SITE SPECIFICITY AND VARIABILITY IN THE MUTATOR AND ANTIMUTATOR EFFECTS OF PHAGE T4 GENE 43 MUTANTS

AMIRAM RONEN, CALOMIRA HALEVY AND NAVA KASS

Department of Genetics, The Hebrew University of Jerusalem, Israel

Manuscript received March 29, 1978 Revised copy received May 30,1978

ABSTRACT

Spontaneous, 2-aminopurine- and 5-bromouracil-induced mutations at six r l l nonsense codons were studied in phage T4 strains possessing wild-type and mutant gene 43 alleles. The mutation pathways studied included inter- conversions and reversions of nonsense codons. The tsCB87 allele, which speci- fies an antimutator DNA polymerase, reduced base-analogue-induced muta- tion frequencies along all pathways. However, GC base pairs were less affected than A T base pairs. The frequency of spontaneous UAA+UAG con- versions was also reduced by tsCB87, but that of spontaneous UAA-UGA conversions was often increased. Mutation in the presence of the mutator allele tsL56 was increased along all pathways, with no preference for either A T or GC base pairs. Mutation frequencies in the presence of the two mutant DNA polymerases were highly variable. A strong correlation was found between 2-aminopurine-induced mutation frequencies in ts+ and tsCB87 phage along the reversion and UAA-UAG (but not UAA-UGA) pathways.

ONSENSE mutations in the rZZ region of bacteriophage T4 are sensitive and Nhighly efficient tools in the study of the specificity of mutation (SALTS and RONEN 1971; RIPLEY and DRAKE 1972; RIPLEY 1975; RONEN and RAHAT 1976). The ochre (UAA) +opal (UGA) interconversion* system (RONEN and RAHAT 1976) is especially useful because one can compare transition frequencies of homologous AT and GC base pairs at different sites along the rZZ gene. Since all these base pairs are flanked by similar (AT and TA) neighbors, any variability in the frequency of their mutation is not attributable to nearest-neighbor effects. On the other hand, the frequencies at which rZZ nonsense codons mutate along other pathways are known to be affected by the nearest neighbors of the base pairs involved (SALTS and RONEN 1971; KOCH 1971; RONEN and RAHAT 1976; RONEN, RAHAT and HALEVY 1976). The mechanism whereby these neighbor effects operate is not clear, but site specificity of the phage DNA polymerase may be an important factor (KOCH 1971; DE VRIES, SWART-IDENBURG and DE WAARD 1972; RONEN, RAHAT and HALEVY 1976).

The three nonsense mutations are referred t o as ochre (UAA), opal (UGA) and amber (UAG). Unless stated other- wise, mRNA language is used to describe DNA sequences. Conversion is the mutation of a nonsense codon to another nonsense codon. Abbreviations: A, T, U, G and C are adenine, thymine, uracil, guanine and cytosine (or 5-hydroxy- methylcytosine), respectively; 2AP and 5BU are 2-aminopurine and 5-bromouracil, respectively.

Genetics 90: 647-657 December, 1978.

648 A. RONEN, C. HALEVY A N D N. KASS

In the present work, we used T4rZZ nonsense mutants to study the specificity of spontaneous, 2-aminopurine- and 5-bromouracil-induced mutations in strains that possess altered DNA-polymerase genes, as the result of mutation in gene 43. DNA polymerase plays a major role in regulating mutation rates, by determining the accuracy of DNA replication (BRUTLAG and KORNBERG 1972; TOPAL and FRESCO 1976). Some temperature-sensitive mutations of gene 43 affect the fidelity of the enzyme even under permissive conditions, thus conferring on it a mutator or antimutator phenotype (SPEYER, KARAM and LENNY 1966; DRAKE and ALLEN 1968). However, in order to understand the mechanism whereby DNA polymerase regulates mutation rates, one needs to know the sequence specificity of the enzyme in the mutation process.

The specificity of the mutator allele tsL56 in spontaneous and baseanalogue- induced reversion of rZZ mutations was studied by BAUTZ-FREESE and FREESE (1967) and RIPLEY (1975). The specificities of various antimutator gene 43 alleles were studied by RIPLEY (1975) and DRAKE and ALLEN (1968). In all cases, however, the rZZ mutants studied were either too few or too ill-defined with regard to the mutational pathway. We undertook this study with six nonsense sites, in order to obtain a broader view of the specificity of mistakes made by DNA polymerase.

MATERIALS AND METHODS

T4rll mutants have been described previously (RONEN and SALTS 1971; SALTS and RONEN 1971).

T4 DNA polymerase mutants are tsL56 (with a mutator phenotype) and tsCB87 (with an antimutator phenotype). Both mutants (derivatives of TIED) were obtained from J. W. DRAKE and introduced into the r l l strains in tbe following way: each ts mutant was backcrossed to T4B four times at an input ratio of 1 ts:20 wild type (wt). The progeny of a single phage from the fourth backcross generation were crossed with each OF the r l l mutants. The various r l l strains (which are derived from T4B) thus acquired little of the T4D genetic background along with the mutant site in gene 43.

Bacterial strains: The K12(A) sufUGA strain used in this work is RN340. I t was constructed by transducing the opal-suppressor gene from strain K223 (RONEN and RAHAT 1976) into a A-lysogenic derivative of K12 thi- lac,, ,2229 (described by LANGRIDGE and CAMPBELL 1969, and obtained from J. LANGRIDGE). Other K12(X) strains with nonsense suppressors used in this work are RN334/1 suf,,, and RN1611 S U + , , ~ (RONEN and RAHAT 1976).

Mdasuring mutation frequencies: Single plaques of the various mutants were suspended in 2 ml of nutrient broth, and the frequency of spontaneous r+ phage in them was measured. Plaques with the lowest frequency of r + phage were used to make lysates, either for treatment with BAP or with 5BU, or as untreated controls. Typically, three or four lysates, each from a separate plaque, were used for each treatment. In some cases, plaques picked from plates seeded with E . coli BB had lower initial concentrations of r f revertants than did plaques from E . coli B plates.

UAA-+UGA and UAA-+UAG conversions were measured on RN34.0 and RN1611, respec- tively. UAA+wt reversion frequencies are average values from measurements on RN34.0 and RN1611 bacteria. U A G w t reversion was measured on RN340. UGA-twt reversion and UGA+UAA conversions were measured on RN334/1. Each value is the geometric mean of at least three measurements involving different phage stocks.

Mutation frequencies and their raiios: In describing the effects of the various mutagenic treatments and gene 43 alleles on mutation frequencies, we follow the convention set forth by

NEIGHBOR EFFECTS O N MUTATION IN T 4 649

RONEN, RAHAT and HALEVY (1976). Thus, Rts+ is the reversion frequency of amber triplets

(through UAG+CAG or UAG+UGC transitions) in the presence of gene 43 tsf, and Rt*L5suAn

is the frequency of UAAdUGA conversion in the presence of tsL56; the effect of a mutant gene 43 allele on a given mutational pathway is described as the ratio, e g., RtsCB87UAA/RtsUAA, of the frequency of mutation in presence of that allele to the frequency of mutation in the presence of the wild-type allele.

UAG

RESULTS

Variability of mutation frequencies: Spontaneous mutation frequencies of the nonsense triplets at six sites, along different pathways, are given in Table 1. 2AP- and 5BU-induced mutation frequencies are given in Tables 2 and 3, respectively.

Mutation frequencies are rather variable in the presence of any of the DNA polymerases. This variability is generally higher after induction with 2AP or 5BU, but its magnitude is similar in mutants carrying the ts+, tsCB87 and tsL56 gene 43 alleles. No pathway seems to have specially high o r low variability of mutation frequencies. Also, the variability in spontaneous mutation frequen- cies along a given pathway does not seem to correlate with that along any other

TABLE 1

Spontaneous mutation of rII mutants in the presence of antimutator, wild-type and mutator D N A polymerase

Mutational pathway Gene 43 rll (1) (2) (3) (4) ( 5 ) (6 )

allele mutant UAA+ wt UAA- UGA UAA-t UAG UAG, wt UGA+ wt UGA-t UAA - tsCB87 YH115 0.037 0.014

ts+

tsL56

YH119 YH122 YH132 YH320 YH341

YH115 YH119 YH122 YH132 YH320 YH341

YH115 YH119 YH122 YH132 YH3U) YH341

0.007 0.018 0.008 0.004 0.002

0.060 0.082 0.12 0.007 0.037 0.26

41. 0.18 1.45 0.50 5.5

15.

0.002 0.00 0.002 0.004 0.002

0.004 0.008 0.00 0.0004 0.006 0.00

0.63 0.023 0.19 1.3 0.94 0.0

0.000 0.67 0.0006 0.11 0.00 0.10 0.000 0.05 0.0006 0.14 0.000 0.26

0.031 0.40 0.007 0.16 0.007 0.34 0.002 0.30 0.003 0.26 0.024 0.10

0.78 20. 0.014 140. 0.06 1.3 0.20 6.5 0.18 100. 0.38 21.

0.79 0.18 0.61 0.28 0.31 0.42 0.76 0.70 0.68 0.31 0.31 0.48

3.2 0.38 0.86 0.015 2.1 0.78 0.67 0.53 1.3 0.34 2.5 0.16

25. 0.25 11. 0.18 61. 0.50 4.6 1.95

50. 2.8 14. 16.

R values are per IO7 phage, and are rounded to two significant figures. A zero in the last decimal place indicates that the value in that place is less than one.

65 0 A. RONEN, C . HALEVY A N D N. KASS

TABLE 2

2-Aminopurine-induced mutation of rII mutants in the presence of antimutator, wild-type and mutator DNA polymerase

Mutational pathway Gene 43 rll (1) (2) ( 3 ) (4) ( 5 ) (6)

allele mulant UAA+ wt ULM+ UGA UAI+ UAG U A G t wt UGA+ wt UGA+ UAA

tsCB87 YH115 YH119 Y H l B YH132 YH320 YH341

t s +

tsL56

YH115 YH119 YH122 YH132 YH320 YH341

YH115 YH119 YH122 YH132 YH320

3.5

9.0 0.85 0.052

59.

14.

280. 350. 330. 94. 14.

330.

1800. 250.

1400. 140. 300.

0.26 12. 0.14 0.060 0.33 3.0 0.091 0.081 0.18 0.018 0.60 14.

6.5 1100. 6.3 19. 2.2 350.

12. 8.8 21. 2.2 17. 490.

260. 6300. 3.1 22. 9.2 1400.

54. 13. 430. 41.

11. 31. 54. 7.3 2.6

85.

1400. 1600. 2700.

630. 430.

5500.

6000. 7000. 7700. 3600. 1600.

49. 11. 44. 0.39 1.2

700.

680. 190. 870. 31. 13.

2100.

3100. 120.

27,000. 130. 670.

4.7 0.68 9.7 0.86

47. 20.

5.8 4.7

7.2 54.

67. 350.

1100. 65.

2600. 810. 170.

YH341 4600. 120. 5200. 22,300. 3900. G O O .

For details, see legend to Table 1.

pathway. Upon 2AP and 5BU treatment, UAA-tCAA and UAA+UAG mutation frequencies in tsL56 phage are correlated (at the P = 0.05 level of significance).

To show the effects of the mutant DNA polymerase on mutation frequencies, the data in Tables 1-3 are represented in Figures 1 and 2 as the ratios, RtSCBB7/ RtS+ and RtsLs6/Rts+, of the mutation frequencies in tsCB87 or tsL56 phage, to those in ts+ .

In the top line of Figures 1 and 2, only interconversions of nonsense codons are considered. The mutation pathways of these interconversions are unambigu- ous, while spontaneous mutation from each of the nonsense triplets to one that gives a wild phenotype can proceed along either transition o r transversion path- ways, which the test system is incapable of distinguishing. Mutation induced with 2AP or 5BU always proceeds along transition pathways. Consequently, in

values are given for all reversion frequencies that are at least five-fold higher than the spontaneous ones.

Antimutator DNA polymerase: The effect of the antimutator gene 43 allele tsCB87 on spontaneous AT-+GC transition frequencies is both pathway- and site-dependent, but highly variable (Figure 1 ) . For example, at site YH115, the frequency of UAA+UAG conversion is increased over 30-fold in the presence

the middle and. bottom lines of Figures 1 and 2, RtSCBB7/Rtsf and RtSL56/RtS+

NEIGHBOR EFFECTS O N MUTATION I N T 4 651

TABLE 3

5-Bromouracil-induced mutation of rII mutants in the presence of antimutator, wild-type and mutator DNA polymerase

Mutational pathway Gene 43 rll (1) ( 2 ) (3) (4) ( 5 ) ( 6 )

allele mutant UAA- wt UAA-t UGA UAA-t UAG UAG+ wt UGA+ wt UGA+ UAA

tsCBS7 YH115 YH119 YH122 YH132 YH320 YH341

I S +

tsL56

YH115 YH119 YH122 YH132 YH320 YH341

YH115 YH119 YH122 YH132 YH320 YH341

0.48 0.83 0.22 0.064 0.38 0.010

3.3

6.3 1.9

2.5

52.

31.

64. 31.

100. 13. 73.

210.

0.066 0.10 0.0004 0.012 0.012 0.002

18. 7.6 2.4 3.8 1.9

17.

5.0

0.56 7.8

1.55

48.

15.

1.41) 1 .o 0.080 1.25 0.82 0.54 0.044 0.M 0.10 1.1 0.036 0.61

53. 16. 13. 98. 19. 24. 2.4 29. 7.1 41.

28. 19.

12. 59. 28. 290. 59. 120. 8.2 77.

50. 2000. 110. 410.

11. 6.6 2.7 16. 5.6 4.7 2.6 3.4 7.0 45. 4.7 7.0

510. 62. 240. 4.0

15. 1.9 190. 100. 24Q. 150. 600. 110.

390. 54. 430. 20.

3000. 51. 220. 44.0.

1300. 160. 700. 5 4 .

~

For details, see legend to Table 1.

of tsCB87, and the frequency of UAA+UGA conversion is increased more than three-fold. At site YH119, the frequencies of both conversions are reduced (12- fold and four-fold, respectively).

The frequency of 2AP-induced A T j G C transitions is reduced by tsCB87 along all mutation pathways.

The antimutator allele also reduces from 2.5- to 104-fold the frequency of SBU-induced AT+GC transitions.

The antimutator effect of tsCB87 on GC+AT transitions (at UGA sites) is weaker than that on AT+GC. I n fact, the effect of the altered DNA polymerase on spontaneous and 5BU-induced GC+AT transition is sometimes that of a weak or even mild mutator. 2AP- and 5BU-induced mutation frequencies of GC base pairs are still 2 to 150 times, and 5 to 150 times, respectively, higher than the spontaneous ones (Tables 1-3).

Mutator DNA pdymerase: The mutator allele tsL56 increases the frequency of spontaneous AT+GC transitions from two- to over 103-fold. As with the anti- mutator DNA polymerase gene, this effect is highly variable. The effect of tsL56 on spontaneous G C j A T transitions is smaller, but also highly variable. Base-analogue- induced RtSLb6/RtS + ratios are, in general, smaller than the spon- taneous ones. tsL56 has a stronger mutator effect on 2AP-induced GC+AT tran-

652

- + w CT c

1

2 w c CT U

0 0

SPONT 3r

-1

2 AP

-1

5BU

A. RONEN, C . HALEVY AND N. KASS

U44

n

n - . il U

a b C d e f FIGURE 1.-The effect of the gene 43 allele tsL56 on mutation frequencies along the various

pathways. Data are from Tables 1 to 3. Mutation pathways are: (a) UAA-twt; (b) UAA+ UGA; (c) UAA+UAG; (d) UAG-twt; (e) UGA+wt; ( f ) UGA+UAA. In each pathway, the rZZ sites are represented by columns; their order (from left to right) is the same as their vertical order in Table 1. The height of each column represents RtsL56/RtS+ values for the corresponding mutational pathway. Sites for which no R t S L 5 6 / R t S + values could be calculated are represented by a broken base line.

sitions than on AT-+GC ones. No site seems to give consistently high or low RtSL56/RtS+ ratios in all pathways. This is even more pronounced in the case of 5BU mutagenesis: at three sites, the effect of tsL56 on induced AT+GC transi- tions is that of an antimutator.

Correlation between the effects of different DNA polymerases: We compared the frequencies at which the various sites mutate along the different pathways in phage with wild-type and mutant DNA polymerases. No correlation was found between either spontaneous o r 5BU-induced mutation frequencies in the presence of the wild-type gene 43 allele, and those in the presence of any of the other two gene 43 alleles. This is not the case, however, with 2AP-induced mutation. Here, UAG-+wt and UAA-+UAG mutation frequencies at the various sites are strongly

NEIGHBOR EFFECTS O N MUTATION IN T4

2-

1-

0 -

-1 -

-2-

653

'LAP

-2 -j -3 U 5 BU

- 4 L

a

%-

U

1 b

pil

LJ

C

w D

d FIGURE 2.-The effect of the gene 43 allele tsCB87 on mutation frequencies along the various

pathways. Data are from Tables 1 to 3. Details are as in Figure 2, but the columns represent RtscB87/RtS+ values.

correlated in the presence of all three gene43 backgrounds (Figure 3 ) . Mutation along the UAA-CAA and UGA+wt pathways is also strongly correlated in the presence of the ts+ and tsCB87 alleles. However, the frequencies of UGA+UAA conversion are only weakly correlated, and those of UAA+UGA conversion are not correlated in the two backgrounds (Figure 3 ) .

The correlations shown in Figure 3 do not result from correlated variability in the efficiency of detection of revertants and convertants in presence of the various gene 43 backgrounds. This is borne out by a reconstruction experiment in which the rZZ nonsense mutants and their T-+ revertants were plated on the appropriate bacterial lawns along with 10i-l Os plaque-forming particles of the deletion mutant rZZ1272. The efficiency of detection (i.e., of plaque formation) of all mutants and revertants was better than 30%. There was no correlation between

6549 A. R O N E N , C. HALEVY A N D N. KASS

4 1 a I b 4 r c 3 -

2 - 0

1 -

mm 0 0

3 -

2 -

1 - 0

0

0

4 r g 5 I h

1

o:, !I., , " , 1 2 3 4 2 3 4 5

l og ( R ~ W O ~ )

FIGURE 3.-Correlation between 2AP-induced-mutation frequencies in six r l l codons between rs+ and tsCB87 phage (a to f) and between IS+ and tsL56 phage (g , h). Correlation coeffi- cients were calculated with logarithmic transformations of the mutation frequencies. Mutation pathways and correlation coefficients (in bracktts) are (a) UAA (0.94); (b) UAA (0.09); (c) UAA (0.93); (d) UAG (0.92); (e) UGA (0.94); (f) UGA (0.81); ( g ) UAA (0.92); (h) UAG (0.97). All except (b) and (f) are significant a t the 0.01 level; (f) is significant a t the 0.05 level. In (e) and (f), two points (half circles) are based on induced R values that are not higher than the spontanems ones, and are thus upper bounds. If one assumed that, in (f), the net 2AP-induced R O B 8 7 value for at least one of these points is less than half its value in Table 2, no significant correlation would be found between RtS+ and RtSCB87. Similarly, no correlation would be found in (f) if one ignxed the two points. On the other hand, in (e) similar procedures would not lower the correlation.

UGA

NEIGHBOR EFFECTS O N MUTATION I N T4 655

the efficiency of detection in phage with ts+ and tsCB87, or ts+ and tsL56 back- grounds. In fact, there was no indication that the variability in the efficiency of detection was more than random experimental variability. These results also rule out the possibility that different efficiencies of detection are responsible for the apparent cases where tsL56 exerts an antimutator effect, or where tsCB87 seems to behave as a mutator.

DISCUSSION

The effect of tsCB87 on frequencies of spontaneous mutation is strongly path- way dependent. For example, spontaneous UAA-+UAG conversion frequencies are reduced in the presence of tsCB87 by as much as two orders of magnitude, while little, if any, antimutator effect is observed with either UAA-UGA or UGA+UAA conversions. tsCB87, in fact, had a fair chance of being classified as a weak or even mild mutator (depending on the rZZ mutant tested) if it were classified by its effects on spontaneous UAA+UGA conversions. These results are in general agreement with those of DRAKE et al. (1969) and RIPLEY (1975), who have shown that tsCB87 reduces spontaneous ochre 4 wild-type mutation up to 20-fold, while it reduces the frequency of GC+AT transitions only by a factor of two.

tsCB87 has a strongly antimutagenic effect on induced AT+GC transitions at all sites. RIPLEY (1975), studying the reversion of two ochre mutants, found that the frequency of 2AP-induced AT-GC transitions was reduced by the anti- mutator DNA polymerase to levels not distinguishable from the spontaneous ones. On the other hand, DRAKE et al. ( 3 969) reported that in the presence of the IsCB87 allele, 2AP-induced reversion frequencies of two presumed AT sites were six and 96 times higher than in untreated phage. We find that in the presence of tsCB87, 2AP-induced AT+GC transition frequencies at ochre sites are still as much as IO4 times higher than the spontaneous frequencies. A similar effect by the antimutator DNA polymerase is found in 5BU mutagenesis: induced AT-GC transition frequencies are as much as 1O3-fo1d higher than the spontaneous ones, despite the antimutator property of the enzyme.

The effect of tsCB87 on induced GC+AT transitions is smaller than that on AT+GC. However, induced mutation frequencies are only as much as 150-fold higher than the spontaneous ones, probably because the latter are rather high. This is in agreement with the results of DRAKE et al. (1969).

The effect of the mutator allele tsL56 on spontaneous mutation is also highly variable. It shows no clear specificity for either AT or GC sites: at four sites, tsL56 has a stronger mutator effect on the frequency of UAA-+UGA conversions than on that of the corresponding UGA+UAA ones, while at two other sites, its effect is stronger on UGA-UAA conversions than on the corresponding UGG-.UGA ones. This disagrees somewhat with the results obtained by BAUTZ- FREESE and FREESE (1967), who found a stronger mutator effect of tsL56 on spontaneous AT+GC than on GC+AT transitions.

The effect of tsL56 on four 5BU-treated AT base pairs is noteworthy. An anti- mutator effect is found on UAA-UGA and UAA-UAG conversions, but not on

656 A. RONEN, C. HALEVY A N D N. KASS

the corresponding U A W w t or UGA-wt reversions, although the latter result from the same transitions, as well as AT transitions, in the first position of the codon. Perhaps antimutator effects at the second and third positions of the opal and amber codons are masked by the strong mutator effect on AT-GC transi- tions at the first position.

In UAAgUGA interconversions, the ATgGC transitions involve base pairs that, at all sites, are flanked by the same nearest neighbors. Therefore, any variability in the frequency of these interconversions does not result from nearest- neighbor effects, but rather from the influences of more remote factors. On the other hand, all other mutation pathways studied here involve base pairs with at least one unknown neighboring pair outside the nonsense codon. It is likely that, at different sites, the unknown neighbors are not the same. Therefore the variability in the mutation frequencies along the UAA-CAA, UAA-t UAG, UAG-.wt and UGA+wt pathways reflects the combined effects of adjacent base pairs and other factors. [The possibility that this variability may reflect different efficiencies of detection was ruled out by RONEN and RAHAT (1976) and RONEN, RAHAT and HALEW ( 1976) 1.

In 2AP-treated phage carrying the ts+ and tsCB87 alleles, UGA-UAA and UAA-tUGA conversions show weak correlation and no correlation at all, respec- tively. This is in sharp contrast to all other mutation pathways, where the vari- ability in ts+ phage is strongly correlated with that in tsCB87. This difference may indicate that in 2AP mutagenesis, the two DNA polymerases have a similar sensitivity to nearest-neighbor effects, but not to more distant ones. This sensi- tivity of the enzyme may differ when replicating AT or GC base pairs.

Israel Academy of Sciences. This research was supported by a grant from the Commission for Basic Research of the

LITERATURE CITED

BAUTZ-FREESE, E. and E. FREESE, 1967

BRUTLAG, D. and A. KORNBERG, 1972

On the specificity of DNA polymerase. Genetics 57:

Enzymatic synthesis of deoxyribonucleic acid. XXXVI. A proofreading function for the 3’+5’ exmuclease activity in deoxyribonucleic acid poly- merases. J Biol. Chem. 247: 241-248.

DRAKE, J. W. and E. F. ALLEN, 1968 Antimutagenic DNA polymerase of bacteriophage T4. Cold Spring Harbor Symp. Quant. Biol. 33 : 339-344.

DRAKE, J. W., E. F. ALLEN, S. A. FORSBERG, R.-M. PREPARATA and E. 0. GREENING, 1969 Genetic control of mutation rates in Bacteriophage T4. Nature 221 1128-1132.

KOCH, R. E., 1971 The influence of neighboring base pairs upon base-pair substitution muta- tion rates. Proc. Natl. Acad. Sci. U.S. 68: 773-776.

LANGRIDGE, J. and J. H. CAMPBELL, 1969 Classification and intragenic position of mutations in the /3-galactosidase gene of Escherichia coli. Molec. Gen. Genet. 103: 339-347.

RIPLEY, L. S., 1975 Transversion mutagenesis in bacteriophage T4. Molec. Gen. Genet. 141: 23-40.

RIPLEY, L. S. and J. W. DRAKE, 1972 A genetic essay for transversion mutations in bacterio- phage T4. Molec. Gen. Genet. 118: 1-10.

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Genetics 84: 423-436. RONEN, A. and Y. SALTS, 1971 Genetic distances separating adjacent base pairs in bacterio-

phage T4. Virology 45 : 496-502. SALTS, Y. and A. RONEN, 1971 Neighbour effects in the mutation of ochre triplets in the T4rII

gene. Mutat. Res. 13: 109-113. SPEYFX, J. F., J. D. KARAM and A. B. LENNY, 1966 On the role of DNA polymerase in base

selection. Cold Spring Harbor Symp. Quant. Biol. 31 : 693-697. TOPAL, M.D. and J. R. FRESCO, 1976 Complementary base pairing and the origin of substitu-

tion mutations. Nature 263 : 285-289. VRIES, F. A. J. DE, CH. J. H. SWART-IDENBURG and A. DE WAARD, 1972 An analysis of replica-

tion errors made by a defective T4 DNA polymerase. Molec. Gen. Genet. 117: 60-71. Corresponding editor: J. W. DRAKE