SPONTANEOUS AND ETHYL METHANESULFONATE-INDUCED CONTROLLING VIABILITY IN DROSOPHILA MELANOGASTER. I. RECESSIVE LETHAL MUTATIONS

OHM1 OHNISHI

Laboratory of , Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Manuscript received December 18, 1976 Revised copy received July 15,1977

ABSTRACT

The efficiency of the adult feeding method for EMS treatment in Dro- sophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effec- tive on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per IO-4~.In addition, about 0.004-0.005 recessive lethals per 10-4 M were found in a later genera- tion in chromosomes that had not shown the lethal effect in the previous gen- eration. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the .

A central problem in population genetics is the relative importance of various factors determining genetic variability in natural populations. Interest in recurrent as a major source of variation has been stimulated by the work of MUKAIand his associates (MUKAI1964; MUKAIet al. 1972), who re- ported a very high spontaneous rate for viability-affecting polygenic mutations in Drosophila melanogaster. From the standpoint of human welfare, the im- portance of mutations is enhanced by the possibility that a number of chemi- cals in our environment may be mutagenic. Ethyl methanesulfonate (EMS) is known to produce base-pair substitutions and chromosome changes. It also induces polygenic mutations affecting viability (MUKAI1970). Mutation and chromosome breaking effects have been reported by ALDERSON(1965) ,EPLER (1966), LIMand SNYDER(1968), JENKINS(1967) , LEE, SEGAand BISHOP(1970), BRINK(1970), YOST,IVES and HALL(1967), ABRAHAMSON,KIRIAZIS and SOBOL(1969), and BISHOPand LEE (1969). For a review of molecular mechanisms, see DRAKE(1969,1970).

Supported in part by grants from the Public Health Service to the University of Wisconsin (GM-08217 and GM-22038).

Genetics 87 : 519-527 November, 1977. 520 0. OHNISHI

Most of the experiments have been done at high concentrations, around 1 0-2 M. The present series of experiments was undertaken to study the nature of mutations produced by feeding with relatively low concentrations of EMS. The main purpose was to study the rate of occurrence and the effects on viability of polygenic mutations. These results are reported in parts I1 and I11 of this series (OHNISHI1977a,b). This paper reports experiments on the induction of lethal mutations as a function of concentration. They establish the linearity of the mu- tation rate with concentration at low doses and offer guidance as to the range of concentrations to be used in the further experiments.

MATERIALS AND METHODS

The wild stock of Drosophila melanogaslei- used in these and all the following experiments was an inbred line established from a natural population in Madison, Wisconsin. The strain was sib-mated for more than three years. A male from this stock was mated with the labora- tory strain In(2) SMI, a12 Cy sp2 / In(2) Pm, dp b Pm dsss7;. From the progeny, a Cy/+ and a Pm/+ male were each backcrossed to the inbred wild-type strain. Again Cy/+ and Pm./+ progeny males were backcrossed to the inbred +/+ strain. This backcrossing procedure was repeated for 20 generations, after which a Cy/+ female and a Pm/+ male were mated to establish a Cy/Pm strain. This strain, except for the second chromosomes, has a genetic background derived from the wild-iype strain. The wild-type strain will he abbreviated +/+, and the marker strain that has background genotype isogenic with the wild-type strain will be designated Cy/Pm. The EMS treatment followed essentially the methods of LEWISand BACHER(1968). The EMS was in a 1% solution of sucrose. A piece of tissue paper soaked with 2.4 ml of this solu- tion was placed in a cylindrical shell vial, 23 x 93". Males 0 to 24 hours old were left in this vial for 24 hours at room temperature (ca 25"), then mated immediately in vials with standard culture medium. Seven concentrations of EMS were used, ranging from 1 x 10-4 to 2.5 x 10-2 M. At each concentratim 160 Pm/+ males were treated, 20 per vial. Each EMS-treated male was immediately mated with two Cy/Pm females. If the brood pattern was to he studied, the male was transferred to new Cy/Pm females on the 4th, 7th, and 10th day after treatment. The mating system for detecting recessive lethal mutations on the second chromosome is the standard method and is shown in Figure 1 as Lethal Test 1. To avoid the possibility of sampling large clusters of lethal mutations from a few males, only two Pm/+ males were sampled from the progeny of an individual treated male. The final mating between Cy/+ males and females (one or two pairs) was made in a vial with heavily yeasted food and kept at 25". The offspring were observed on the 12th or 13th day. The cultures in which no wild- type flies appeared by the 13th day were kept for three more days to make a final decision of lethality. If less than one percent of the progeny were +/+, the treated chromosome was clas- sified as lethal. In some experiments recessive lethals not manifested until a later generation (so-called mosaic mutations) were also studied (AUERBACHand KILBEY1971). The scheme to detect these is also shown in Figure 1 as Lethal Test 2. These tests involved one further generation of back- crossing before the chromosome was made homozygous. A chromosome that was not classified as lethal in Test 1, but was lethal in Test 2, was designated as a mosaic lethal. This method for detecting mosaic mutations does not answer the question of whether a given normal chromo- some carries a mosaic mutation, hut it gives an unbiased estimate of the frequency 3f lethals among the progeny of nonlethal F, chromosomes. The reason for treating Pm/+ rather than +/+ males was to obtain results comparable to those of Part I1 and TI1 of these experiments, where the type 3f male was treated in suc- EMS-INDUCED MUTATIONS IN DROSOPHILA 521 dEMS treatment -cy x --(id)Pm' Pm I +

-cy x p" (16) Pm I + cy c'y +!T

Lethal Test 1 cycy +i+ Lethal Test 2 FIGURE1.-Mating scheme to detect complete and mosaic recessive lethal mutations on chromosome 11. cessive generations (OIINISHI 3 977a,b), However, in order to compare mutation frequencies induced in the two genotypes, some experiments were also carried out with treated +/-I- males.

EXPERIMENTAL RESULTS Although there is a report by ALDERSON(1965) that feeding adult flies with EMS causes some death, there was 100 percent survival in all the experiments reported here. However, there was sonie sterility, particularly in the higher con- centrations. About 10 percent of the males were sterile after treatment with 2.5 X M, and 5% and 3% with 1 X and 5 X M respectively. The percentage of sterility is roughly proportional to EMS concentration. The observed frequencies of chromosomes containing one or more recessive lethals induced by EMS are shown in Table 1. Those from the Pm/+ first brood data are converted to the number of lethal mutations per chromosome, assuming a Poisson distribution, in Table 2. Figure 2 shows the data in graphical form. It is apparent that the increase is very nearly linear, except €or a slight down- ward curvature at high doses. It might be expected that some of the effects of high concentrations might be to produce multiple-hit rearrangements, leading to an upward curvature. The failure to find this might be due to a compensatory effect stemming from a small fraction of flies that €ailed to feed on the EMS. PELECANOSand ALDERSON(1964) reported that at very high doses about 15 percent of the flies yielded no mutations, presumably because they failed to ingest any EMS. Table 2 shows a further analysis of the data. If 15 percent of the flies fail to feed during the treatment, as suggested by the results of PELECANOSand ALDER- SON (1964), we can correct the data accordingly. Assuming that this is true in my experiments, the expected number of mutants among those flies that feed 522 0. OHNISHI

TABLE 1 Frequency of recessive lethal chromosomes induced by EMS

Pm/+ 0 368 2 0.54 I XIO-4 209 3 1.44 5 x 10-4 206 IO 4.85 1 X 10-3 259 35 13.51 222 38 17.12 2% 25 12.20 182 11 6.04 2.5 X lk3 276 83 30.07 269 98 36.43 215 63 29.30 236 27 11.44 5 X IO-3 280 118 42.12 211 106 50.24 190 88 46.32 211 28 13.27 1 X IC2 156 94 60.26 +/+ 2.5 X le2 192 155 80.73 2.5 x 10-3 156 53 33.97 5 X lW 140 66 47.14

(a) Number of chromosomes tested. (b) Number of lethal chromosomes. (c) % of lethal chromosomes.

on the EMS is given in the last two columns. As can be seen, with this correction the estimated number of EMS-induced mutations is almost exactly linear, and the number induced per M is nearly constant for all concentrations tested. I have no direct evidence that some of the males fail to feed, but the improved linearity after making such a correction suggests that this may be true. The recessive lethal mutation rate per M is 0.0105, if all points are equally weighted, 0.01 16 if the two highest concentrations (where the nonlinearity begins) are omitted, and 0.0135 if it is assumed that 15 percent of the flies were not exposed to the mutagen. I shall take 0.012 as the best estimate for future comparisons, where the same treatment method was used.

TABLE 2 Recessive second-chromosome lethals induced by various concentrations of EMS in mature spermaiozoa

Prop. of Estimated mean number of induced lethals per chromosome* ElIS conc. lethal Poisson correction 15% not feeding X 10-4 mol chromosomes Number per lo4 mol Number per mol 1 0.0144 0.0095 0.0095 0.0121 0.0121 5 0.0485 0.0447 0.0089 0.0538 0.0108 10 0.1351 0.1401 0.0146 0.1681 0.0168 25 0.3007 0.3527 0.01141 0.4316 0.0171 50 0.4212 0.5418 0.0108 0.6792 0.0136 100 0.6026 0.3178 0.0092 1.2292 0.0123 250 0.8073 1.6416 0.0066 2.9860 0.0119 Unweighted means 0.01 (45t 0.0135

* Corrected for a spontaneous mutation rate of 0.005. -f If only the first 5 items are averaged, the value is 01.0116. EMS-INDUCED MUTATIONS IN DROSOPHILA 523

W I: 1.5 - 0 Complete recessive lethal mutation 0 I: 0 Mosaic mutation lx0 I U 1.2 - n z 0 U :0.9- \ v) Z t

FIGURE2.-Frequency of recessive lethal mutations induced by EMS and its 95% confi- dence interval.

ALDERSON(1965) and LIM and SNYDER(1968) observed 41.5% X-linked lethal mutations at 0.01 M and 53.6% at 0.02 M. Considering the relative lengths of the X and second chromosomes, my results are in good agreement. On the other hand, MUKAI’S(1970) result, giving a mutation rate of 42% at 0.025 M, seems to be abnormally low. The spontaneous rate of 0.005, although based on only two mutants, agrees with the earlier results of MUKAI(1964) and WALLACE (1956). Comparison of the rate in the last two rows in Table 1 for +/+ males with the same concentrations for Pm/+ males shows that they are very similar (xI2= 0.95 and 0.70). There is no evidence for any effect of the Pm inversion. The frequency of mutations in successive broods from the same male can be compared from the data in Table 1, and the number of lethal mutations per chromosome converted from the data by the assumption of a Poisson distribution are shown graphically in Figure 3. It is clear that for all three concentrations tested there is no important difference among the first brood, treated as mature spermatozoa, the second, treated as spermatozoa and spermatids, and the third, treated as spermatids. The frequency decreases drastically, however, in the 4th brood, where spermatocytes and spermatogonia were treated (COOPER1950). The pattern of effectiveness in different broods agrees with that found for X-ray treatment (AUERBACH1954; THOMPSON1960), acridine feeding (ALDERSON 524 0. OHNISHI

6C

2 -0 c 2.5~10-~ a \ c 3 2 4c -1 a I I- W -1 LL O 2c 8

0 I 1 I I 1 2 3 4 BROOD FIGURE3.-The brood pattern of EMS-induced recessive lethal mutations. and KHAN 1968), and diethyl sulphate (ALDERSONand PELECANOS1964). it suggests that in order to keep a consistently high efficiency of EMS mutagenesis, the early broods should be used; although treating only mature sperm implies the analysis of the chromosome-breaking effects of EMS, rather than the base- replacement aspects of the mutagen. The frequency of mosaic lethals is given in Table 3 and plotted in Figure 2.

TABLE 3 Frequency of mosaic lethal muiaiions induced by EMS

No. of No of Mean number Mean number Ciinc of EMS chromosomes mosaic lethal PC1 induced (b1) tested chromosomes Percent cliromosome per ni 5 x 10-3 162 28 17.28 0.190 0.0037 2.5 x 10-3 193 20 10.36 0.109 0.0042 I x 10-3 223 I2 5.38 0.055 0.0052 5 x 10-4 192 5 2.60 0.026 0.0046 0 278 1 0.36 0.03 EMS-INDUCED MUTATIONS IN DROSOPHILA 525

TABLE 4 Frequency of recessiue lethal chromosomes induced by EMS after the staruation treatment

No. of No of Mean number Mean number Conc. of EMS chromosomes lethal per induced (M) tested chromosomes Percent chromosome per M 5 x 10 140 91 64.29 1.030 0.021 1 x 10 275 103 37.45 0.469 0.047 5 x 10 282 64 22.70 0.258 0.052 1 x 10 21 9 14 6.39 0.0166 0.066 0 213 0 0.00 0.000 -

It can be seen that the frequency of lethal chromosomes due to mosaic mutations in chromosomes that were not lethal in the previous generation is about 0.004 per second chromosome per M. This is a little less than half the frequency of complete lethals, 0.012. Lethal mutation frequencies were also estimated from accumulation experi- ments to be reported in Part I1 of this series (OHNISHI 1977a). Males were treated in successive generations, so that the same chromosome was repeatedly exposed. The estimated lethal frequency was about 0.013 per chromosome per lo4 M (see Table 4 in Part 11). The accumulation experiments included both complete and mosaic mutations, so that this value should be compared to 0.012 f 0.004. The experiments agree within the margin of uncertainty. It should be mentioned here that a minor variation in the experimental pro- cedure can have a large effect on mutation rate. In an experiment where young males were starved for I2 hr in a vial containing only tissue paper moistened with water before the ELVIS treatment, the lethal mutation rate increased. The observed frequency of recessive lethal chromosomes for the first brood increased from 2 to 5 times, as shown in Table 4. The number of lethal mutations per chromosome converted by the assumption of a Poisson distribution is approxi- mately linear, except at the highest concentration, as in the case of nonstarvation experiment.

DISCUSSION AND CONCLUSIONS It is clear that EMS, fed to Drosophila adults, is a very effective mutagen at doses considerably lower than customarily employed. The main purpose of this experiment was to determine the concentrations to use in measuring the fre- quency and effect of polygenic mutations. For the interpretation of future experi- ments, the most important result is the internal consistency of the mutation rates and the linear relation between concentration and mutation frequency, at least at low concentrations. It is also important for interpreting the results of experiments in the later papers (OHNISHI 1977a,b) that single treatments give the same effect as the same total concentration given in repeated treatments at lower concentrations over several generations. Because of the brood effect, all later experiments were done with flies treated when less than 24 hr old and mated immediately after treatment, so that all progeny come from gametes 526 0. OHNISHI whose chromosomes were exposed in the mature sperm stage. Further reassur- ance of the generality of the results comes from the fact that wild-type chromo- somes heterozygous with the Pm inversion produced the same effect as when homozygous. Although the experiments reported here gave very consistent results, it is important to note that minor variations in the experimental procedure can have a large effect. One point already mentioned is the necessity of having a homo- geneous sample of sperm treated at the same stage. Another factor is the way in which the flies feed on the EMS, as demonstrated in the starvation experiment. Other factors such as counting procedure and culture condition may be consid- ered, particularly with regard to the difference between MUKAI’S (1970) results and my own. Counting flies at the 13th day is sufficient for classifying lethals, and this was confirmed by keeping the culture with no wild-type flies for three more days before making the final decision of lethality. Overcrowding, as in the present experiment, may act to inflate the estimated number of lethal chromosome. I suspect, however, that a technical difference in EMS treatment, treating many flies in a milk bottle in MUXAI’Sexperiment and 20 flies in a small vial in my case, may be responsible for the difference. Since the amount of solution actually taken in by the flies is unknown. we give results only in terms of the concentration in the solution to which the flies are exposed. However, by standardizing the procedure it is possible to get quite consistent results. The most important conclusions from this study are: (1) Feeding young adult Drosophila with EMS induces recessive lethal mutations in direct proportion to the concentration, at least at low and moderate doses. (2) EMS is most effective on mature spermatozoa and spermatids, but considerably less effective on earlier stages. (3) Complete recessive mutations are about twice as frequent as lethals that appear one generation later from chromosomes that did not manifest the lethal earlier. (4) The complete recessive mutation rate is about 0.012 per sec- ond chromosome per 10-4 M. (5) Anticipating the results of Part TI (OHNISHI 1977a), we note that the number of mutations produced is proportional to the product of the concentration and the number of generations that the chromosome is exposed.

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