale, S. S. Springhorn, and S. A. Lacks. 1987. Proteins encoded by the DpnII restriction gene cassette: two methylases and an endonuclease. J. Mol. Biol. 196:457-469.qtk, 百拇医药
Ephrussi-Taylor, H., and T. C. Gray. 1966. Genetic studies of recombining DNA in pneumococcal transformation. J. Gen. Physiol. 49:part2.,211-231.qtk, 百拇医药
Ephrussi-Taylor, H., A. M. Sicard, and R. Kamen. 1965. Genetic recombination in DNA-induced transformation of pneumococcus. I. The problem of relative efficiency of transforming factors. Genetics 51:455-475.qtk, 百拇医药
Fishel, R., M. K. Lescoe, M. R. S. Rao, N. G. Copeland, N. A. Jenkins, J. Garber, M. Kane, and R. Kolodner. 1993. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75:1027-1038.
Fox, M. S. 1957. Deoxyribonucleic acid incorporation by transformed bacteria. Biochim. Biophys. Acta 26:83-85.$il(in$, 百拇医药
Fox, M. S. 1960. Fate of transforming deoxyribonucleate following fixation by transforming bacteria. II. Nature 187:1004-1006.$il(in$, 百拇医药
Fox, M. S., and M. K. Allen. 1964. On the mechanism of deoxyribonucleate integration in pneumococcal transformation. Proc. Natl. Acad. Sci. USA 52:412-419.$il(in$, 百拇医药
Ghei, O. K., and S. A. Lacks. 1967. Recovery of donor deoxyribonucleic acid marker activity from eclipse in pneumococcal transformation. J. Bacteriol. 93:816-829.$il(in$, 百拇医药
Goodgal, S. H., and R. M. Herriott. 1957. Studies on transformation of Haemophilus influenzae, p. 336-340. In W. D. McElroy and B. Glass (ed.), The chemical basis of heredity. Johns Hopkins Press, Baltimore, Md.$il(in$, 百拇医药
Griffith, F. 1928. The significance of pneumococcal types. J. Hyg. 27:113-159.$il(in$, 百拇医药
Haber, L. T., P. P. Pang, D. I. Sobell, J. A. Mankovich, and G. C. Walker. 1988. Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae. J. Bacteriol. 170:197-202.
Håverstein, L. S., G. Coomaraswamy, and D. A. Morrison. 1995. An unmodified heptadecapeptide induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 92:11140-11144.]]v^, 百拇医药
Hotchkiss, R. D. 1954. Cyclical behavior in pneumococcal growth and transformability occasioned by environmental changes. Proc. Natl. Acad. Sci. USA 40:49-55.]]v^, 百拇医药
Hotchkiss, R. D. 1957. Criteria for the quantitative genetic transformation of bacteria, p. 321-335. In W. D. McElroy and B. Glass (ed.), The chemical basis of heredity. Johns Hopkins Press, Baltimore, Md.]]v^, 百拇医药
Lacks, S. 1962. Molecular fate of DNA in genetic transformation of pneumococcus. J. Mol. Biol. 5:119-131.]]v^, 百拇医药
Lacks, S. 1965. Genetic recombination in pneumococcus, p. 159-164. In M. Kohoutova and J. Hubacek (ed.), The physiology of gene and mutation expression. Academia, Prague, Czechoslovakia.]]v^, 百拇医药
Lacks, S. 1966. Integration efficiency and genetic recombination in pneumococcal transformation. Genetics 53:207-235.
Lacks, S. 1970. Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J. Bacteriol. 101:373-383.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1967. Deoxyribonucleases of Pneumococcus. J. Biol. Chem. 242:3108-3120.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1973. Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 114:152-163.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1975. A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA. J. Biol. Chem. 250:4060-4066.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114:153-168.r.f)gs, 百拇医药
Lacks, S., and M. Neuberger. 1975. Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 124:1321-1329.
Lacks, S., and R. D. Hotchkiss. 1960. A study of the genetic material determining an enzyme activity in pneumococcus. Biochim. Biophys. Acta 39:508-517./v*, 百拇医药
Lacks, S., and R. D. Hotchkiss. 1960. Formation of amylomaltase after genetic transformation of pneumococcus. Biochim. Biophys. Acta 45:155-163./v*, 百拇医药
Lacks, S., B. Greenberg, and M. Neuberger. 1974. Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc. Natl. Acad. Sci. USA 71:2305-2309./v*, 百拇医药
Lacks, S., B. Greenberg, and M. Neuberger. 1975. Identification of a deoxyribonuclease implicated in genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 123:222-232./v*, 百拇医药
Lacks, S. A. 1989. Generalized DNA mismatch repair—its molecular basis in Streptococcus pneumoniae and other organisms, p. 325-339. In L. O. Butler, C. Harwood, and B. E. B. Moseley (ed.), Genetic transformation and expression. Intercept, Wimbourne, England./v*, 百拇医药
Lacks, S. A., J. J. Dunn, and B. Greenberg. 1982. Identification of base mismatches recognized by the heteroduplex-DNA-repair system of Streptococcus pneumoniae. Cell 31:327-336.
Lacks, S. A., B. M. Mannarelli, S. S. Springhorn, and B. Greenberg. 1986. Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism. Cell 46:993-1000.jz(-{oi, 百拇医药
Lerman, R. S., and L. J. Tolmach. 1957. Genetic transformation. I. Cellular incorporation of DNA accompanying transformation in pneumococcus. Biochim. Biophys. Acta 28:68-82.jz(-{oi, 百拇医药
Litman, R. M. 1961. Genetic and chemical alterations in the transforming DNA of pneumococcus caused by ultraviolet light and by nitrous acid. J. Chim. Phys. 58:997-1004.jz(-{oi, 百拇医药
Lopez, P., M. Espinosa, B. Greenberg, and S. A. Lacks. 1987. Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme. J. Bacteriol. 169:4320-4326.jz(-{oi, 百拇医药
Lopez, P., M. Espinosa, D. L. Stassi, and S. A. Lacks. 1982. Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology. J. Bacteriol. 150:692-701.
Mandell, J. D., and A. D. Hershey. 1960. Anal. Biochem. 1:66-.$1a[, 百拇医药
Marmur, J., and D. Lane. 1960. Strand separation and specific recombination in deoxyribonucleic acids: biological studies. Proc. Natl. Acad. Sci. USA 46:453-461.$1a[, 百拇医药
Morrison, D. A., and W. R. Guild. 1972. Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor. J. Bacteriol. 112:1157-1168.$1a[, 百拇医药
Morrison, D. A., and W. R. Guild. 1973. Breakage prior to entry of donor DNA in pneumococcus transformation. Biochim. Biophys. Acta 299:545-556.$1a[, 百拇医药
Muckerman, C. C., S. S. Springhorn, B. Greenberg, and S. A. Lacks. 1982. Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae. J. Bacteriol. 152:183-190.$1a[, 百拇医药
Priebe, S. D., S. M. Hadi, B. Greenberg, and S. A. Lacks. 1988. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 170:190-196.
Reenan, R. A. G., and R. D. Kolodner. 1992. Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins Genetics 132:963-973.syhp, 百拇医药
Rosenthal, A. L., and S. A. Lacks. 1977. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal. Biochem. 80:76-90.syhp, 百拇医药
Schaeffer, P. 1961. Doctoral thesis. University of Paris, Paris, France.syhp, 百拇医药
Shoemaker, N. B., and W. R. Guild. 1974. Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation. Mol. Gen. Genet. 128:283-290.syhp, 百拇医药
Stassi, D. L., and S. A. Lacks. 1982. Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae. Gene 18:319-328.syhp, 百拇医药
Stassi, D. L., P. Lopez, M. Espinosa, and S. A. Lacks. 1981. Cloning of chromosomal genes in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 78:7028-7032.syhp, 百拇医药
Tiraby, G., and M. S. Fox. 1973. Marker discrimination in transformation and mutation of pneumococcus. Proc. Natl. Acad. Sci. USA 70:3541-3545.syhp, 百拇医药
Tomasz, A., and R. D. Hotchkiss. 1964. Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc. Natl. Acad. Sci. USA 51:480-487.syhp, 百拇医药
Vovis, G. F., and S. Lacks. 1977. Complementary action of restriction enzymes Endo R. DpnI and Endo R. DpnII on bacteriophage fl DNA. J. Mol. Biol. 115:525-538.(Sanford A. Lacks)
In these retrospective comments I hope to accomplish several objectives. I shall briefly review the early history of bacterial transformation, with an emphasis on breakthroughs and personalities. Then, I shall describe my own role in the development of the field with its curious dependence on the interplay of rational experimentation and serendipity. In this context, I shall discuss the importance of mentoring, cooperation, chance, and revelation. My title refers to my good fortune in having support that enabled me to ramble over various aspects of transformation, although from time to time I was obliged to scramble to obtain grant funding, to produce publications, and to generally compete in the field. To achieve within these few pages the multiple objectives mentioned above, I shall be rambling and scrambling once again.*6'(0d;, 百拇医药
EARLY HISTORY*6'(0d;, 百拇医药
Fred Griffith in 1928 reported that heat-killed encapsulated pneumococci could transfer the ability to make a capsule and, hence, to infect mice, when injected together with live, unencapsulated and nonpathogenic pneumococci (18). He termed the phenomenon "transformation." At the time, it was not realized that bacteria contained genes, let alone that DNA was the genetic material. Griffith conjectured that a seed of capsular polysaccharide was perhaps transferred from the heat-killed bacteria, but he went one step further in wondering whether an enzymatically active protein might be the agent transferred. Few people know that Griffith, a medical doctor, was killed, together with a colleague at the Ministry of Public Health, during the German bombing of London in 1941 (5).
Soon after Griffith's discovery, Oswald Avery at the Rockefeller Institute in New York took up the problem, and most of the subsequent developments were connected to his laboratory. In the 1930s Dawson and Sia achieved transformation in vitro as opposed to in mice (8), and Alloway was able to extract the active principle from heat-killed cells (2). However, proof that the transforming principle was DNA awaited the landmark paper of 1944 (3). That work had enormous impact in demonstrating that the genetic material consisted of DNA.?jy, 百拇医药
QUANTITATIVE TRANSFORMATION?jy, 百拇医药
Although capsular transformation was effective in revealing the phenomenon of transformation, it depended on screening for smooth (encapsulated cell) and rough (unencapsulated cell) colonies. For precise quantitation, selectable markers are preferable. Rollin Hotchkiss, a member of Avery's group who carried on the investigation of pneumococcal transformation at Rockefeller, obtained drug resistance markers and devised methods for the quantitative measurement of transformation frequencies. The value of quantitative measurements for understanding molecular mechanisms of transformation, regulation of recipient cell competence, processes of DNA degradation, and genetic analysis by transformation is apparent from the results of Hotchkiss and those influenced by his laboratory. In 1957, using a streptomycin resistance marker, Hotchkiss showed that transformants increased linearly with added marker DNA, until a saturation level was obtained, thereby demonstrating a discrete number of DNA uptake sites on the recipient cells (22). Competence for DNA uptake, measured as transformation frequency, was found to vary systematically during the culture growth cycle (21). Later it was shown that competence depended on accumulation of an extracellular polypeptide (20, 55).
Particularly noteworthy was the demonstration by Marmur and Lane of strand separation on denaturation of the native, duplex DNA and the renaturation of its transforming activity after thermal denaturation by annealing the separated strands at submelting temperatures (44). Although this work was accomplished at Harvard University, Marmur had been a postdoctoral fellow and Lane had been a technician in the Hotchkiss laboratory. Another outstanding contribution from that laboratory was Maury Fox's discovery of the eclipse of donor marker transforming activity immediately after DNA uptake. When DNA is extracted from recipient cells just after uptake, it is unable to transform tester cells, but the ability is fully restored within 10 min at 37°C (14). Several investigators, including Fox (13), Goodgal and Herriott (17), and Lerman and Tolmach (39), pioneered the use of 32P-labeled DNA to demonstrate the physical uptake of DNA in transformation. Sol Goodgal was instrumental in organizing the first Transformation Meeting in 1956. It is to Leonard Lerman that we owe the name of the Wind River Conference; he organized the Transformation Meeting at the Wind River Ranch, Estes Park, Colo., in 1959.
My own work in transformation began in 1955, as the first graduate student of Rollin Hotchkiss. Rollin fostered self-determination in his students, and he agreed to my proposal to study recombination between nutritional mutants—to see if we could analyze genetic fine structure by transformation. For my thesis I isolated and analyzed eight mutants unable to use the sugar maltose (32). Even from this small number of mutants, several significant facts emerged. Three maltose-negative mutations were multisite and ultimately shown to be deletions; one covered the entire locus, and the other two overlapped from opposite ends. Their transformation efficiencies depended inversely on deletion length. (Transformation efficiencies are expressed as the ratio of a maltose marker transformation frequency to that of a standard streptomycin resistance marker to account for variations in culture competence.) Five mutations recombined with each other and appeared to be mutated at single sites; they presumably corresponded to base changes. What was surprising was that these single-site mutations showed transformation efficiencies with wild-type DNA that varied over a 10-fold range. Later, such variation in transformation efficiency was shown to result from differential action of a base mismatch repair system (11, 25). We were able to measure production of the enzyme amylomaltase, product of the malM gene, immediately after DNA uptake by the various mutants. Surprisingly, the amount of enzyme produced was not dependent on the amount of DNA taken up but was proportional, rather, to the transformation efficiency of the marker (33). This, too, was a manifestation of the eclipse; not only was the incoming DNA unable to transform another cell, but it was also unable to direct production of a protein prior to its integration.
MOLECULAR MECHANISM OF DNA UPTAKEmoz-%ew, 百拇医药
In searching for a molecular explanation of the eclipse phenomenon, my initial hypothesis was that DNA was fragmented during entry and that the double-stranded fragments were later connected, presumably while aligned on a recipient chromosomal template, to restore both transforming and transcribing activity. My idea was to label the donor DNA with 32P, break open recipient cells after uptake, and look for 32P-DNA fragments with a methylated-albumin coated kieselguhr (MAK) column. Mandell and Hershey had recently shown that fragments of T-even coliphage DNA were separated on MAK columns in a size-dependent manner (43). However, when I carried out the experiment, ~ 40% of the 32P counts were immediately eluted and only ~ 5% were eluted by the salt gradient, which eluted even the high-molecular-weight recipient DNA; most of the radioactivity was missing, and I was puzzled over the low recovery. That evening I had a moment of "eureka": the remaining counts must have been retained by the column! On returning to the laboratory, I detected, in fact, radioactivity still on the MAK column. When eluted with 0.1 N NaOH, the counts amounted to ~ 50% of the total. Upon more carefully reexamining the paper by Mandell and Hershey, I saw that they had mentioned in passing that denatured DNA sticks to the column. Apparently, the DNA was converted to single strands on uptake. This conclusion was soon confirmed by separation of newly entered and resident DNA in a CsCl density gradient that separated single- from double-stranded DNA.
In reporting my findings in 1962, I proposed that one strand of the duplex DNA was degraded as the other strand entered the cell (23). Such conversion to a single-stranded form explained the eclipse, inasmuch as denatured DNA was known to be poorly taken up by cells and could not serve as a template for transcription. Furthermore, conversion to a single strand would facilitate interaction with the recipient chromosome. In fact, radioactive label passed rapidly from single strands into double stranded, presumably chromosomal DNA (23). Later, it was shown by density labeling that single-stranded segments of donor DNA were indeed incorporated into the transformed chromosome (15).xl}78, http://www.100md.com
I considered the determination of the fate of DNA on entry to be an important new finding for the understanding of transformation. Therefore, I was quite surprised to read, afterward, in a thesis of Pierre Schaeffer published in 1961 the following concluding statement, "Une hypothèse est proposée d'après laquelle la pénétration de la molécule d'ADN dans la bactérie requiert une structure à double chaîne, alors que la synapse, dont dépend l'intégration, requiert au contraire une chaîne polynucléotidique unique" (50a). Translated, this reads "A hypothesis is proposed according to which penetration of the DNA molecule into the bacterium requires a double-stranded structure, while synapsis, on which integration depends, conversely requires a single polynucleotide strand." Schaeffer was a medical doctor, a mature investigator, who had for some years investigated the competition between different DNAs in the transformation of various bacterial species. As was the custom in France, such researchers often took an additional degree of Docteur Sciences later in life. Schaeffer based the uptake part of his hypothesis on his own experimental work, but the conclusion that a single strand was needed for integration was the result of careful thought. He reasoned that only single-stranded DNA, with its bases exposed, could readily interact with recipient DNA. It was sobering for me that these thoughts were presented before publication of my own work. In fact, it is possible that they were expressed during Schaeffer's thesis defense, which I attended in Paris, France, in 1958, when I was working in the laboratory of François Gros, another very fine mentor, at the Pasteur Institute. Despite its proposal on theoretical grounds, I comforted myself in the novelty of my work showing that the strand conversion process occurred during DNA entry. However, I never forgot that scientific understanding is a large edifice to which many contribute.
Because the amount of low-molecular-weight 32P found in the cells approximately equalled the amount in single strands, it appeared that one strand was degraded as the other strand entered (23). However, Morrison and Guild showed that the amount of DNA breakdown inside the cell depends on the size and quality of the donor DNA; more intact DNA gave fewer breakdown products so that a larger proportion of DNA taken up was in the form of single strands (45). In the early 1970s, they and Bill Greenberg and I independently discovered that the breakdown products of the strand complementary to the one that enters appear external to the cells (28, 46). Early on, I hypothesized that a cellular DNase acted on donor DNA to facilitate entry of one strand by degrading the opposite strand (23). Investigation of the pneumococcal nucleases revealed two enzymes active on DNA (27). In vitro, EndA, which was found to be membrane bound (31), degraded DNA to give oligonucleotide products, and ExoA (like ExoIII of Escherichia coli) gave mononucleotides and single strands. To see which, if either, enzyme facilitated entry, I sought mutants lacking them. Useful in this approach was a method for detecting colonies that lacked nuclease activities by growing them in agar containing DNA and methyl green, which stains only intact DNA (26). Colonies around which the DNA was digested showed colorless zones that were absent around mutant colonies (Fig. 1).
fig.ommitted+#k], http://www.100md.com
Nuclease detection in nutrient agar plates containing DNA and methyl green. Cells of S. pneumoniae wild-type strain R6 (endA+ exoA+) and mutant strain 593 (endA1 exoA+) were plated in a ratio of 1:10 and incubated at 37°C for 30 h.+#k], http://www.100md.com
Before discussing another technique helpful in analyzing the DNase mutants, I shall divert briefly to discuss mentoring. I personally was fortunate to have as a mentor Rollin Hotchkiss, who not only advised me wisely as a graduate student but also remained a friend and inspiration throughout my life. Still, I recall that when I first began to look for nutritional mutants of pneumococcus, the medium used in the Hotchkiss laboratory was only semidefined—a good wallop of yeast extract was added to the otherwise defined mix. I set about to find out what was essential in the yeast extract, and after mastering several new techniques, was able to identify the substance as glutamine. "Aha!" said Rollin, "Adams [and Roe (1), who formulated the medium] did add glutamine, but we thought it too unstable to make a difference, and to get good growth we added yeast extract." Earlier, I had a similar experience in my senior research at college. I was to obtain and study petite mutants of yeast, but I could not grow the cells to any significant turbidity in the medium for which I was given the formula. After two weeks of frustration I returned to my mentor, and he discovered an error in his formulation that made the medium a hundredfold too dilute. Next, it was my turn. When a postdoctoral fellow, Allan Rosenthal, who was characterizing DNase mutants, pointed out to me the potential utility of revealing enzyme activity in situ after electrophoresis of extracts in a denaturing gel, I told him to forget about it because it would be impossible to remove sodium dodecyl sulfate (SDS) from the enzyme to renature it. However, when I was away from the laboratory for a week, he tried the experiment, and now we all know how useful such enzyme detection after denaturing electrophoresis can be (50). The importance of mentoring in developing new generations of scientists cannot be overstated; however, skepticism on the part of a student is also warranted.
The in situ detection method helped show conclusively that null mutants of EndA reduced uptake and transformation by a factor of a thousand (35). ExoA mutants had no effect on transformation. Both its membrane location and finding that the external products of in vivo degradation were oligonucleotides (34) supported a role for EndA in bringing one strand into the cell as it degraded the other.3kw'c{^, http://www.100md.com
fig.ommitted3kw'c{^, http://www.100md.com
DNases in wild-type and mutant strains of S. pneumoniae detected after electrophoresis in polyacrylamide gels containing SDS and DNA. Wells contained 25 µg of protein from SDS lysates of S. pneumoniae as follows: wild-type R6 (a), exoA5 (b), endA1 (c), and endA14 (d). (e) This well contained pancreatic DNase I and micrococcal nuclease. Electrophoresis was anodal in the downward direction. After washing to remove SDS, the gel was incubated for 64 h, stained with ethidium bromide, and photographed with UV illumination. Size markers (in thousands) were from a parallel lane of proteins stained with Coomassie blue (not shown). Dark bands correspond to DNases.
DpnI AND DpnII RESTRICTION SYSTEMS8+7:{, 百拇医药
Elimination of the major DNases from the pneumococcal cell still left a weak DNase activity, which gave no acid-soluble products but could be detected by its reduction of DNA viscosity. This enzyme turned out to be DpnI, an unusual restriction endonuclease that cleaves only DNA with adenine methylated at GATC sites (29, 56). Normally, restriction enzymes cleave unmethylated DNA, and DNA is protected from cleavage by methylation. The contrary is true for DpnI. Its discovery was fortuitous, in that E. coli DNA, which happens to be methylated at GATC sites, was used as the substrate for testing DNase activity, and not DNA from the pneumococcal strain, which was unmethylated. Interesting as this enzyme was, it was only the first part of the story.8+7:{, 百拇医药
The concept of serendipity, proposed by Horace Walpole in 1754, derives from an ancient tale entitled The Three Princes of Serendip. In the story, three young men from Serendip, which is an old name for Sri Lanka, set off on a trip abroad and encountered many adventures. By a combination of chance and sagacity, they fared well in their travels. In a personal instance of serendipity, a culture of a strain that should have made DpnI produced instead a different enzyme, DpnII, which is complementary to DpnI and cleaves only at unmethylated GATC. We wondered if by some genetic switch, perhaps related to DNA methylation, the DpnI-producing strain had converted itself into a DpnII-producing strain (30). This turned out not to be the case. This DpnII strain was a contaminant streptococcus that outgrew the original inoculum. However, different isolates of pneumococcus from nature could produce either DpnI or DpnII (47), and the responsible genes were found to constitute mutually replaceable genetic cassettes (38). Similar findings for capsular genes were foreshadowed by this result. An additional unexpected feature of the DpnII system was the presence of two methyltransferases (9), one of which preferentially methylated single-stranded DNA and was able to prevent the restriction of unmethylated plasmid entering a cell by the transformation pathway (6).
CLONING IN STREPTOCOCCUS PNEUMONIAE2o, http://www.100md.com
Among the tools and methods developed in my laboratory, one of the most important was the pneumococcal cloning system using derivatives of the plasmid pMV158 (53). We were forced to turn to this system by our inability to clone many important pneumococcal genes, such as malM, endA, and hexA, in the available E. coli systems, apparently due to the strong promoters of S. pneumoniae and the toxicity of the gene products in E. coli (52). The pneumococcal cloning system was elaborated in a relatively short time by the intense effort of Diane Stassi, Paloma Lopez, and Manolo Espinosa. This followed an initial suggestion by Francis Barany in a telephone conversation with me. Walter Guild provided pMV158; he obtained it from its discoverer, Vickers Burdett. There again, we see the interwoven undergarment of scientific progress.2o, http://www.100md.com
Cloning in pneumococcus not only allowed the analysis of otherwise unclonable genes, but it also provided a facile method for exchanging chromosomal segments present in a recombinant plasmid. Such exchange resulted from chromosomal facilitation of plasmid establishment, in which an entering linear strand from a recombinant plasmid must interact with the chromosome in order to circularize (42). That the DpnI and DpnII system genes resided at the same position in the chromosome was first discovered by the substitution of a DpnI gene segment on transformation of a DpnI cell by a recombinant plasmid containing the DpnII gene segment (38). Cloning in S. pneumoniae of a chromosomal segment containing sul-d, a mutation which conferred sulfonamide resistance, enabled us to carry out the first genetic analysis of folate biosynthesis in any organism (41).
DNA MISMATCH REPAIR%v, 百拇医药
A major thrust of my work has been in DNA mismatch repair. This phenomenon was first discovered in S. pneumoniae, a decade before it was found in E. coli, by examination of transformation frequencies of different mutational markers at the amylomaltase locus. In my doctoral work (circa 1957) I had found different mutations to be transformed at characteristic frequencies or efficiencies (32). Perhaps influenced by both the periodic table, with its categories of elements, and astrophysics, with its grouping of stars by luminosity, I undertook a classificatory approach, grouping mutations of similar transformation frequency. By 1965, I had over 70 single-site mutations in the malM gene, which clustered in four efficiency groups, and I had found that reciprocal transformation of markers gave identical frequencies (24, 25). These findings allowed the attribution of the four efficiency groups, I to IV (ratios of 0.05, 0.2, 0.5, and 1.0), to the six possible classes of mutations giving base mismatches. On the basis of mutagenic origin and changes at the same site, I proposed the following correspondence of efficiency class to mutations giving rise to mismatches in the heteroduplex product of transformation: I, ATGC, GCAT; II, ATTA; III, GCCG; IV, ATCG, GCTA (25). These proposed assignments proved to be generally correct when cloning and sequencing of the malM gene was achieved (37).
I had been impressed by the colligative properties of the efficiency groups with respect to parameters other than transformation efficiency. Marker sensitivity to UV light (24, 40) and speed of recovery from eclipse (16) were both inversely correlated with efficiency. Both effects result from dependence of donor marker excision on single-strand breaks prior to ligation of the donor segment. Repair of UV damage produces subsequent strand breaks that result in additional mismatch repair (36). The apparent earlier recovery of low-efficiency markers reflects their later loss by repair, as was evident from early peaking of their recovery (51). These correlations helped support a model for mismatch repair in which an entire donor strand segment that would normally be integrated in transformation is excluded from integration (7, 25, 36).h^r#5]x, 百拇医药
With important contributions by various investigators, the different transformation efficiencies and other properties were ascribed to a cellular mismatch repair system. A major contributor was Harriett Ephrussi-Taylor. In 1958, she invited me to give a talk at her laboratory in Gif, near Paris. Harriett was very interested in the malM mutant transformation frequencies and recombination results. With Michel Sicard she devised another gene system, based on aminopterin resistance, for analyzing mismatch repair (11). She was the first to propose that the pneumococcal system removed mismatches by an enzymatic mechanism (10). Unfortunately, Harriett died soon afterward; however, Michel and, later, his student Jean-Pierre Claverys, continued to contribute importantly to the field. Our discovery of the hex mutants, in which all markers transformed at high efficiency (26), supported the concept of an enzymatic system for detecting and repairing DNA mismatches, in which large tracts of donor DNA containing the mismatched base were removed. Using such hex mutants, Tiraby and Fox showed that the mismatch repair mechanism also prevented spontaneous mutations (54). T. S. Balganesh in my laboratory (4) was the first to clone a mismatch repair gene, hexA, and that enabled determination of its nucleotide sequence (48). Sequence comparison with mismatch repair genes from other species indicated that the system was universally found in living cells (19, 48, 49). Strikingly, a homologous system in human cells prevents colon cancer (12) (Fig. 3).
fig.ommitted&:$gr, 百拇医药
. From left to right, Bill Greenberg, Sanford Lacks, and Sylvia Springhorn examine autoradiograms that reveal the DNA sequence of genes encoding the restriction enzymes of S. pneumoniae.&:$gr, 百拇医药
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.&:$gr, 百拇医药
ACKNOWLEDGMENTS&:$gr, 百拇医药
I thank the scientists who were my mentors, my collaborators, and those who came to work in my laboratory as visiting scientists, postdoctoral associates, students, and technical associates. Although everyone made important contributions, I regret that I was able to mention only a few by name in this memoir. I am indebted to two associates who worked with me for very long periods, Bill Greenberg and Sylvia Springhorn; they are pictured in&:$gr, 百拇医药
REFERENCES&:$gr, 百拇医药
Adams, M. H., and A. S. Roe. 1945. A partially defined medium for cultivation of pneumococcus. J. Bacteriol. 49:401-409.
Alloway, J. L. 1931. The transformation in vitro of R pneumococci into S forms of different specific types by the use of filtered pneumococcus extracts. J. Exp. Med. 55:91-99.}5e/, http://www.100md.com
Avery, O. T., C. M. MacLeod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J. Exp. Med. 89:137-158.}5e/, http://www.100md.com
Balganesh, T. S., and S. A. Lacks. 1985. Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: Cloning and expression of the hexA gene. J. Bacteriol. 162:979-984.}5e/, http://www.100md.com
British Medical Journal. 1941. Obituary. Br. Med. J. 1941-I:691.}5e/, http://www.100md.com
Cerritelli, S., S. S. Springhorn, and S. A. Lacks. 1989. DpnA, a methylase for single-strand DNA in the DpnII restriction system, and its biological function. Proc. Natl. Acad. Sci. USA 86:9223-9227.}5e/, http://www.100md.com
Claverys, J. P., and S. A. Lacks. 1986. Heteroduplex deoxyribonucleic acid base mismatch repair in bacteria. Microbiol. Rev. 50:133-165.
Dawson, M. H., and R. H. P. Sia. 1931. In vitro transformation of pneumococcal types. I. A technique for inducing transformation of pneumococcal types in vitro. J. Exp. Med. 54:681-699.qtk, 百拇医药
de la Campa, A. G., P. Kale, S. S. Springhorn, and S. A. Lacks. 1987. Proteins encoded by the DpnII restriction gene cassette: two methylases and an endonuclease. J. Mol. Biol. 196:457-469.qtk, 百拇医药
Ephrussi-Taylor, H., and T. C. Gray. 1966. Genetic studies of recombining DNA in pneumococcal transformation. J. Gen. Physiol. 49:part2.,211-231.qtk, 百拇医药
Ephrussi-Taylor, H., A. M. Sicard, and R. Kamen. 1965. Genetic recombination in DNA-induced transformation of pneumococcus. I. The problem of relative efficiency of transforming factors. Genetics 51:455-475.qtk, 百拇医药
Fishel, R., M. K. Lescoe, M. R. S. Rao, N. G. Copeland, N. A. Jenkins, J. Garber, M. Kane, and R. Kolodner. 1993. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75:1027-1038.
Fox, M. S. 1957. Deoxyribonucleic acid incorporation by transformed bacteria. Biochim. Biophys. Acta 26:83-85.$il(in$, 百拇医药
Fox, M. S. 1960. Fate of transforming deoxyribonucleate following fixation by transforming bacteria. II. Nature 187:1004-1006.$il(in$, 百拇医药
Fox, M. S., and M. K. Allen. 1964. On the mechanism of deoxyribonucleate integration in pneumococcal transformation. Proc. Natl. Acad. Sci. USA 52:412-419.$il(in$, 百拇医药
Ghei, O. K., and S. A. Lacks. 1967. Recovery of donor deoxyribonucleic acid marker activity from eclipse in pneumococcal transformation. J. Bacteriol. 93:816-829.$il(in$, 百拇医药
Goodgal, S. H., and R. M. Herriott. 1957. Studies on transformation of Haemophilus influenzae, p. 336-340. In W. D. McElroy and B. Glass (ed.), The chemical basis of heredity. Johns Hopkins Press, Baltimore, Md.$il(in$, 百拇医药
Griffith, F. 1928. The significance of pneumococcal types. J. Hyg. 27:113-159.$il(in$, 百拇医药
Haber, L. T., P. P. Pang, D. I. Sobell, J. A. Mankovich, and G. C. Walker. 1988. Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae. J. Bacteriol. 170:197-202.
Håverstein, L. S., G. Coomaraswamy, and D. A. Morrison. 1995. An unmodified heptadecapeptide induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 92:11140-11144.]]v^, 百拇医药
Hotchkiss, R. D. 1954. Cyclical behavior in pneumococcal growth and transformability occasioned by environmental changes. Proc. Natl. Acad. Sci. USA 40:49-55.]]v^, 百拇医药
Hotchkiss, R. D. 1957. Criteria for the quantitative genetic transformation of bacteria, p. 321-335. In W. D. McElroy and B. Glass (ed.), The chemical basis of heredity. Johns Hopkins Press, Baltimore, Md.]]v^, 百拇医药
Lacks, S. 1962. Molecular fate of DNA in genetic transformation of pneumococcus. J. Mol. Biol. 5:119-131.]]v^, 百拇医药
Lacks, S. 1965. Genetic recombination in pneumococcus, p. 159-164. In M. Kohoutova and J. Hubacek (ed.), The physiology of gene and mutation expression. Academia, Prague, Czechoslovakia.]]v^, 百拇医药
Lacks, S. 1966. Integration efficiency and genetic recombination in pneumococcal transformation. Genetics 53:207-235.
Lacks, S. 1970. Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J. Bacteriol. 101:373-383.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1967. Deoxyribonucleases of Pneumococcus. J. Biol. Chem. 242:3108-3120.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1973. Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 114:152-163.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1975. A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA. J. Biol. Chem. 250:4060-4066.r.f)gs, 百拇医药
Lacks, S., and B. Greenberg. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114:153-168.r.f)gs, 百拇医药
Lacks, S., and M. Neuberger. 1975. Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 124:1321-1329.
Lacks, S., and R. D. Hotchkiss. 1960. A study of the genetic material determining an enzyme activity in pneumococcus. Biochim. Biophys. Acta 39:508-517./v*, 百拇医药
Lacks, S., and R. D. Hotchkiss. 1960. Formation of amylomaltase after genetic transformation of pneumococcus. Biochim. Biophys. Acta 45:155-163./v*, 百拇医药
Lacks, S., B. Greenberg, and M. Neuberger. 1974. Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc. Natl. Acad. Sci. USA 71:2305-2309./v*, 百拇医药
Lacks, S., B. Greenberg, and M. Neuberger. 1975. Identification of a deoxyribonuclease implicated in genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 123:222-232./v*, 百拇医药
Lacks, S. A. 1989. Generalized DNA mismatch repair—its molecular basis in Streptococcus pneumoniae and other organisms, p. 325-339. In L. O. Butler, C. Harwood, and B. E. B. Moseley (ed.), Genetic transformation and expression. Intercept, Wimbourne, England./v*, 百拇医药
Lacks, S. A., J. J. Dunn, and B. Greenberg. 1982. Identification of base mismatches recognized by the heteroduplex-DNA-repair system of Streptococcus pneumoniae. Cell 31:327-336.
Lacks, S. A., B. M. Mannarelli, S. S. Springhorn, and B. Greenberg. 1986. Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism. Cell 46:993-1000.jz(-{oi, 百拇医药
Lerman, R. S., and L. J. Tolmach. 1957. Genetic transformation. I. Cellular incorporation of DNA accompanying transformation in pneumococcus. Biochim. Biophys. Acta 28:68-82.jz(-{oi, 百拇医药
Litman, R. M. 1961. Genetic and chemical alterations in the transforming DNA of pneumococcus caused by ultraviolet light and by nitrous acid. J. Chim. Phys. 58:997-1004.jz(-{oi, 百拇医药
Lopez, P., M. Espinosa, B. Greenberg, and S. A. Lacks. 1987. Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme. J. Bacteriol. 169:4320-4326.jz(-{oi, 百拇医药
Lopez, P., M. Espinosa, D. L. Stassi, and S. A. Lacks. 1982. Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology. J. Bacteriol. 150:692-701.
Mandell, J. D., and A. D. Hershey. 1960. Anal. Biochem. 1:66-.$1a[, 百拇医药
Marmur, J., and D. Lane. 1960. Strand separation and specific recombination in deoxyribonucleic acids: biological studies. Proc. Natl. Acad. Sci. USA 46:453-461.$1a[, 百拇医药
Morrison, D. A., and W. R. Guild. 1972. Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor. J. Bacteriol. 112:1157-1168.$1a[, 百拇医药
Morrison, D. A., and W. R. Guild. 1973. Breakage prior to entry of donor DNA in pneumococcus transformation. Biochim. Biophys. Acta 299:545-556.$1a[, 百拇医药
Muckerman, C. C., S. S. Springhorn, B. Greenberg, and S. A. Lacks. 1982. Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae. J. Bacteriol. 152:183-190.$1a[, 百拇医药
Priebe, S. D., S. M. Hadi, B. Greenberg, and S. A. Lacks. 1988. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 170:190-196.
Reenan, R. A. G., and R. D. Kolodner. 1992. Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins Genetics 132:963-973.syhp, 百拇医药
Rosenthal, A. L., and S. A. Lacks. 1977. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal. Biochem. 80:76-90.syhp, 百拇医药
Schaeffer, P. 1961. Doctoral thesis. University of Paris, Paris, France.syhp, 百拇医药
Shoemaker, N. B., and W. R. Guild. 1974. Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation. Mol. Gen. Genet. 128:283-290.syhp, 百拇医药
Stassi, D. L., and S. A. Lacks. 1982. Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae. Gene 18:319-328.syhp, 百拇医药
Stassi, D. L., P. Lopez, M. Espinosa, and S. A. Lacks. 1981. Cloning of chromosomal genes in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 78:7028-7032.syhp, 百拇医药
Tiraby, G., and M. S. Fox. 1973. Marker discrimination in transformation and mutation of pneumococcus. Proc. Natl. Acad. Sci. USA 70:3541-3545.syhp, 百拇医药
Tomasz, A., and R. D. Hotchkiss. 1964. Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc. Natl. Acad. Sci. USA 51:480-487.syhp, 百拇医药
Vovis, G. F., and S. Lacks. 1977. Complementary action of restriction enzymes Endo R. DpnI and Endo R. DpnII on bacteriophage fl DNA. J. Mol. Biol. 115:525-538.(Sanford A. Lacks)