Chaos and Order in Spontaneous Mutation

Abstract

T HIS 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 muta-tional 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 GÁC / AÁT 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. Phages and antimutators: 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 frame-shift 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. 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 l 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 1