Evidence for an inducible repair-recombination system in the female germ line of Drosophila melanogaster. III. Correlation between reactivity levels, crossover frequency and repair efficiency.

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Evidence for an inducible repair-recombination system

in the female germ line of Drosophila melanogaster. III.

Correlation between reactivity levels, crossover

frequency and repair eﬀiciency.

Anne Laurençon, Françoise Gay, Judith Ducau, Jean-Claude Bregliano

To cite this version:

Anne Laurençon, Françoise Gay, Judith Ducau, Jean-Claude Bregliano. Evidence for an inducible

repair-recombination system in the female germ line of Drosophila melanogaster. III. Correlation

between reactivity levels, crossover frequency and repair eﬀiciency.. Genetics, Genetics Society of

America, 1997, 146 (4), pp.1333-44. �hal-03079384�

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Copyright 0 1997 by the Genetics Society of America

Evidence for an Inducible Repair-Recombination System in the Female Germ

Line of Drosophila

melanogaster. 111. Correlation Between Reactivity Levels,

Crossover Frequency and Repair Efficiency

Anne Laurenqon, Franqoise Gay, Judith Ducau and Jean-Claude Bregliano

Institut de Biologte du Deueloppement de Marseille, 13288 Marseille Cedex 9, France

Manuscript received August 28, 1996 Accepted for publication April 24, 1997

ABSTRACT

We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of Zfactor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the Xchromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it

_-

VAMOS for - variability modulation system. _-

I

N previous articles we reported data constituting evidence that a regulatory system of DNA repair, with unusual genetic behavior, takes place in oogenesis of Drosophila melanogaster, and that its expression is modulated by several factors, especially aging and DNAdam- aging agents. These induced changes are maternally heritable over several generations (BREGLIANO et al.

1995; LAURENCON and BREGLIANO 1995).

The starting point of our work is the I-R system of hybrid dysgenesis. In this system, transposition of the I retrotransposon occurs at a high frequency in germline of F1 daughters (denoted SF) from a dysgenic cross between females of reactive stocks and males of inducer stocks. Reactive flies do not bear any active I elements in their genome, whereas in inducer flies there are active I elements on the chromosomes. Transposition is regulated by a cellular state taking place in the oocytes of the reactive females and transmitted to their SF

daughters. This cellular state may exhibit different levels of expression, called reactivity levels, that can be measured by the hatching percentage of eggs laid by

SF females. These levels are transmitted over several generations in a complex way involving both chromosomal determinants and a maternally inherited c o m p e nent (BUCHETON and PICARD 1978). Also, reactivity levels are liable to undergo heritable changes, cumulative over generations but always reversible, due to the effects of nongenetic factors on the maternally inherited com- ponent. Among these factors, aging and thermic treat-

Corresponding author: J. C. Bregliano, Institut de Biologie du Devel- oppement de Marseille, Case 907 Marseille-Luminy, 13288 Marseille Cedex 9, France. E-mail: breglio@lgpd.univ-mrs.fr

Genetics 1 4 6 1333-1344 (August, 1997)

ments of late oogenesis are known to decrease reactivity levels (reviewed in BREGLIANO and KIDWELL 1983).

In two previous articles we proposed the hypothesis that the biological function underlying reactivity levels might be one manifestation of a stress-response system whose biological role would be comparable to that of the SOS response in bacteria. We reported data showing that medium reactivity levels are enhanced by agents that are known to induce the SOS response in Escherichia coli, namely inhibitors of nucleotide synthesis and gamma rays. As for aging and temperature, this effect is maternally heritable, cumulative over several generations and reversible (BREGLIANO et al. 1995). We showed also that different reactivity levels are related to different sensitivity to gamma rays. This sensitivity was estimated on fecundity (number of eggs laid), fertility (hatching percentage of eggs), and larval-to-adult viabil-

ity (LAURENCON and BREGLIANO 1995).

However, these fitness parameters may be the out- come of various genetic events. To further investigate the DNA metabolic activities related to reactivity levels, it was necessary to characterize more precise events linked to recombination and repair activities. In the present report we show that in common genetic backgrounds, reactivity levels are correlated with crossover frequencies, at least in some chromosomal regions, and also with chromosome losses and recessive lethal mutations occurring after irradiation.

MATERIALS AND METHODS

Fly, stocks and culture conditions: Some specific D . melano- gasterstocks were used: stM, a strongly reactive stock bearing

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1334 A. Laurencon et al.

the ebony mutation; Paris, a reactive stock caught in that city in 1952; e ry, a weakly reactive stock originating from crosses between an ebony stock and a rosy 506E stock; seF8, a strongly reactive stock bearing the sepia mutation; Cha-(RC+), an inducer strain originating from a strong reactive wild-type stock “Charolles” contaminated by I factor by the use of crosses with inducer balanced stocks (PELISSON and BREGLIANO 1987); h215 (RC+), an inducer strain, originating by a similar procedure from the h215 reactive marked stock, which bears the st, sr and ry phenotypic mutations; Canton-S and B2’, standard inducer stocks used to measure reactivity levels (the former is used in most experiments reported here except when stated otherwise); s t M SG and Cha-(RC+) SG lines, bred with short generation pattern, ie., from young mothers only (1-3 days old). Lines issued from stM and Cha-(RC+) stocks and denoted LG x are bred with long generation pattern, from mothers aged from 30 to 45 days, x indicates the number of long generations. Lines denoted LG x-y were bred with long generation pattern during x generations then put back with short generation pattern during y generations. Lines denoted stM LGA and stM LGB are sublines originating from the same s t M LG line but separated from the 21st long generation; line denoted stM LGC was isolated sepa- rately. All marked stocks used for recombination experiments or chromosome losses are reactive stocks provided either by the Middle American Drosophila Stock Center or by the UMEA Drosophila Stock Center. Mutations and balanced chromosomes are described in LINDSLEY and ZIMM (1992). All stocks used in this work are devoid of P elements.

Due to the extreme sensitivity of the reactivity levels to

breeding conditions, several parameters were accurately con- trolled, especially temperature (20 2 0.5”) and fly age. Flies were reared on the axenic food described by DAVID (1959), under uncrowded conditions, with a normal lightdark cycle. Irradiation with gamma rays was done with a 6oCo source, with dose rate of 3,4 rad/sec.

Measure of reactivity levels: Sets of five females were mated

in vials with seven inducer males and transferred to fresh food every day during 1 month. The 24hr-old SF daughters were

put on fresh food with sib males and allowed to lay eggs during another 24 hr. Then they were discarded and 2 days later the percentage of nonhatching eggs was scored. Values are given for 10-day-old reactive females except when stated otherwise. As stated in the introduction, the reactivity level is essentially maternally inherited, however the paternal genome has a slight influence. In consequence, the reactivity levels of the F1 flies from a same maternal strain may exhibit some differences.

Measure of crossover frequencies: The same experimental

procedure was used for every recombination experiment. Young females of the selected maternal line were mated with males of the marked reactive stock; 1 day after eclosion, six sets of four F, females were backcrossed and transferred to new vials every day during nearly I month to measure the crossover frequency over a 25-3Oday period. Because this kind of experiment is labor-intensive, it was not possible to

count the progeny from every day. Data presented in most tables are for the same 2-day periods, appearing as the most informative. Each value is established on the progeny of 1

day only within each period (except when stated otherwise), depending on which data were available. As shown by several authors (see, for example, BROADHEAD and KIDWELL 1975), the recombination fraction is normally distributed. Confi- dence intervals on percentages were calculated after angular transformation, statistical comparisons between different values were made with the Student t-test.

RESULTS

Heritable maternal aging effect on recombination:

Chromosome 3: The most detailed analysis of the correla-

:

i o 1 5

Age of

F1

females (days)

2 0

FIGURE 1.-Crossover frequency between the st and sr loci on chromosome 3 for sth4 strain. Results are shown as a function of F1 females age, with lines maintained with short generations (0), with 17 long generations ( 0 ) and 20 long generations ( W ) . Mean values with CL are presented in Ta- ble l .

tion between reactivity levels and crossover frequency was performed on a pericentromeric interval between 79B and 85E, using the h215 stock, bearing the st, ry

and sr markers, mapped at 44, 52 and 62, respectively. It was not possible to seek a correlation on the distal chromosome arms because we did not find reactive stocks bearing suitable markers.

The first experiment was performed using, as maternal parent, two lines of the strong reactive stock stM, bred either after a short generation pattern (the strongly reactive line s t M SG) or after a long genera- tion pattern (the weakly reactive line stM LG). With the latter, the frequency of crossing over was measured after

17 and 20 long generations (LG 17 and LG

20).

Five sets of four F1 females were crossed with Canton-

S males to check for their reactivity levels.

Recombination data are plotted on Figure 1 and mean values with confidence limits (CL) are presented in Table 1. They show a clear correlation between reactivity levels and crossover frequencies; however this correlation is not evenly distributed throughout the life of the flies. It is very strong within the first week and from day 13 to 18, and weaker between these two periods. The effect is roughly similar on the two intervals st-

y and ry-sr (data not shown). Note that for the three categories of flies, the recombination rate decreases from day 1 to day 12; this is in agreement with the decreasing effect of female age on crossing over described since the beginning of the century (BRIDGES

1927). With the SG line, recombination rises again after day 14, this increase is greatly reduced with LG

17

and

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Inducible DNA Repair in Drosophila 1335 TABLE 1

Effect of short and long generation patterns on crossover frequencies in the &srinterval of third chromosome

Maternal Reactivity Time interval analyzed

line level (%) Days 2-4 Days 8-10 Days 11-13 Days 15-17

s t M SG 95 38.8 ? 3.7 29.2 2.4 26.2 2 3 32.5 ? 2.8

s t M LG 17 70 31.5 ? 1.6** 28.2 t- 0.5 24.4 ? 3.8 26.9 2 3.2*

s t M LG 20 30 26.3 ? 2.7*** 21.0 2 2.6** 23.8 ? 4.0 21.3 t- 1.4***

~

Recombination values are means ? confidence limits. Data are from experiment presented on Figure 1; they are scored on six sets of four females. SG is a short generation line, LG 17 has undergone 17 long generations, LC 20 is LC 17 with three more long generations. Asterisks indicate the level of significance when compared to the SG line in the same time interval: * P

<

0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Reactivity level is measured on 10day old F1 females, and is expressed as the mean of the fly sets.

completely absent with

LG

20. After day 20, the frequencies yielded by the three lines are not very different and exhibit mainly random variations. For this reason they are not plotted on the graph. The late rise had been reported by some authors, at least for the same genomic region (NEEL 1941; REDFIELD 1966), and no satisfactory explanation was proposed. In our work, we have enough information to relate this late rise with develop- mental stages. All adult progeny were counted, the F1

females were dissected at the end of the experiment to count their ovariole number (on average 48 per female) and the egg-to-adult viability was measured (it was between 80 and 90%). This allows us to conclude that during the first 12-14 days of their life, females lay all eggs whose oogenesis initiated during their pupal life. After this period, they lay eggs whose oogenesis initiated after adult eclosion. Thus the late rise in crossover frequency is very likely due to hormonal changes linked with eclosion. Why this physiological state enhances recombination only in short generation breeding is not understood, but it means that a heritable aging effect may induce biochemical changes that interact with de- velopmental functions.

It is also noteworthy that the recombination fraction between the markers used here is always higher than the published values. For the line with the highest efficiency, it vanes between 26 and 42%, for the others it varies between

21

and 34%, whereas the published value is 18% (LINDSLEY and ZIMM 1992). This is a good illustration of the great range of variation that this phenomenon may undergo, depending on environmental factors and genetic backgrounds.

X chromosome: It is known that the variations in crossover frequency on the X chromosome, which is under the effect of several factors, are complex. With female aging, the distal part of this chromosome exhibits in- creasing recombination, whereas the medial part stays the same, and the most proximal part exhibits a decrease as do pericentromeric regions of chromosomes 2 and 3 (LUNING 1983). To test the effect of short and long generations on the various parts of the X chromosome, we used males of three marked stocks, y ct v for

the first experiment, y cv

f

carfor the second experiment and

y

d

m

f

carfor the third experiment. The published locations for these markers from the distal to the proximal ends are as follows:

y

= 0, cv = 13.7, ct = 20, v = 33, m = 36, f = 56.7 and car = 62.5. The strongly reactive females were from lines atM

SG;

the weakly reactive ones are from line stM

LGA

27

for the first experiment, from line a t M

LGB

43 for the second experiment and from line a t M

LGB

49 for the third. Data are presented in Figure

2

and Tables

2

and 3. In the y-ct interval the recombination frequency increases with age with both

SG

and

LG

lines; the difference between the two lines agrees with a cumulative effect of maternal aging. The ct-v interval does not exhibit any clear change with aging

5 1 0 15 2 0 25

Age

of

F1 females (days)

FIGURE 2.-Crossover frequency within the yct, ct-v and f

cur intervals on X chromosome for stM strain. Results are shown as a function of F1 females age, with lines maintained with short generations (0) and long generations ( 0 ) . Full and bold lines represent the yct interval. The dotted lines represent the ct-v interval. Simple lines represent the values for the Scar interval. Mean values with CL are presented in Table 2.

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1336 A. Laurencon et al.

TABLE 2

Effect of short and long generation patterns on crossover frequencies on the X chromosomes

Chromosome Maternal Reactivity Time interval analyzed

interval line level (%) Days 2-4 Days 8-10 Days 11-13 Days 15-17

y-ct ct-v Experiment 1 s t M SG 70 19.7 t 3.5 20.1 2 1.6 18.2 2 2.5 s t M LGA 27 5 21.0 2 2.9 24.2 2 2.9*** 23.3 2 2.4"" s t M SG 70 12.0 2 1.2 12.0 2 1.2 13.2 2 1.8 12.5 2 2.3 s t M LGA 27 5 12.7 t 3.5 14.2 ? 1.5* 15.4 2 2.8 13.7 +- 4.1 Experiment 2 fcar stM SG 55 10.4 2 1.9 5.4 2 1.5 5.4 2 1.8 4.5 2 1.5 atM LGB 43 3 7.1 2 1.6* 3.6 2 1.0* 4.3 2 0.7 6.4 ? 1.4 Recombination values are means 2 confidence limits. Data are from experiment presented on Figure 2; they are scored on six sets of four females. For experiment 1 data on y-ct from days 8-10 and 11-13 are pooled because they are very similar in their mean and standard error values. Reactivity level was measured with strong inducer males (Canton-S) for experiment 1 and with medium inducer males (B2') for experiment 2. LGA and LGB are two sublines separated since the 21st long generation. Asterisks indicate the level of significance when compared to the SG line in the same interval: * P < 0.05, **P < 0.01, ***P <

0.001 (Student's t-test).

and, in accordance, there is no cumulative effect, only one period (days 8-10) gives a significant difference at P = 0.05 between the

SG

and the

LGA

lines. The fcar interval shows a significant decrease with aging for both

SG

and

LG

series, and the differences between the two series until day 13 are in agreement with a cumulative effect. The third experiment shows that in the m-finter- Val there is no effect due to females aging and no effect due to long generation pattern. Thefcarinterval exhib-

its a slight but significant aging effect within the very first days only; accordingly we observe a slight effect of long generation breeding within the same period (Ta- ble 3). Note that in these experiments on X chromosome there is no great discrepancy between the observed values and the published map.

Chromosome 2: The experiment was performed with a paternal stock bearing the al, dp, b,

p,

cn markers with published locations as follows: a1 = 0, dp = 13, b = 48.5, pr = 54.5 and cn =

57.5.

Data are presented in Table 4. In the dpb interval there is a moderate aging

effect and no effect of long generation pattern, the

b

cn interval exhibits a strong aging effect with a late rise at days 20-22 for the atM

SG

line, but not for the

L G

13

and

L G

40 lines. There is no effect of long generation

breeding except a highly significant difference at days 20-22 between the

L G

40

line and the two others. Note that of all chromosomal regions investigated in this paper, the left arm of chromosome 2 is the only region that exhibits an effect of female aging, without exhib- iting a correlative effect of long generation pattern within the same period. However, the strong depressing effect exhibited by

LG

40

at the 20th day, compared to the other lines, is reminiscent of that observed on the third chromosome between 13 and 18 days (Figure 1

and Table 1).

The heritable maternal aging effect on recombma-

tion is reversible: To investigate further the correlation between reactivity and recombination, it was necessary to test whether the cumulative aging effect on crossover frequency was reversible as it is for reactivity levels. For

TABLE 3

Third experiment on the effect of short and long generation patterns on crossover frequencies on the distal part of X chromosome

Chromosome Maternal Reactivity Time interval analyzed

23.1 -t 2.5 25.8 t 3.2 s t M LGB 49 4 26.3 2 4.8 22.1 2 3.5 23.1 lr 4.4 25.0 t 1.9

fcar s t M SG 50 8.4 2 1.3 5.4 2 2.2 3.4 lr 1.1 5.2 t 2.3

stM LGB 49 4 5.7 2 1.6" 5.1 2 1.5 4.1 t 1.7 6.3 2 1.5

nz-f

StM SG 50 22.1 2 0.8 24.2 2 2.3

Recombination values are means lr confidence limits; data are scored on six sets of four females. SG is a short generation line, and GLB 49 is a long generation line. Asterisk indicates that the value is significantly different at P = 0.05 from the SG value in the same time interval. Forf<arinterval, the value obtained within days 2-4 is significantly different from the following ones. Reactivity is measured with medium inducer males (B2').

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Inducible DNA Repair in Drosophila 1337 TABLE 4

Effect of short and long generation pattern on crossover frequencies on the left arm of the second chromosome

Chromosome Maternal Reactivity Time intervals analyzed

dPb ~ ~~~ ~~~ s t M SG 90 36.9 2 2.7 31.4 t 3.8 28.4 C 4.1 30.1 C 3.2 s t M LG 13 80 34.2 +- 1.5 29.4 ? 3.4 29.1 2 3.1 30.6 2 3.6 s t M LG 40 5 34.9 +- 4.3 29.5 t 2.6 29.4 5 3.4 28.1 t 3.8 b-cn s t M SG 90 15.7

+-

3.9 7.7 5 1.6 6.0 ? 2.9 13.6 C 4.0 s t M LG 13 80 14.7 +- 2.9 11.0 ? 3.4 9.7 t 4.1 10.6 2 2.8 stM LG 40 5 14.0 2 3.1 9.1 5 2.4 7.1 5 1.8 7.3 5 1.3** Recombination values are means 5 confidence limits. Data are scored on six sets of four females. SG, short generation; LG 13, 13 long generations; LG 20 is the same line as LG 13 with seven more long generations. For both d p b and b-cn intervals, all mean values at days 2-4 are significantly different from mean values at days 11-13. Asterisks indicate the level of significance when compared to SG line: *P

<

0.05, **P < 0.01 (Student’s t-test). No progeny from the 15-17day period were available in this experiment.

~ ~~ ~~ ~ _ _ _ _ _

this purpose, we performed experiments with s t M LG lines put back in short generation breeding. We tested the pericentromeric region of chromosome 3 that exhibited the strongest effect of long generation breeding on crossovers; two experiments were performed at a 1- year interval. Experiment I involved stM SG, stM LGA 35 and s t M LG 21-30 (bred with 21 long generations followed by 30 short generations). The crossover percentages were scored only from days 3 to 5, previously shown to be one of the most sensitive periods (Figure 1). The reactivity level was measured with Canton-S as

the inducer strain. Data are presented in Table 5. The percentage of recombinants was identical for SG and LG 21-30; the difference with LGA 35 was highly significant.

Experiment I1 was performed 1 year later with SG, LGB 46, LGA 37-23 and LGA 3331 lines. The reactivity

level was measured using males from the B2’ stock that

is a weaker inducer than Canton-S. The crossover per-

centages were scored in both “sensitive” periods: days 3-5 and days 15-17 (Table 5). Values obtained with line LGB 46 were significantly different from those from the three other lines, which were not significantly different from each other. However between days 15 and 17, the LGA 37-23 line had a lower value than the two

other strong reactive lines; this was due only to the st-

9

interval which was 7.5 ? 1.2% (CL), a value similar to that of LGB 46 (7.2 ? 2.0%) and very different from that of the other lines, which were very close to each other (12.5 2 2.8 and 12.3 2 3.0%). Thus, for the LGA 37-23 line, the st-? interval had no reversible effect within days 15-17, whereas the y s r interval had one, the difference with LGB 46 on the whole st-sr segment being significant at P = 0.05. We have no simple explanation for this result.

Another discrepancy appears when comparing the values of the first experiment described above on chromosome 3 (Figure 1) and those of Table 5 . In the for- TABLE 5

Effect of short and long generations patterns on recombination fraction on the third chromosome, reversibility of the cumulative maternal age effect

Time interval analyzed

Experiment Reactivity level (%) Days 3-5 Days 15-17

I s t M SG 98 29.9 2 0.72 stM LGA 35 55 22.8 t 1.08*** s t M SG 67 28.1 t 1.2 26.7 t 2.2 stM LGA 33-31 77 29.5 5 12 26.2 ? 1.7 s t M LGB 46 2 23.9 ? 1.1* 19.1 t 1.8** s t M LG 21-30 93 30.1 2 0.95 I1 s t M LGA 37-23 65 27.8 2 0.7 23.2 5 1.7

Recombination values are means C confidence limits. Asterisks indicate the level of significance of recombination fraction when compared to SG lines: *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s &test). Reactivity level is given as the mean of the data for IOday-old females; for experiment I it is measured with strong inducer males (Canton-S), for experiment I1 with medium inducer ones (B2’). LGA and LGB are two sublines separated from the 21st long generation. For LG lines, the second number indicates the number of short generations following the long ones.

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1338 A. LaurenCon et al. mer case, for the 3-5-day period, the crossover percent-

ages were 39% for the SG lines and 28-30% for LG lines, whereas in the present experiments, the corre- spondingvalues are 30 and 23%, respectively. The same difference was true for the 15-17-day period. The first experiment was performed 1 year before experiment

I of Table 5 and 2 years before experiment 11. The differences observed probably reflect genetic drift of some determinants of crossover frequency. At this point, it may be of interest to note another variation. All data presented above were obtained with some isofemale sublines of the h215 marked strain; occasionally, other sublines were used for the same experimental procedures and gave different results. With these sublines, the crossover frequency exhibited a very sharp decrease with age and stabilized to low values, whatever the reactivity level of the RstM line used as the mother. We did not study in detail this problem, but the most likely explanation is that the h215 stock was polymor- phic for a dominant crossover suppressor; when this suppressor is present in flies, the recombination efficiency is worn down and, as a consequence, the effect of strong reactivity levels is erased. The argument sup- porting this explanation is that from the low h215 s u b lines it was possible to select, over several generations, isofemale lines with enhanced recombination rates and again we observed differences between RstM LG and RstM SG flies.

Correlation between crossover frequencies and reac- tivity levels can be found independently of heritable aging effect: The above results show that crossover fre-

quency, at least in some parts of the genome, undergoes the same kind of heritable aging effect as reactivity levels. Now we address the question ofwhether this similarity is the consequence of an identical aging effect on

two independent mechanisms or is the result of the aging effect on a unique system controlling both reactivity level and recombination efficiency. For this purpose, we took advantage of a polymorphism that appeared in a reactive strain named Paris. From 1970 to the end of the 1980s, this stock had been very weakly reactive

(BUCHETON and PICARD 1978); then it became heterogeneous for the reactivity level and it was possible to isolate isofemale lines with very clearcut reactivity levels. This provided a very good opportunity to seek a correlation between reactivity levels and recombination efficiencies, in very similar genetic backgrounds, independently of aging effect. For the reasons already mentioned in the above section, the test was performed on the pericentromeric region of chromosome 3.

The comparison was made between a moderately strong reactive line named PF2 (reactivity = 70%), a weakly reactive line named

Pw

(reactivity = 10%) and a medium reactive line named P2 (reactivity = 40%), all bred with a short generation pattern. The marked stock used as males was again h215. Figure 3 shows the variation of crossover frequency with age of heterozy-

-

d , . . . ) . . . . , . . . . , . . . . , . .

0 5 1 0 i s 20

Age of F1 females (days)

FIGURE 3.-Crossover frequency between the st and ST loci

for Paris strain. Results are shown as a function of F, females age, with the weaker reactive line Pw ( A ) , the medium line P2

(A)

and the strong reactive line PF2 (0). Mean values with CL are presented in Table 6.

gous females, and data are presented in Table 6. The strongly reactive flies roughly exhibited the same general kinetics as the strong RstM flies (see Figure 1) with a very sharp rise after day 15; the two other lines did not exhibit any rise in this period. The percentage of recombinants was in good correlation with the reactivity level; however there are some differences with the data on RstM, the largest difference between the three lines is not within the 0-5-day interval, but between days 6

and 14. Other experiments with other strong and weak reactive Paris lines gave the same kind of results with little variation. Thus these data strongly support the conclusion that there is a close relationship between reactivity levels and crossover frequencies, including the late rise that, as suggested above, is possibly related to hormonal effects.

It may be noted that we also used these Paris lines to look for a correlation in the b-cn interval of chromo-

some 2; but no difference was found (data not shown).

Different “reactivity” levels might exist in inducer strains: We took advantage of the close relationship

between reactivity levels and crossover frequencies on the pericentromeric region of chromosome 3 to have an assay other than dysgenic crosses to compare reactivity levels in different categories of flies with common genetic background. More particularly, we took this opportunity to address the question of whether different reactivity levels also exist in inducer stocks. Indeed, if these levels are closely related to repair-recombination activities, and therefore part of a fundamental biological mechanism, they are expected to also exist in inducer flies. Thus, we tried to use the crossing-

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Inducible DNA Repair in Drosophila

TABLE 6

Comparison of crossover frequencies in the sbsr interval of third chromosome between strong and

weak reactive lines of the Paris stock

1339

Maternal line Reactivity level (%) Days 2-4 Days 8-10 Days 11-13 Days 15-17

PF2 P2 Pw 70 48 9 32.0 -C 3.3 31.0 ? 1.3 26.0 2 2.2 23.8 -C 1.8 32.8 2 2.0 25.8 ? 1.4*** 24.3 ? 2.7 17.0 2 2.7** 33.1 ? 4.5 21.9 t 2.0*** 20.2 ? 3.5* 16.8 ? 3.4** Recombination values are means +- confidence limits. Data are from experiment presented in Figure 3; they are scored on six sets of four females. Asterisks indicate the level of significance of recombination fraction obtained with P2 and Pw maternal lines when compared to the values obtained with PF2. * P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

over criterion to address this question, in spite of the difficulties inherent to the complex control of recombination.

To avoid any disturbing effect of transposition, we crossed two inducer stocks: Cha-(RC+) and h215 (RC+) (see MATERIALS AND METHODS). Two lines of Cha-(RC+) were used: Cha-(RC+) SG bred in short generation pattern and Cha-(RC+) LG 31 that had undergone 31 long generations. Females of these lines were crossed with h215 (RC+) males and the F1 daughters backcrossed to score for recombinant percentages (Figure 4). The crossover frequency with the LG 31 line was significantly lower than with the SG line (Table 7). This result supports the view that reactivity levels can be measured in inducer strains. We have tested the reversibility of the aging effect; for this purpose we used a Cha-(RC+) LG 31-34 line simultaneously with the SG line. Within the first days of their lives, F1 females

10 2 0

Age of F1 females (days)

FIGURE 4.-Crossover frequency between the st and sr loci

for Cha-(RC+) strain. Results are shown as a function of F, females age, with lines maintained with short generations (0) and with 31 long generations

(e).

yielded recombinant percentages similar to that obtained with the SG line during the same period, but from day 8 they yielded values similar to that with LG 31 (Table 7). We suppose that within the period that separated the reversibility experiment from the other

(34 short generations, i.e., nearly 1.5 years), the h215 (RC+) line underwent a drift analogous to that reported above for some h215 lines.

Therefore, this experiment supports the argument for different reactivity levels in inducer strains, but it cannot be considered as conclusive. This question will be pertinently addressed only when we have molecular markers for reactivity levels, allowing a more direct approach to this phenomenon.

Chromosome losses and recessive lethals: In a previous article, we showed that different reactivity levels were related to different levels of sensitivity of oogenesis to gamma rays (LAURENCON and BREGLIANO 1995). The parameters tested were fecundity, fertility and larval-to- adult viability. Here, we present data on more precise anomalies: Xchromosome losses, which result from un- repaired double-strand breaks, and recessive lethals, which reflect erroneous reunion of chromosomal breaks (see among others GRAF et al. 1979).

The experimental scheme used for scoring both events is presented in Figure 5. For chromosome loss experiments, we scored the y ct u f males that are X0

males and are produced either by chromosome loss or by X nondisjunction during oogenesis. Bar stone (@) daughters are

XXYP

females that result from either X X gametes produced by maternal nondisjunction events or from XU@ gametes issued from paternal nondisjunc- tion. In the first case, they have two wild Xchromosomes and yield only wild-type male progeny; in the second case, they are heterozygous for a y ct u f Xchromosome and yield about half sons of this phenotype. This allowed us to distinguish between maternal and paternal nondisjunction. Wild-type X0 males that receive from their father a gamete devoid of Y@ result from a pater- nal nondisjunction reciprocal to that providing the first category of

@

daughters. Those X0 males are not shown in the data presented.

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1340 A. Laurenqon et al. TABLE 7

Effect of short and long generation patterns on crossover frequencies in the st-m interval of third chromosome of the Cha-(RC+) inducer strain

Maternal line Days 2-4 Days 8-10 Days 11-13 Days 15-17

Cha-(RC+) SG 32.5 ? 2.9 27.2 +- 2.3 22.5 ? 3.5 19.8 ? 2.5

Cha-(RC+) LG 31 28.4 ? 1.7" 19.9 ? 1.8*** 19.0 ? 1.9' 15.9 ? 3.6* Cha-(RC+) LG 31-34 32.2 ? 2.6 22.1 ? 3.7" 16.9 +- 1.2** 17.2 ? 3.1

Recombination values are means +- confidence limits. Data are from experiment presented in Figure 4. They are scored on six sets of four females. Asterisks indicate the significance level when compared to the Cha-(RC+) SG line within the same time interval. * P < 0.05, **P< 0.01, ***P < 0.001 (Student's t-test). SG, short generation; LG 34, 34 long generations; LG 31-34, 34 short generations after 31 long generations.

era1 lines of the atM stock: SG lines, with reactivity of 100% at the time of this experiment, LGC

7

(90%) and LGA 37 (55%) lines. Virgin females 48hr-old were irradiated at 40 Gy, then mated with y ct v f / Y @ males

(15

sets of four females each with six to eight males for the treated series and 10 sets for the controls). In paral- lel, five sets of four females each were crossed with the inducer stock Canton-S to check for the reactivity level. Data are presented in Table 8. The frequency of X0 males was much higher in the irradiated series than in the controls, whereas the frequency of Barfemales was identical. Therefore, we can infer that the great major- ity of these X 0 males resulted from X-chromosome loss during oogenesis. As previously reported (KELLEY and LEE 1983), we observed that mature eggs are the most

Q

X+/X+

irradiated or control X Scoring for absence of

6 X + / Y

sensitive to irradiation, this sensitivity progressively de- creased for earlier stages.

When pooling the data for 1 month, we note a significant difference between the stronger reactive line SG and the weaker line LGA 37. The difference is highly significant between days 7 and 10. The data of line LGC

7

are not significantly different from the two others, but we note they are intermediate, as is the reactivity level. Considering the egg-laying rate of the three lines, we infer that the eggs laid between days

7

and 10 origi- nate from egg chambers irradiated at stages 4-7 according to King's nomenclature (see ASHBURNER 1989). At those stages the synaptonemal complex begins to disappear (CARPENTER 1975). According to more re- cent works in yeast, this disappearance is concomitant

X

I

Q X + l y c t v f [+I

6

X+IYBS [ B S ] Normal progeny

Q

[ B S ] (') Non-disjunction

6

b c t v f

3

Chromosome loss

Measurement of recessive lethal mutations. Measurement

of chromosome anomalies.

FIGURE 5.-Scheme of crosses designed to quantify chromosomal aberrations in response to gamma rays. Wild-type flies of known reactivity levels were mated in two different ways. To check for recessive lethal mutations they were crossed with males bearing the Binscy balancer chromosome with Bar ( B ) mutation. To observe X-chromosome segregation those flies were mated with males bearing X and Y marked chromosomes. (1) The Bar stone (@) females were generated by nondisjunction from either female or male; in the first case, their progeny was only wild type for the markers of the Xchromosome. These events also generated wild-type males among those exceptional offspring. (2) The sterility of [y ct U J males was checked; all were

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Inducible DNA Repair in Drosophila

TABLE 8

Chromosome anomalies after irradiation of weak and strong reactive lines

1341

Frequency of X 0 Exceptional males

( x ~ o - ~ )

in

Total offspring the period analyzed Frequency

Reactivity normal Female Male Days Days of female

level (%) offspring [Bar1 [y ct u p 7- 10 1-30 nondisjunction s t M SG C 100 35,111 27 50 < 10-5 I 100 27,045 24 204 25.5 15.3 < 10-3 C 93 31,552 41 40 < 10-5 I 98 27,314 30 246 32.2 18.3 < 1 0 - ~ C 58 23,751 9 20 < 10-5 I 70 18.445 12 161 46.7** 19" < 10-3 atM LGC 7 s t M LGA 37

Period analyzed: days 1 - 10

seF8 X e r y C 55 6,552 5 0 I 50 7,281 2 2 0.54 C 3 7,537 1 2 I 4 6,995 2 11 2.92* e ry X seF8

In the first experiment with the stM stock, adults 48 hr old are irradiated with a 40-Gy dose. In the second experiment with seF8 and e ry stocks, 48-hr-old pupae are irradiated at 30 Gy. Bar females come from female or male nondisjunction events (see text and Figure 5). The number of X 0 males due to male nondisjunction (wild-type males) is not presented on this table. The frequency of [y ct u J I males is given as the fraction of those X 0 males on total number of females plus X 0 flies. Asterisks indicate the level of significance for the differences between irradiated strong and weak reactive flies ( s t M SG and stM LGA 37 in the first experiment): * P < 0.05, **P < 0.01. C, control; I, irradiated.

to generation of mature recombinant molecules from recombination intermediates (PADMORE et al. 1991). This process needs repair activities that appear correlated with reactivity levels.

Second experiment: Another way to have isogenic fe- males with different reactivity levels is to use F1 females from reciprocal crosses between strong and weak reac- tive stocks. We used seF8 as a strong reactive stock and e

y

as a weak reactive one. In this experiment, the whole experimental scheme of Figure 5 was utilized to measure the frequency of both X0 males and X-recessive lethals. Reactivity was measured using the B2' inducer stock instead of Canton-S. F1 females were irradiated as 48-hr-old pupae at 30 Gy (at higher doses, the pupae gave adults with partial sterility). The irradiation of pupae instead of adults was done to see whether the first steps of meiosis were also sensitive to differences in reactivity levels. Data on chromosome losses (Table

8) show that within the first 10 days there is a significant excess of X0 males in the progeny of F1 females from the e y X seF8 cross, in which reactivity is very weak compared to that of the F1 females from the reciprocal cross. Data for days 11 -30 are not shown because there is no difference in X0 males frequencies, neither between the two irradiated series nor between irradiated and control series.

The frequencies of recessive lethals were scored in the progeny issued from eggs laid by F1 females between days

2

and 5. As shown in Table 9, there is a highly significant excess of lethal mutations in the offspring of the seF8 x e y cross, i.e., in the progeny of F1 females with highest reactivity level.

DISCUSSION

Reactivity level is one of the factors controlling cross- over frequency: Striking similarities with reactivity levels appear when one reads the literature on recombination in D. melanogaster published since the beginning of the century. In MORGAN'S group, it was shown that crossover frequency varies widely with various factors, such as the temperature at which females develop, their age and interchromosomal effects of chromosome rear- rangements, the greatest variations being generally ob-

served in the centromeric regions of chromosomes 2

and 3 (PLOUGH 1917; STEIN 1926; BRIDGES 1927). These features have been confirmed by several authors (HAY-

MAN and PARSON 1960; GRELL 1978) and other kinds of variation were described such as the great interindi- vidual variability of the crossover frequency at identical age and breeding conditions (BROADHEAD and KIDWELL

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1342 A. Laurenqon et al. TABLE 9

Frequency of recessive lethal on X chromosome scored after irradiation of strong and weak reactive females

No. of No. of Origin of F1 chromosomes recessive

females analyzed lethals ( R L ) % RL Cross seF8 X e 9

Control 2,226 9 0.40

Irradiated 2,145 52 2.42

Control 2,102 9 0.43

Irradiated 2,456 31 1.26

Females were irradiated at pupal stage 48-hr old with a 30- Cy dose. The experimental scheme is shown on Figure 5. Difference in recessive lethals produced by the two reciprocal F1 females after irradiation is highly significant ( P < 0.01). The reactivity of the hybrids are 55% from the cross seF8 X

e ry and 3% for those from the cross e ry X seF8.

Cross e ry X seF8

behavior of reactivity levels (for a review see BREGLIANO

and KIDWELL 1983). The data reported here strongly support the view that there is a relationship between these phenomena, however in some cases, it may be confounded by other undetermined factors. The relationship is quite clear in the pericentromeric region of chromosome 3. In this case the correlation can be seen either by the way of the heritable, cumulative and reversible aging effect, or by the use of the strong and weak reactive variants of the Paris stock, all bred with short generations.

Concerning the aging effect, what emerges from our data is that when there is an effect of female age on recombination, there is also an effect of long generation breeding in the same direction, i e . , the effect of the female aging is heritable and cumulative over generations. When there is no aging effect there is no effect of long generation pattern (ct-v and m-fintervals on X chromosome). The only exception to this rule is with the second chromosome. Leaving apart this exception, we may simply assume that in some chromosomal regions the recombination efficiency is dependent on the same biochemical function as reactivity level, while in other regions it is not. When the two functions are connected they can be correlated positively, as on the pericentromeric regions of X and third chromosomes, or negatively as on the distal part of the Xchromosome. This situation is not surprising given the very complex control of crossover frequency, already pointed out by several authors (CHARLESWORTH and CHARLESWORTH

Considering now the results on the second chromosome, the situation is more puzzling. There is a female aging effect but it is not heritable, thus there is only a partial similarity with reactivity levels. This is especially true in the b c n interval from day 0 to 13 where the aging effect is strong. We have no clear explanation for this fact, however two points may be noted. First, this 1985a,b; PARSONS 1988).

situation is reminiscent of that observed on the third chromosome with some h215 sublines, therefore we cannot exclude some interfering effect of crossover sup- pressors. Second, on the b-cn interval we observed a strong late effect of long generation pattern, near the 20th day (Table 4).

In a more general way, it must be noted that all our experiments deal only with crossovers. Crossing over is only one of the two results of homologous recombination that can be analyzed; the other result is conversion. Unfortunately, data about gene conversion cannot be obtained for the various regions of the genome in Dro- sophila and has only been reported in a few works (CAR-

PENTER 1982; HILLJKER et al. 1991). At the rosy locus it has been reported that the percentage of conversion events without crossing over is nearly 80% of total recombination events (CARPENTER 1982). Therefore we may imagine that some factors acting on recombination modify the balance between the two kinds of events without modifying the whole frequency of events or, conversely, that some factors act on the achievement of only one type of event. Thus, in our case, no effect of reactivity levels on crossovers does not mean no effect at all on homologous recombination. It would also be of interest to know what kind of genetic event is respon- sible for the sudden appearance of heterogeneous reactivity in the Paris stock after at least 20 years of stable low reactive state. We have no definitive answer however, as it was relatively easy to select stable isofemale lines with two extreme levels (100% and nearly 0 % ) . We propose that this event is a mutation involving one major chromosomal determinant of reactivity level. Unfortunately, because of the existence of the maternally inherited state, it has not been possible to map this determinant. In the future, this situation may provide a useful tool for a molecular approach to reactivity.

Reactivity levels and DNA repair: The two experiments on induced chromosome losses and recessive lethals provide convergent data. It appears that chromosome losses are correlated with weakly reactive backgrounds whereas recessive lethals are correlated with strongly reactive one; in this latter case it seems that repair of breakages does occur but is often illegitimate. In a previous paper we reported data indicating that medium reactive level is the most efficient condition for correct repair of damage after germ-line irradiation of females. Weak reactive levels led to low fecundity and low embryo viability, which we interpreted as the result of loss of genetic material. Strongly reactive levels led to good fecundity but low viability of embryos, which agrees with few losses but many chromosomal rearrangements leading to aneuploid gametes through meiosis (LAURENCON and BREGLIANO 1995). The data presented here on recessive lethals strengthens this inter- pretation, although it is not known if these lethals are associated with rearrangements or are due to point mutations. In either case they indicate error-prone repair

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Inducible DNA Repair in Drosophila 1343 that is reminiscent of the mutagenesis in E. coli that

occurs when the SOS network is strongly induced. In summary, the available data make a strong case for reactivity levels being closely related to repair of DNA breakage.

The mechanisms of DNA repair in higher organisms are still poorly understood, thus it does not seem profitable to discuss in detail the possible significance of our results with regard to the molecular mechanisms underlying reactivity levels. However, many repair-recombination mutants have been described in Drosoph- ila and it may be of interest to look for possible analo- gies with the phenomenon described here; moreover, this may provide interesting clues for a molecular approach to the analysis of reactivity levels. First we may notice that the link between reactivity and both meiotic recombination and repair suggests that it is related either to a repair mechanism involving homologous recombination, such as post-replication repair or double- strand break repair described in both bacteria and yeast (SZOSTACK et al. 1983; KUSANO et al. 1994), or to a repair process interfering secondarily with recombination. In the first case the mei-41 gene may be involved because it has been described as required for post-replication repair (BOYD et al. 1987). The second possibility may involve mei-9, as it is required for excision-repair and also has a function in resolving the recombination intermediates into crossovers during meiosis (CARPENTER

1982). Other DNA repair genes are probably involved, but mei-9 and mei-41 are especially interesting to con- sider because they are involved in meiotic recombination and they have recently been cloned

(m

et al. 1995; SEKELSKY et al. 1995). A second kind of comparison can be made. With regard to their effect on both frequency and distribution of exchange events, the already characterized loci involved in repair-recombination in Drosophila fall into two general categories: those that are involved in the control of both the nonrandom distribution of exchange events along the chromosomes and their normal frequency (precondition for exchange loci) and those necessary only for the normal frequency of events (exchange loci). mei-41 belongs to the first category along with other known meiotic loci, whereas mei-9 belongs to the second class. Considering the opposite effects of aging along the X chromosome and assuming that these effects are mediated through reactivity levels, we may assume that reactivity acts on preconditions for exchanges. This feature leads to bring it together with the mei-41 category rather than with mei-9. Some repair genes identified in Drosophila have been described as genes with a prolonged maternal effect, lasting until late larval development ( G w and WURCLER 1978; BAKER et al. 1982), but no heritable maternal transmission of the phenotype has been reported. The possibility that these genes are phenotypi- cally expressed in a nonconventional way, such as that described for reactivity level, cannot be excluded; ex-

periments are underway to address this question. It must also be pointed out that whatever may be the recombinational step to which reactivity is related, we cannot know whether this function consists of true repair enzymes or of other kind of proteins secondarily influencing repair and recombination processes because they might be involved in the regulation of meiotic gene expression or chromatin structure. The second possibility might help to understand the complex inheritance of reactivity levels by suggesting epigenetic mechanisms involved in chromatin structure (MEYER et al. 1992; DILLIN and &NE 1995).

Reactivity levels and Z factor: In the past, the term reactivity was defined as “the permissive condition for I factor transposition at high frequencies” (PELISSON and BREGLIANO 1987). More recently, LACHAUME and

PINON (1993) showed that reactivity levels regulate expression of I factor. In a previous article, we pointed out the fact that reactivity level being closely related to inducible repair activities, it very likely depends on a host function (LAURENCON and BREGLIANO 1995). Therefore it must be dissociated from the regulation by &encoded repressor that prevents transposition in inducer strains (PELISSON and BREGLIANO 1987;

MCLEAN et al. 1993). Within this line of reasoning, we may assume that the same variable repair system takes place also in inducer flies. The cumulative aging effect on recombination described in this report with the Cha- (RC+) inducer stock is in agreement with this assump- tion although, as already mentioned, it is not possible to get definitely conclusive data on this point as far as we lack molecular markers for reactivity levels.

Thus the term reactivity, as previously defined, covers at least two different mechanisms: the regulation that operates in inducer strains, very likely depending on I factor-encoded functions, and the repair-recombination system related to reactivity levels. To clarify the situation, we think it necessary to now use a proper appellation for this variable repair-recombination function. We propose to call it VAMOS, which stands for variability modulation system. This acronym stresses the fact that thismechanism modulates genomic variability under influence of several endogenous and exogenous factors.

Depending on the different factors described (age, temperature, DNA-damaging agents) oocytes are able to repair DNA damage and to produce recombinant gametes with different efficiencies. The transmission to the offspring of these variable efficiencies certainly makes the VAMOS very important for adaptation and evolution. The main potential biological implications of this system will be discussed further elsewhere.

We especially thank ~ U R I Z I O GAITI for very useful discussions and critical reading of the manuscript, we are very grateful to RAYMOND

DEVORET, JEAN-LUC ROSSIGNOL and A W N PELISSON for commentS on the first draft and very useful discussions on the whole work. We also thank YANNICK Azou, CHRISTOPHE DE LA ROCHE SAINTANDRE

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1344 A. Laurencon et al. and KIM MCKIM for very useful comments on the manuscript. We

also thank the Centre d’Immunologie d e Marseille-Luminy for kind access to the gamma-ray source. This work was supported by the Centre National de la Recherche Scientifique, the UniversitC d e la MediterranCe and the Association pour la Recherche sur le Cancer.

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