The 2-aminopurine-induced transition,A T --G C, was studied at particular sites in bacteriophage T4 as a function of the nearby base-pair composition of the DNA. Changing a base pair changed the transition rate at the adjacent base pair up to 23-fold, and at the next base pair by a lesser amount. Destabilization was achieved by replacing an A -T base pair by a G C base pair.The base-pair substitution mutation rate at a particular genetic site is often thought to depend not only upon the base pair at that site, but also upon the molecular environment determined by nearby base pairs. This idea seems to have originated as an explanation of the "hot spots" observed in the pioneering studies of mutability at the r1I locus of bacteriophage T4 (1, 2). The main difficulty in interpreting the spectra of forward-and reverse-mutation rates arises from indefinite knowledge of which degenerate codon occurs at a given DNA locus, and about what amino acids are acceptable at particular positions in the polypeptide. Determinations of the amino acid compositions of many revertants of amber mutations located in the T4 head-protein gene, however, revealed differing rates of specific base-pair substitution at different positions within the cistron (3). Furthermore, T4rII ochre mutants were induced by hydroxylamine (CAA UAA) at rates that varied by as much as 20-fold at different sites (4). The ultravioletinduced reversion of ochre mutants within the yeast iso-icytochrome c gene differs at different positions: although glutamine is acceptable at two particular positions, for instance, it was induced in 11 of 11 revertants at one, and in 0 of 11 at another (5). While strongly suggestive of neighboring-base effects, results of this type are also compatible with an alternative interpretation, namely, that mutation rates vary within a cistron because of extrinsic factors such as direction of gene replication, direction of transcription, proximity to control elements, and so on. In fact, only a small number of externally determined gradients of mutability would be necessary to explain the variations in specific base-pair substitution mutation rates thus far observed. A convincing demonstration that nearby base pairs do in fact influence base-pair substitution mutation rates therefore requires that a mutation rate be experimentally altered by a nearby base-pair substitution. RATIONALEThe amber (UAG) and ochre (UAA) chain-terminating codons are mutationally interconvertible by a transition in the third position (Fig. 1). Mutation rates at the first two positions of such homologous codons can be measured, providing that the 773 phenotype corresponding to each possible genotype can be recognized with confidence. These measurements are possible using the T4rII system. First, the possible types of base-pair substitutions have been restricted by using the mutagen 2-aminopurine, which produces exclusively transitions. It is already clear that 2-aminopurine readily produces transitions, and the following evidence shows that it does not pro...

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HE apparent mutation rate depends upon a number of contingencies: the Tprimary error rate, the probability o€ repair, error avoidance by the apparatus of DNA replication, and the probability of detection. The probability of detection of those base pair substitutions which produce an amino acid substitution appears to be highly variable, and presumably reflects the sensitivity of polypeptide functions to small variations in primary structure.Fine-scale mapping has often revealed a highly nonrandom distribution of mutations among sites. In the T4rZZ system, for instance, 809 of the first 1609 independently isolated mutants were observed to map into only two sites (BEN-ZER 1961). Genetic data indicate that the number of rZZ sites capable of mutation by base pair substitution is about 1700 (EDGAR et al. 1962;STAHL, EDGAR and STEINBERG 1964), and this estimate is supported by quasi-chemical measurements (GOLDBERG 1966). When frameshift mutations are excluded from consideration, however, fewer than 3% of these potential sites have been identified (FREESE 1959;BENZER 1961;ORGEL and BRENNER 1961; see Chapter 5 of DRAKE 1970). What then is the nature of the apparently immutable sites?Two possible answers deserve serious consideration: many sites are extremely weakly mutable, or else many amino acid substitutions are undetected in standard screening systems because they fail to exert significantly deleterious effects upon protein function. The first possibility is supported by the observation that

Temperature-sensitive mutations in bacteriophage T4 gene 30 (polynucleotide ligase) were examined for their effects on spontaneous and proflavine-induced frameshift mutagenesis in the rII and ac (acridine resistance) cistrons. Only small (fourfold or less) effects on mutation rates were observed, even when selection artifacts involving suppression of gene 30 mutations by rII mutations were taken into account. The deoxyribonucleic acid ligase gene of T4 therefore appears to be only a minor determinant of frameshift mutation rates. This result is consistent with the particular nature of frameshift mutagenesis in bacteriophage T4.

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.