As sentences in scientific papers go, this was guaranteed to raise eyebrows: "We describe here. experiments and some circumstantial

Transcription

1 DIRECTED MUTATIONS by Billy Goodman That bacteria may be able to mutate in response to environmental cues could add a wrinkle to evolutionary theory but is likely to do it no serious damage. As sentences in scientific papers go, this was guaranteed to raise eyebrows: "We describe here. experiments and some circumstantial evidence suggesting that bacteria can choose which mutations they should produce." The sentence appeared in a 1988 Nature paper, 'The Origin of Mutants," by John Cairns, Julie Overbaugh, and Stephan Miller of the Harvard School of Public Health. The paper inflamed passions in the genetics and evolutionarybiology communities and reopened an issue that biologists had considered long settled. Must mutations arise spontaneously, independent of natural selection and without regard to their potential usefulness? the Harvard researchers asked. Or do mutations sometimes arise as a specific response to the current needs of an organism? For nearly half a century, the unequivocal answer has been that mutations are spontaneous and random. According to the neo-darwinian view of evolution, variation created in this fashion is then acted on by the forces of evolution, chiefly natural selection, to produce change. One blunt sentence and what the authors referred to as "three or four rather ambiguous experiments" are not enough to overturn the mutational cornerstone of neo-darwinism, in the minds of most biologists. After all, the alternative view, that mutations happen in direct response to need, harks back to the theories of Jean Baptiste de Lamarck, the French forerunner of Charles Darwin. Lamarck's views often are caricatured simplistically as "inheritence of acquired characteristics." No one in this debate about mutation is abandoning natural selection as the prime shaper of evolution. But Cairns and some supporters suggest that evolutionary theory must incorporate a new wrinkle. They say that some mutations may occur more often when they are advantageous than when they are not. (Some call this contention neo-lamarckian, but Cairns professes no interest in such a label) Cairns's report in Nature was provocative but short on experimental details and data. Several biologists have since obtained experimental results, mainly with the bacterium Escherichia coli, that they say support Cairns and what has come to be called directed mutation. Meanwhile, some criticize the results on theoretical grounds. A few have produced experimental results that contradict at least some of the observations supporting directed mutation. The blunt language of Cairns's paper and its challenge to long-held views stirred controversy and debate that continue with each new paper published. For Sahotra Sarkar, a Boston University philosopher of biology, the uproar is appropriate: "Science working as it should; claims have led to skepticism, which has sparked people to do further experiments." The Luria-Delbruck distribution Demonstration of the spontaneity of mutation was one of the triumphs that ushered in the modern age of molecular biology, ten years before the elucidation of the structure of deoxyribonucleic acid, or DNA, by James Watson and Francis Crick. The credit goes largely to Salvadore Luria at Indiana University and physicist-by-training Max Delbriick at Vanderbilt University, who were studying bacterial viruses called phages. 24 MOSAIC Volume 23 Number 1 Spring 1992

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3 In the early 1940s, bacteriologists were largely in the dark about the nature of heredity and mutation in bacteria. However, they easily,could select for mutants, such as bacteria resistant to attack by phages. When abacterial culture was spread on a plate containing an abundance of phage particles, typically only a rare colony, representing a single resistant bacterium and its descendants, would arise after incubation. The obvious question was: Did contact with the phages cause bacteria to become resistant, at some low frequency? Or did bacteria spontaneously become resistant while growing in the test tube before contact with the phages? In that case, phages simply would be revealing preexisting mutants by killing sensitive cells. The answer came in a series of elegantly simple experiments conceived by Luria, analyzed mathematically by Delbriick, and widely regarded as the birth of bacterial genetics. Luria and Delbriick presented their conclusions in a famous 1943 report in Genetics: "We consider the... results as proof that in our case the resistance to virus is due to a heritable change of the bacterial cell which occurs independently of the action of the virus. It remains to be seen whether or not this is the general rule." Goodman is a free-lance science writer based in Brooklyn, New York. He was also the author of'toward a Pump, Not a Filter," Mosaic Volume 22 Number 2. The method of proof was indirect. They relied on a statistical argument. Luria said later that the argument occurred to him when, as he watched a slot machine at a faculty dance, ; he noticed that most coins fed into the machine yielded nothing. Occasionally, small returns were forthcoming. Rarely, one coin produced a jackpot. Luria realized that the slot machine might be a model for spontaneous mutation, in which a mutation to phage resistance could occur at any time during the history of a growing culture. If a mutation occurred early, subsequent cell division ; would lead to a large clone of resistant cells. When plated in the presence of phages, many resistant colonies would appear, even though only one mutation had occurred. Such a situation would mimic the rare jackpot from the slot machine. Late-occurring spontaneous mutations would produce smaller clones. A series of cultures would show great variability in the number of resistant cells. This clonal statistical distribution is generally called the Luria- Delbriick distribution. The last bastion By contrast, if mutations to phage resistance occur only after exposure to phages, results of plating replicate cultures would be quite different. If each cell has the same small chance of being made resistant and if roughly the same number of cells are exposed on each plate, then each plate would contain similar numbers of mutants and no jackpots. The mutants would represent a Poisson distribution around a mean value. That value is the product of the number of bacteria exposed and their probability of becoming resistant. This is known as the fluctuation test, because it examines the fluctuation in the number of mutants per plate. The results clearly demonstrated the existence of jackpots, so mutations must have been occurring spontaneously during the growth of the cultures before they were challenged with phages. The Luria-Delbriick analysis contained clear caveats ("in our case" and "it remains to be seen"). It also stimulated others to look for more direct ways of distinguishing spontaneous from environmentally induced adaptive mutations. Joshua Lederberg, then at the University of Wisconsin, confirmed and extended the Luria-Delbriick results. Lederberg and his colleagues showed that mutations for resistance to some antibiotics occurred spontaneously. The researchers ingeniously revealed that cells that never had been exposed to the antibiotic still could be resistant. Lederberg says that his work and that of Luria, Delbriick, and others "brought bacteria into the [neo-darwinian] fold. Bacteria had been the last bastion of Lamarckian ideas." Since these famous experiments of the 1940s and 1950s, almost no one seriously had believed that mutations may occur in response to the need of an organism. No one, that is, until John Cairns. Challenge... A molecular geneticist interested in the rate-limiting steps of mutations that lead to cancer, Cairns recognized that Luria, Delbriick, and the others never had really tested whether the environment could selectively induce mutations. For example, for a bacterial cell to become resistant to phages, the cell must have a mutation before it comes in contact with the virus. Phages bind to cell-surface receptors and kill sensitive cells immediately, before they replicate and have a chance to mutate. When a mutation makes a cell genotypically resistant, its surface still will bear phage 26 MOSAIC Volume 23 Number 1 Spring 1992

4 receptors that had been made previously. The cell will remain sensitive. Only after several cell divisions, which eliminate the receptors by dilution, will the progeny be phenotypically resistant. As a result of phenotypic lag, "these classical experiments could not have detected (and certainly did not exclude) the existence of a non-random, possibly product-oriented form of mutation," Cairns and his colleagues wrote. "If you want to know whether creatures can turn on a mutation they need," John Cairns says, "it's not fair to kill them. You want to know if they can turn on a mutation by worrying about the environment." To give bacteria something to worry about, and time to respond adaptively, Cairns and his colleagues imposed nonlethal selection. In separate experiments, they plated strains of E. coli that shared an inability to use lactose as an energy source (and thus are designated Lac-) on agar plates with lactose as the only energy source. The conditions were sharply selective for Lac+ cells. Under such conditions Lac- cells do not die, however. They enter a starvation state known as the stationary phase. It has been known for several decades that mutations can occur in stationary-phase bacteria. This is paradoxical. For a mutation to become part of the genome, a damaged DNA strand the incipient mutation must be replicated before it is corrected by DNA repair systems. Yet stationary-phase bacteria no longer replicate their DNA or divide. How can mutations happen in such bacteria? Stanford University's Philip Hanawalt says that limited DNA repair activity is believed to take place in stationary-phase bacteria. Stationary-phase bacteria are hardly quiescent; many of their genes are specifically expressed. One experiment typifies what Cairns and his colleagues found. Some Lac+ revertants formed colonies within 48 hours of plating, providing evidence that they probably formed spontaneously in the growing cultures before plating. Other revertants continued to appear on the plates as the bacteria starved. This is a key observation, which Cairns was not the first to make, but which he was the first to exploit Cairns also noted that revertants did not accumulate when lactose was absent, a circumstance in which the mutations would not have conferred any advantage.! The shape of the distribution of mutants also suggested to Cairns and his colleagues that some mutations occurred spontaneously during the growth phase and that other mutations occurred during the stationary phase. The shape was similar to that predicted by models in which mutations happen randomly during the growth of a culture, followed by additional mutations in the final generation, after exposure to the selective agent has taken place. All these facts led Cairns to conclude that the selective agent was inducing mutations in the stationary- phase E. coli cells on his plates.... and response As befits a boldly written paper in a prominent journal, Cairns's article galvanized evolutionary biologists. Critical letters poured into Nature pointing out that Cairns and his colleagues had failed to rule out simpler alternatives to directed mutation. Barry Hall of the University of Rochester, who has published a series of papers that are widely regarded as the best evidence for directed mutation, has written that Cairns's paper caused controversy for two reasons. "First, it challenged the orthodox view of the complete separation of mutation and selection; second, it suggested the existence of unknown mechanisms by which the environment could somehow instruct a cell as to which mutations should be generated or retained," Rochester's Barry Hall wrote. Hall was inspired by discussions with Cairns before the publication of Cairns's Nature article. Then at the University of Connecticut, Hall began working with a system that required two mutations to occur before E. coli could use the sugar salicin. One of the mutations, he documented in a 1988 Genetics article, is so rare in growing cells that he failed to detect it. But plated on salicin, Sal+ revertants appeared after a delay of about 12 days. Their frequency was orders of magnitude greater than expected if the two mutations were occurring independently and spontaneously. Furthermore, the rarer spontaneous mutation happened first This mutation was excision of an insertion sequence. It was followed relatively quickly by the second mutation, which only then permitted transcription. When salicin was absent, Sal+ revertants had no advantage, and Hall was unable to detect excision mutants. Among Cairns's critics, Bruce Levin of the University of Massachusetts is typical in acknowledging that Cairns's paper "made me aware that the alternative hypothesis to random mutation hadn't been rejected." Levin is one of a handful of biologists who use microbes to probe evolution. He says that the distribution of mutants on plates can shift from the clonal arrangement expected of spontaneous mutations toward the Poisson distribution expected of specifically induced mutations for a variety of simple biological reasons. He has pursued theoretical and experimental work on this topic with postdoctoral associate David Gordon in his laboratory and mathematician Frank Stewart of Brown University in Providence, Rhode Island. Following the lead of earlier workers who had studied the Luria-Delbriick distribution, the trio presented what Levin calls "a general method for incorporating virtually any form of biological reality into the distribution of mutants." The key conclusion, he says, is that almost any biological complication, such as mutants growing faster or slower than the parental strain, shifts the distribution to ward the Poisson. "The implication of this is that one can't, or shouldn't, use MOSAIC Volume 23 Number 1 Spring

5 deviations from Luria-Delbruck distributions as arguments for directed mutation, as Cairns and colleagues did." Nonetheless However, Levin's arguments about statistics do not address Cairns's observation of accumulation of mutants on plates. Levin suspects that this accumulation was not a result of mutations in specific response to the selection pressure. Instead, most of the mutants on the plates were replication-dependent, spontaneous mutants. For a variety of physiological and ecological reasons, some occurred in the growing cultures but were delayed in their appearance on the plates. Others occurred on the plates themselves as a result of growth by cross feeding. For example, a Lac- cell can divide on a plate containing lactose as a carbon source, by using metabolites excreted by Lac+ colonies that are already on the plate. Patricia Foster, a bacterial geneticist at the Boston University School of Medicine who has done some further experiments with John Cairns, says that growth on plates does not account for the accumulation of all revertants. In her work, the bacterial population on the plate never grows enough to account for the number of revertants seen. Another possible explanation for the observations is that the stress of starvation could cause a general increase in mutation rates, rather than a specific directed response. That is what was suspected by Richard Lenski, an evolutionary biologist at the University of California at Irvine, and his graduate student, John Mittler. "We're not convinced by the control experiments published so far or by the bookkeeping that certain mutations are occurring more often in the presence of favored selection than not," Lenski says. Shortly after Cairns's paper appeared, Lenski and Mittler took aim at one of the key examples. That example was based largely on a 1984 paper by University of Chicago microbiologist James Shapiro. Shapiro, in fact, is the unsung protagonist of this entire field. His paper foreshadowed Cairns's 1988 work, referring explicitly to the problem that phenotypic lag presented for the Luria-Delbrtick paradigm. Using the techniques of Chicago colleague Malcolm Casadaban, Shapiro worked with an E. coli strain which was engineered to study hybrid proteins. Shapiro's construct had an arabinose regulatory gene followed by an arabinose structural gene into which was inserted a Mu prophage. Further downstream was the laczgem that codes for beta-galactosidase, the enzyme that degrades lactose. This arrangement, in which the strain could grow on neither arabinose nor lactose, is designated Lac (Am)-. However, appropriate excision of the Mu element fused the initial region of the ara gene to the beginning of the laczgene, allowing the bacterium to grow as long as lactose and arabinose are present. Shapiro demonstrated that ara~lac fusions occurred only after plating on a selective medium, with both arabinose and lactose, and only after a long delay. Cairns confirmed these results. The interpretation of Cairns's work on this system is still debated by his supporters and critics. As Boston University's Foster sees it, "Cairns showed that saturated cultures that had exhausted their carbon source did not produce fusions unless and until lactose (and arabinose) was added." To Cairns and his coauthors, the events seem to be "another example of the production of appropriate mutations in response to selection." On the other hand, John Mittler says, he and Richard Lenski were concerned that the experiments of Shapiro and Cairns did not adequately distinguish between the effects on the rate of Mu excision caused by cell starvation and those caused by the presence of lactose and arabinose. Mittler and Lenski therefore designed an experiment to focus on the effect of starvation on the rate of Mu excisions. They plated Lac (Ara) - cells on plates with minimal nutrients and no added sugars. They sprayed some plates immediately with lactose and arabinose. Any Lac (Ara) + colonies that appeared within two days of spraying were considered to have been preexisting and to have formed spontaneously. When the plates were sprayed at the time of plating, no Lac(Ara)+ colonies appeared within two days. However, the longer the bacteria were allowed to starve on the plates, the more Lac (Ara) + colonies were revealed within two days. After controlling for cell death and growth, 28 MOSAIC Volume 23 Number 1 Spring 1992

6 Mittler and Lenski concluded that the increased rate of excision of Mu elements could be attributed entirely to the stress of starvation. Among starving cells excision mutations occurred at comparable rates in the presence or absence of lactose and arabinose; there was no need to invoke directed mutation to explain the excisions. Another route After his salicin paper appeared, Hall chose to work on E. coli with base-substitution mutations that rendered the bacteria unable to synthesize the amino acid tryptophan. Bacteria that are unable to synthesize an amino acid are called auxotrophs because they must be supplied with a nutrient to grow, tryptophan in this case. In growing culture with tryptophan supplied, the bacteria that Hall used reverted to prototrophy at the rate of about one every ten billion cell divisions. When Trp- cells were incubated on plates without tryptophan, some revertants appeared within four days. These were judged to be preexisting. Revertants continued to appear after the fourth day, for as long as ten days (since extended to 28 days). In an important experiment, Hall used an E. coli strain that was auxotrophic for the amino acids tryptophan and cysteine to test the specificity of the mutagenic response to starvation. Colonies that were Trp-Cys- were starved for either tryptophan or cysteine. Those starved for tryptophan reverted to Trp+, but did not revert to Cys+. Those starved for cysteine reverted to Cys+ but did not revert to Trp+. These results demonstrated that the delayed mutations were specific to the selection that was imposed upon them. Hall believes that he eliminated the possibility of growth on plates by using ampicillin to kill growing cells. He measured the death rate of cells on selective plates in the presence and absence of ampicillin. With ampicillin, cells could die, but new cells could not arise by cell division. The observed death rate equals the actual death rate minus the growth rate. If growth were occurring, the observed death rate would be greater in the presence of ampicillin than in its absence. However, the observed death rates were identical. Despite strong confirmation of preferential production of advantageous mutations, Hall was not ready to accept a truly directed mechanism. In the 1988 Nature paper, Cairns and his colleagues had sketched three possible mechanisms for their observations. One required some form of information flow from protein back to the DNA, counter to the central dogma of molecular biology. Cairns speculated that a bacterium under stress might make many mistakes in transcribing its DNA. For any gene, a variable set of messenger RNAS would exist Using a still-unidentified organelle, the cell then would sense which mrna made the best protein and would reverse- transcribe the messenger. Aside from the fact that no direct evidence has been found to support such a model, there are "about 47 problems" with it, quips Hall. "I accepted [John Cairns's] argument about the limits of the Luria-Delbriick work, but I was firmly rooted in conventional dogma," Hall says. John raised the question of a specific response to the environment. I kept asking, 'Could an underlying random process account for what I'm seeing?'" In fact, Cairns and his coauthors had suggested random processes for their second and third mechanisms, in a single sentence that attracted much less attention than the more speculative first mechanism. Error-prone stages Franklin Stahl, of the Institute of Molecular Biology at the University of Oregon, gave a more detailed version of one of Cairns's random mechanisms in a "News and Views" report that accompanied Cairns's paper in Nature. Stahl's model is based on the knowledge that the first steps of DNA synthesis are error prone. Those steps create thousands of mispairings and other incipient mutations. Mismatch-repair systems largely correct those errors before replication can proceed to stabilize them as mutations. In the absence of mismatch repair, DNA synthesis has an error rate of about one per million bases, which shrinks to about one per billion bases after mismatch repair. Stationary cells do not replicate their DNA, but there is thought to be limited DNA turnover or repair synthesis, as polymerases remove stretches of DNA and put in patches. Mistakes occur during MOSAIC Volume 23 Number 1 Spring

7 sluggishness, of the repair system has no consequence for incipient mutations that do not permit the cell to grow, Stahl says. As long as the cell remains In sta- tionary phase, such mutations eventu- ally will be repaired. Another model, again relying on well- known molecular biology, was proposed In 1989 In the Proceedings of the National Academy of Sciences by bacterial physi- ologist Bernard Davis, retired from the Harvard Medical School. Davis calls his mechanism transcriptional bias. He rea- this process. In what Stahl terms the one speculation of his model, he suggests that mismatch repair in starving cells Is sluggish and depends on a slow trickle of energy, perhaps from cannibalizing the cell's own building blocks. If by chance a useful error occurs, the cell Is able to grow In Its current environment. Then the cell will replicate Its DNA and divide. If replication occurs faster than mismatch repair can correct the error, the mutation becomes a permanent part of the cell's genome. The soned that transcription Itself might be mutagenic, because the double helix of DNA unzips partly during transcription in order to permit one strand to form a template In making mrna. "Single-stranded DNA is much more vulnerable to mutation," Davis says. "During transcription there is always a region of about 12 single-stranded bases behind the RNA polymerase," he says. Normally, transcription proceeds at a pace of about 45 bases a second, but Davis speculates that It may be much slower as a cell starves for nutrients, leaving the window of single-stranded bases open longer. What would account for mutations being directed at just the gene or operon that a cell needs fixed? Take the lactose operon as an example. Although a mutation In the lacz gene may render the enzyme inoperative, its operator should still function to turn the operon on. According to the Davis model, if bacteria are plated on lactose, their lac- operons should hold temporary regions of singlestranded DNA. A trigger In a 1990 Genetics paper, Hall proposed his own heuristic model along the lines of the Stahl and Davis models. Essentially, according to Hall's model, when life is tough, cells mutate like crazy, in hopes that something good will happen. Hall wrote that the problem "is to explain how selective conditions could Increase the frequency of useful mutations without increasing the frequency of mutations at other loci." He suggested that during prolonged starvation, some cells in a colony enter a hypermutable state. In this condition, both extensive random DNA damage and error-prone repair occur. By chance, an error introduced into the DNA during this process might solve the problem facing the cell at the time. The cell then would 30 MOSAIC Volume 23 Number 1 Spring 1992

8 replicate Its DNA and stabilize the advantageous mutation. The cell then would exit the hypermutable state. If none of the incipient mutations proved useful, the cell would die. It wouldn't "hang around for you to look for other, nonselected mutations," Hall says. Foster points out that Hall's hypermutable-state model, Stahl's slow-repair model, and one of the random models proposed In Cairns's 1988 paper share the same underlying idea: "There is a reversible process for creating variation, that only results In permanent genetic change if the cell is successful." The hypermutable-state model predicts that mutations at nonselected loci should be found In successful mutants. Hall screened 110 trp+ revertants and discovered two other auxotrophic mutations. This frequency of auxotrophic mutations "Is about 600-fold higher than expected If the two mutations (trp reversion and an auxotrophic mutation) were independent events," Hall wrote. Joshua Lederberg, who played a leading role In starting the field of microbial genetics nearly a half century ago, was one of those Inspired to investigate the molecular biology underlying apparently directed mutation. Until a few years ago, Lederberg was president of Rockefeller University. Now he and a colleague, David Thaler, are beginning to examine the Issue from a mechanistic perspective. Mechanistic Lederberg finds Davis's views "very congenial" to his own. He suggests that there is "a good case that differences in secondary structure of DNA will result In differential mutability." In other words, the DNA double helix Is hardly homogeneous. There are all manner of landmarks along Its length that could be targets for mutagenesis. Those landmarks are related to its supercoiling and the proteins with which It interacts. In Davis's model, It Is the single-stranded region around the RNA polymerase that is the target. MOSAIC Volume 23 Number 1 Spring

9 The little evidence that addresses the transcriptional-activation model is not supportive. Cairns says he excluded it with an experiment reported in 17 words in the 1988 paper. The experiment employed isopropyl beta-d-thiogalactoside (IPTG). This substance is known as a gratuitous inducer because it can induce the lactose operon to begin transcription, but it cannot be degraded by betagalactosidase. Thus it provides no energy to the cell Cairns reported that Lac+ mutants did not accumulate when Lac- cells were grown in the presence of IPTG. If transcriptional activation alone were enough to stimulate mutation, Lac+ revertants should have accumulated with IPTG. Cairns returned to the laboratory with Patricia Foster. In 1991, the pair published work on reversion to Lac+ in a strain of E. coli that contains a frame shift rendering it Lac- but in which the lactose operon is permanently switched on. Foster says their work disproved transcriptional activation in this case. The operon was always transcribed, whether or not lactose was present, and yet Lac+ revertants accumulated only when lactose was present. Foster and Cairns found preliminary evidence that what they call adaptive mutation depends on a function of the reck gene, which is part of the recombination-repair system of DNA. In their experiments, mutations in reck had no effect on spontaneous mutation during growth. However, the mutations in reck greatly reduced the occurrence of mutations appearing on the plates after lactose had been added. Transcription Meanwhile, others remain convinced that transcription is playing a role in the phenomenon. Thomas Cebula, chief of the molecular biology branch of the division of microbiology of the United States Food and Drug Administration, and Michael Prival of the agency's genetic toxicology branch, have found evidence for late-appearing, apparently directed mutations in the common bacterium Salmonella typhimurium. They studied missense mutations in the histidine operon. Reversions to His+ could occur intragenically, by base substitutions in the codon for a crucial amino acid. Alternatively, reversions could occur extragenically, by a suppressor mutation that causes transfer RNA to make a mistake in translation that cancels out the mutation- causing histidine auxotrophy. Cebula says that both kinds of reversions appear to have happened spontaneously in growing cultures. However, only intragenic events were observed in colonies that had starved for more than two days. The absence of late-arising suppressors is consistent with a transcriptionalactivation hypothesis, says Cebula. In addition, he says, the kinds of mutational events that they uncovered are consistent with Stahl's model of mismatch repair being slow. However, they have not measured repair rates directly. Philip Hanawalt, an expert on DNA repair at Stanford University, is also convinced that both transcriptional activation and DNA repair are playing some role in the phenomenon of directed mutation. Recent work in his laboratory shows that repair may be more efficient in active genes than in inactive ones. Hanawalt also suspects that the mismatch repair system is not working in starving cells. As Hanawalt sees it, a starving cell may use repair synthesis to put in a 32 MOSAIC Volume 23 Number 1 Spring 1992

10 patch of DNA, perhaps where an RNA polymerase got hung up at a kink in the DNA. If mismatch repair is not operating, the error rate during repair synthesis would be about 1,000-fold higher than during normal DNA replication. 'The mere act of putting in a patch entails the possibility of making a mistake, because polymerases are not perfect," Hanawalt says. A direct test The transcriptional-activation model and its variants continue to attract experiments, but so far the only direct test of Hall's model has been by Hall himself, in experiments published in Proceedings of the National Academy of Sciences in He measured the reversion to trp+ of a strain that was doubly mutant, trpatrpb~. He found double mutants (revertants to tryptophan prototrophy) approximately 100 million times more frequently than expected if the mutations were independent Hall calculated the probability of each base substitution occurring, given that the cell had entered a hypermutable state. The conditional probability for each mutation worked out to about That figure should apply to other bases in the genome, according to the hypermutable-state model. But "that's mayhem," Hall says. "That won't fly. You can't have a base-substitution rate of one in 25 and live to tell the tale." Hall wondered whether such a high rate might apply only to a small region of the genome around the trpa and trpb loci, so he sequenced 700 base pairs around the two loci in 11 double revertants. The hypermutable-state model predicts that other base substitutions should occur among those 700 base pairs. He failed to find any, and concludes that in its simple form, the hypermutable-state model is not correct. Barry Hall argues that the analysis also applies to the Stahl and Davis models, making them incorrect in their simple versions as well. Many biologists, though by no means all, are convinced that some mechanism in bacteria allows at least some incipient mutations to be incorporated into the genome more frequently when they are advantageous than when they are not. The production of variation continues to be regarded as a completely random phenomenon, although many interpreted the 1988 Nature paper as an argument for nonrandom production of variation. As they try to document and probe the incorporation of mutations from all angles, some biologists speculate on its importance for evolutionary biology. A question of significance Cairns himself, recently retired from the Harvard School of Public Health, says there may be no evolutionary significance. "We're looking at reversion of defects, which is somewhat unnatural," Tie declares. ~ - On this point, at least, Bruce Levin agrees. To accept the existence of directed mutation, we have to believe that, in addition to random mutation, organisms have mechanisms to produce specific modifications of their genomes to repair genes that are normally functional and very rarely mutate to nonfunctional states," Levin says. Could directed mechanisms work in organisms other than bacteria? Foster thinks that is unlikely. "Organisms with a germ line have a tremendous barrier," she says. 'The whole game is to protect the genome from the environment." (In late 1991, there were preliminary reports, as yet unpublished, of similar phenomena in yeast, a one-celled eucaryote.) Nevertheless, the possibility that one or more mechanisms may increase the rate at which beneficial mutations occur in starving cells has drawn increased attention to the stationary phase and new respect for E. coli and other bacteria in their natural habitats. Starving on a laboratory plate is far more typical of the conditions under which most bacteria evolved and live than the optimal laboratory conditions in which many bacteria can grow exponentially: aerated liquid cultures in 0.2 percent glucose solutions kept at 37 degrees Celsius. "E. coli has another life," Boston's Patricia Poster says. "It's not over when the log phase ends." The National Science Foundation has contributed to the support of the research described in this article principally through its Genetics program. MOSAIC Volume 23 Number 1 Spring