Chaos and Order in Spontaneous Mutation

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove THIS story starts with one of those unanticipated turning points that mark the lives of most of us. Around 1960 I was working at the University of Illinois at Urbana using Newcastle disease virus (NDV), a tractable animal virus that infects chickens rather than humans. (Working previously with polioviruses, I had acquired extraordinarily high serum titers against all three strains.) The NDV plaque assay required that chicken embryos of the correct age be harvested and dissociated into single cells, which were then used to produce monolayers that became confluent in a few days. Virus samples were adsorbed to the monolayers, which were then incubated for a few more days while plaques formed. The regimen was cumbersome and contained day-long gaps that, as it turned out, were incompletely filled with the planning and analysis of experiments, the preparation and delivery of lectures, and the miscellaneous duties of a bottom-rung academic. As a graduate student I had followed the antics of the Caltech phage group and had also become aware of the mutagenic base analog 5-bromouracil. Over the next few years I was increasingly impressed by the early work on mutation by Ernst Freese and Seymour Benzer using phage T4. Ernst in particular was dissecting out mutational pathways with mutagens that seemed to be highly specific in their actions, for instance, the base analogs 5-bromouracil and 2-aminopurine that induced transitions in both directions and hydroxylamine that induced only G·C → A·T transitions. I often had occasion to UV irradiate NDV, and I wondered if the mutagenic specificity of UV irradiation could be ascertained by performing reversion tests on UV-induced mutations with base analogs, hydroxylamine, and proflavin, the specificity of the last having been demonstrated by Benzer and Sydney Brenner. Starting with a protocol developed by Raymond Latarjet for phage T4, I began to intercalate phage experiments into the free days provided by the NDV protocols. I quickly found that phages produced interesting results at least an order of magnitude more frequently than did NDV. There followed a burst of investigations into the mutation process in phage T4. A key component of these investigations was reversion analysis of rII mutations, which tended to reveal that the mutants I was making—not only with UV, but also arising spontaneously in free phages and induced by photosensitizers—contained several different kinds of mutations. Here, however, a barrier arose. Transitions of both types could be recognized, as well as many frameshift mutations (which could be reverted by proflavin), but no agent was known to induce only transversions. The answer, I hoped, might lie in an altogether different kind of mutagen. Joe Speyer (1965) had just reported that two temperature-sensitivity mutations in T4 gene 43 (which encodes the viral DNA polymerase) were also strong mutator mutations. Perhaps some such mutators would make transversions specifically. To that end, I acquired the entire Caltech collection of gene-43 mutants. Elizabeth “B. J.” Allen hunted among these for mutators and found many. Unexpectedly, she found others that seemed to be antimutators.2 These were much more interesting, so we quickly changed course. I write “seemed to be” because most of the initial antimutator candidates were selected because of deleterious effects of the gene-43 mutations that rendered the mutants that Allen was scoring difficult to detect, a frequent artifact in the history of mutation research. Our first report on antimutators was at a Cold Spring Harbor Symposium (Drake and Allen 1968) and contained a formal description of the antimutators plus some general observations based on the literature. One of the latter was that the phages T4 and λ and the bacteria Escherichia coli and Salmonella typhimurium shared similar genomic mutation rates of roughly 0.2% per chromosome replication. In a huge leap, I wrote that “This result suggests that mutation rates are usually about as large as can be tolerated” (p. 339). It was only several years later that I discovered an important article by A. H. Sturtevant (1937) and contemporary (1960s) work by Motoo Kimura, addressing both the issue of downward pressures on mutation rates and the limits of such reductions. When the formal antimutator story was published soon thereafter, it was divided (by the journal editor) into two parts: one an article about the T4 work (Drakeet al. 1969) and the other a short note (Drake 1969) on comparative genomic mutation rates in diverse organisms. The note presented the phage/bacteria rate concordances but also cited a rate about tenfold lower for Neurospora crassa (which eventually turned out to be an underestimate) and a value for Drosophila melanogaster that must have confused many readers because it was a rate (of about 0.8) per sexual generation rather than per germ-line cell division. The same table and its legend allowed astute readers to estimate that the rate per germ-line cell division was about 1.4%. Although this number was based on several components now known to be inaccurate, the result was close to a later calculation based on somewhat better information. Not yet having discovered Kimura's articles on the subject, I wrote that “Mutation rates may be adjusted by natural selection to achieve a balance among the mostly deleterious effects of mutation, the need for variation, and the cost of suppressing mutation” (p. 1132). Well, two out of three was perhaps not too bad for a beginner; Kimura (1960, 1967) had already pointed out that selection for downmodifiers of mutation rates would be balanced by the cost of further reducing rates and that selection for upmodifiers of mutation rates hitchhiking with rare advantageous mutations was rapidly obviated by recombination. I soon became attached to the possibility of a universal genomic mutation rate. As reports appeared offering numerical orts that could reasonably be converted into genomic mutation rates, my table was adjusted and expanded and published serially in several chapters and reviews in the 1970s and 1980s. The data of best quality were those for DNA viruses, bacteria, and fungi, and by the early 1990s I was able to produce a compilation that showed that all the DNA-based microbes for which data were available shared a genomic rate of spontaneous mutation of close to 0.003 (mean 0.0033; range 0.0019–0.0046, excluding two formally defined outliers) (Drake 1991). This result fascinated me because it implied that the action of powerful evolutionary forces was capable of finely tuning the rate of spontaneous mutation. Both Kimura and I had imagined a mutation rate balanced between the deleterious effects of most mutations and what Kimura called the “physiological” cost of further reducing mutation rates. However, the defining microbes are extraordinarily diverse in both their genomics and their life histories: a tiny phage with single-stranded DNA, a genetically complex and completely lytic phage pair, a lysogenic phage (whose rate, however, was based on its lytic cycle), a bacterium, a yeast, and a filamentous fungus. Why should such diversity not lead to different optimal rates in such different microbes? I concluded that the balancing forces must be very deep, but they remain mysterious to this day. In an effort to expand the range of DNA microbes exhibiting this standard rate, more examples were slowly added to the table. The most recent was for a DNA virus that is also a human pathogen, herpes simplex virus (Drake and Hwang 2005). The most bizarre was for an archaeon adapted to growth in hot acid, Sulfolobus acidocaldarius (Groganet al. 2001). Both organisms display typical genomic rates, but the Sulfolobus rate of 0.0018 may have been slightly lower than usual for reasons to be discussed later. Half a century of work in many laboratories has revealed such a plethora of mechanisms that it is now abundantly clear that mutagenesis reflects the sum of many ways in which a complex consortium of fidelity processes can go wrong. This being the case, it is unlikely that any single mutation could lower the genomic mutation rate very much. I argued thusly against the existence of general antimutator mutations of any considerable strength and free of strongly deleterious pleiotropic effects (Drake 1993a) and supported the argument with the experimental demonstration that all of the nine antimutators in phage T4 gene 43 and one in gene 45 exhibit mutation rates the same as or slightly greater than that of the wild type when assayed using reporter genes containing roughly 3600 bp; clearly, while mutation rates at some sites were decreased in these antimutators, rates at other sites were increased. This result had been modestly anticipated by some of the values posted by Drakeet al. (1969) and strongly anticipated by Lynn Ripley when she developed the first T4 system for scoring transversions (Ripley and Drake 1972; Ripley 1975). In fact, the T4 antimutators all seem to do the same thing: reducing the rate of A·T → G·C transitions while modestly increasing rates of transversions and small indels.3 In an extensive series of investigations, Roel Schaaper has had a similar experience: antimutators of modest effect can be selected in E. coli but they are again rather pathway specific. The only exception that one would expect would be an organism that already harbors a mutator mutation, perhaps not known to the investigator; reversion or suppression of that specific defect could then produce a strong antimutator effect, but would simply lower the rate toward, but not below, the standard value. Curiously, the term “indel” (meaning insertions and deletions, frequently without regard to size), although in widespread use by evolutionists, is still often unfamiliar to students of the mutation process. It is, of course, superior to “frameshift mutations” because it includes indels of a size that is a multiple of three nucleotides or resident in sequences that do not encode proteins. Until recently, tabulations of mutation rates in textbooks tended to present a chaotic picture. The rates were rarely genomic, so the overall regularities were obscured; microbial by with the result that and rates by at least that much. rates also because of large in gene size and of rapidly base the of base huge This became the very first T4 rII published by and reflects not only such as al. but also the in which a base of the latter also T4 rII the mutation rate to → → by more than the rII and This result not only that rates on but also that the the two base work on the DNA and or revealed large in their on In extensive of fidelity in showed that different DNA produce different of rates, the with its many to DNA in the of a as a of rates. The also turned out to exhibit strong DNA further although phage T4 not to have The most discovered of is the of at least the and the that and single-stranded DNA al. 2005). Why natural selection the of high by which some genes a of their deleterious mutations at a single This is two The first is simply some sites are so The answer, I is that the a number of different short sequences and simply to rates at all of when other such as and must be The is selection against sequences not mutational the high of in the the is may be on but more often the may be that selection against high at single sites is simply As Benzer published more T4 rII mutational it became clear that most sites were by this because their mutation rates were those of the were the sites those at which simply to produce a This was by and Drake a graduate student was to a Starting with two rII mutations that had only a at the mutants with to produce diverse rII mutations. then the for strong mutants at and mutational In to the produced a number of mutations at previously sites the rII When the mutations, the induced mutations had so that they would have many sites were discovered that it was that most or all sites were simply the result of very One me if could do a on the while working on that we had but about the of the I A few later a of an article appeared on my It was using 2-aminopurine to the mutations to A·T → G·C had the first experimental demonstration that mutation rates on their in this their base A discovered an between two rII in which one both spontaneous and mutation rates at the other while tests that the were at least several base in but this work was by al. and later that the were a nucleotides and Drake These showed that mutation rates could be not only by but also by somewhat more base which further to the huge in mutation rates are of the sum of diverse small and large When we the Sulfolobus mutations, we that most of were small had been that most were by A of a considerable collection of data that, most of the roughly of mutations are only about were for Sulfolobus (Groganet al. at had both and selection for but the of in Sulfolobus was in the two which argued against an high of the being mutants that It to me that the mutation might be more deleterious at a high than in a a that found but was then an mutational would to in a a cost perhaps the of downmodifiers of One further result would be to the of mutations to mutations the of This was by al. found that the of to in of was about lower in than in of they were or unanticipated this work was the based on an of work group and that rates of are among and As a Caltech graduate student in I had become aware the work of that tended to be highly the few mutants that could be high The few mutants that were were later found to multiple mutations. the the high of had become al. but was based on rates of and on mutation rather than on mutation rates. by the of the standard genomic rate for DNA I to what the had to quickly became The first was the of a of viral it with a as in the of and or a such as To this I converted into rates using the for both and and the of the two which usually by only a The was the data for selection were was to be between and most important of the reporter sequences were usually such as a single pathway at a single and to the large in already well for genes of The result (Drake was a of rates a a an and virus The value of the genomic rates was but the range was a so that this analysis showed only that rates could be and were very However, because most of the values seemed too large to I an perhaps only high mutation tended to be This did not later in the further of the me that by the the initial to that in However, the of the for into mutation rates became to me only in the first of on a to I turned for to to a of nine mutation and The genomic rate per chromosome was and the range was (Drake and still but at least than in The experimental any of an rate among these The rate still seemed high to because it that the mutation a single of was about so that only about a of would be free of mutations. experiments had that were by increasing the mutation by about al. which would seem to that the mutation would have to be as high as to achieve the highly of and the of many sites in important However, as we below, the mutation of a may important among the seem to have genomic mutation rates than do (Drakeet al. and are slightly more to mutagenesis and The rate is the of three very different kinds of of a then → DNA, and then DNA → with to the rate. The next in mutation virus al. A of an entire the first mutational most or all deleterious mutations could be and by in the The genomic mutation rate this system was about the lower of the for animal The mutational contained the usual of base and However, as with the base were in a which might a deleterious of in these because of the of in life often mutations The also contained several examples of a of mutations that I had not been insertions of the mutation a number of the mutants contained two or three mutations, an to be later. It has been to the high mutation rates of to their need to To this seemed unlikely because the has a mutation rate (Drake while the DNA has a DNA mutation rate about lower (Drake 1991). recently, al. used a system to that the of mutation rates did not with rates of that viral is not by but rather that high rates result in However, report al. a very different that can be both by the mutation rate and by the of the the value of high mutation rates an It is also to the high mutation rates of to the of a of course, to the of which is often In it has seemed more to me that life their high mutation rates, the particular mechanisms simply the values are There is a DNA-based organisms. T4 and E. coli in mutation rate per base by about this reflects the of in T4 plus a modestly rate of than in E. but the the T4 rate is not of but rather the evolutionary forces that the standard genomic microbial rate. lower mutation rates were that is, if their were more than by their they could be by increasing the fidelity of base selection by a kind of has been al. and might be acquired by a is the component of the life history that the high mutation I a specific is highly to at and this is by of such as and al. al. it is difficult to and As a high rates and large of be required high mutations rates may become mutational are for genomic mutation rates because only such can that the reporter gene is in an germ-line simply do not yet for In I a with Crow and and to mutation rates in all of organisms (Drakeet al. to this article of to I was by my that this rate was on very and I rates for and that selection on mutation rates would be to mutations in we to use an size of those base in which most mutations that is, were rates for or for per sexual among these but rates per per germ-line cell division showed of and However, the of three of these values to the rate for microbes is because values were not adjusted for mutations of small and are is such a standard rate for it not be defined instance, is increasing that selection on large of DNA, which to of the size of the As as I the size in microbes is close to However, the of that is deleterious not only in but also in extensive is important to gene and to to these values are not to be this for the might a because the rate of in the recent evolutionary to be roughly than in other of al. 1991). Why should mutation rates be rather than lower in these to DNA when mutation rates an entire sexual generation often values in the of The most is that the of mutations should be lower in a than in a selection for downmodifiers of mutation rates. most of the in these organisms lives for instance, a for the of is a but one of experimental of mutation have for a more of mutation one based on in DNA rather than on However, a recent such report al. was It a genomic mutation rate in to per germ-line cell about than the rate by Drakeet al. In the mutations general were a different the usual and different the for a roughly of in the more (Groganet al. at among the indels was a of of the more of the of in is also with some of their it be important to these the rate and of mutation in or the is not yet the When early articles about mutation in cells, I used to about the mutagenic and might be and I still well this has been In any case, that to the more interesting one of mutation rates might be lower in than in cells, a evolutionary When mutation began to become available using E. coli it soon became clear that samples in about mutations than were in diverse a in some such data I if the of in the samples called and that it might be about A calculation that would then have mutation roughly lower than article soon appeared with the a tenfold al. This appeared to be a tuning of mutation rates, but were more The mutation for was and for plus plus it was but for type A the was an can a mutation be by a of There is a of between type A and type but could could be some other kind of selection against such cells, as selection for rather than in the of human mutations al. This is a for which mutation rates is than was to a burst of and than these mutations with this of more recently, many years of scoring spontaneous mutations in the also revealed a burst of and the mutation rate per sexual generation a large such a then the for presented in Drakeet al. would be However, if this were to the then only that value would be and the value in to be than the other the of several rather large experiments the of DNA we that some mutants produced in and many produced in contained two or more mutations. It was that the multiple mutants were usually more frequent than the the is and the mutations are to a then the number of mutations per mutational is a mutants are the number of is the number of is and so When I published it soon became clear that most contained too many (Drakeet al. 2005). This is with diverse DNA in with viral and microbial with cells, and with and human There were strong that the were not produced by a of mutator but by with too many can be as the sum of two or more a with a lower mutation and one or more with much mutation This may important instance, some of mutations that are or deleterious but when and these are much more by mutational than by single mutations. more mutations than can be produced by the typical mutation rate. seem to a mutator mutation early in their but others do not and may be by of In most the of to the mutation rate is but in it to be so that the of the has a mutation lower than the As a may be somewhat more genetically than of have been in but not are to diverse of such as mutator by of or As I the of a of life rather than I to these I and for their on the This was supported in by the of the of of