radiation-sensitive mutants of caenorhabditis elegans - Europe PMC

Copyright 0 1982 by the Genetics Society of America

RADIATION-SENSITIVE MUTANTS OF CAENORHABDITIS ELEGANS PHILIP S. HARTMAN'

AND

ROBERT K. HERMAN

Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108 Manuscript received January 21, 1982 Revised copy accepted June 4, 1982 ABSTRACT

Nine rad (for abnormal radiation sensitivity) mutants hypersensitive to ultraviolet light were isolated in the small nematode Caenorhabditis elegans. The mutations are recessive to their wild-type alleles, map to four of the six linkage groups in C. elegans and define nine new games named rad-1 through rad-9. Two of the mutants-rad-1 and rad-2-are very hypersensitive to X rays, and three-rad-2, rad-3 and rad-4-are hypersensitive to methyl methanesulfonate under particular conditions of exposure. The hypersensitivity of these mutants to more than one DNA-damaging agent suggests that they may be abnormal in DNA repair. One mutant-rad-5, a temperature-sensitive sterile mutant-shows an elevated frequency of spontaneous mutation at more than one locus; rad-$, which shows a cold-sensitive embryogenesis, reduces meiotic X-chromosome nondisjunction tenfold and partially suppresses some but not all mutations that increase meiotic X-chromosome nondisjunction; the viability of rad-6 hermaphrodites is half that of rad-6 males at 25'; and newly mature (but not older) rad-8 hermaphrodites produce many inviable embryo progeny. Meiotic recombination frequencies were measured for seven rad mutants and found to be close to normal.

ADIATION-sensitive mutants have played an important role in the develR opment of current models of DNA repair, recombination and mutation in bacteria, including the view that these processes are interrelated (for reviews, see HANAWALT et al. 1979; HANAWALT, FRIEDBERG and Fox 1978). The general approach of identifying and characterizing radiation-sensitive mutants-as well as mutants hypersensitive to chemical mutagens, an overlapping class-has been extended to single-celled eukaryotes such as yeast (for reviews, see LEMONTT 1980; PRAKASH and PRAKASH 1980; REYNOLDS and FRIEDBERG 1980) and to the metazoan Drosophila (for reviews and recent references, see SMITH, SNYDER and DUSENBERY 1980; BAKERet al. 1976, 1980; BOYDet al. 1980, 1981; BOYDand HARRIS1981). The eukaryotic work has shown that at least some radiation-sensitive and mutagen-sensitive mutants are affected in DNA repair and that certain mutants are affected in one or more other processes such as recombination, mutation and chromosome behavior in meiosis and mitosis. The small, free-living nematode, Caenorhabditis elegans, is receiving attention as a model for studying the genetic basis of animal development, largely because each individual animal has a small number of cells that are produced through extremely precise patterns of cell lineage during development (for review, see VON EHRENSTEIN and SCHIERENBERC 1980) and because the organism possesses ' Permanent address: Department of Biology, Texas Christian Genetics 102 159-178 October, 1982.

University, Fort Worth, TX 76129.

160

P. S. HARTMAN, AND R. K. HERMAN

several advantages for genetic manipulation (BRENNER 1974; HERMAN and HORVITZ 1980). In this paper we describe the isolation and genetic characterization of nine mutants of C. elegans showing hypersensitivity to ultraviolet light. We also describe how the mutants are affected with respect to the following properties: sensitivity to X rays, sensitivity to the chemical mutagen methyl methanesulfonate (MMS), recombination frequency, spontaneous mutability, meiotic chromosome disjunction and general viability and fertility. MATERIALS AND METHODS

Strains and general procedures: Wild type (designated N2) and many mutant strains of C. elegans and were cultured as described by vac. Bristol were obtained from the laboratory of S. BRENNER BRENNER (1974). Nomenclature is as described by HORVITZ et al. (1979). The following genes and alleles were used: LG (linkage group) I: him-l(e879), dpy-5(e61), unc-l3(e52), unc-76(~950),unc-54(el90), rad-3(mn157), rad-8(mnl63), rad-l(mnl55), him-2(~1065) LG 11: dpy-lO(el28). unc-4(~120),him-S(e2487) LG 111: dpy-l(el), him-lO(el511), unc-36(~251), unc-86(e1416), unc-32(el89), dpy18(e364), rad-9(mn162), rad-5(mnl59), rad-6(mnl60) LG IV: unc-5(~53), dpy-4(~1266), him-3(~1147), him-6(~1423), rod-7(mnl61), him8(e1489) LG V: unc-60(~677), him-7(el480), dpy-11(~224), unc-42(~270), him-5(el467), unc76(e911), rad-4(mnZ58), rad-Z(mn156) LG X: dpy-7(~88),nuc-l(el392), unc-3(e152), fIu-2(t203), unc-58(~665),him-4(~1267) The map positions of these genes, based on information gathered by the Caenorhabditis Genetics Center (M. SWANSON and D.RIDDLE, personal communication) and a personal communication from M. FINNEYand R. HORVITZconcerning unc-86, are shown in Fig. 1. In addition, the unmapped mutation rec-l(sl80) was used. All experiments were conducted at 20’ except where noted. Mating procedures were as described previously (BRENNER 1974; HERMAN 1978). Mutant isolation: All rad mutants were isolated after EMS (ethyl methanesulfonate) mutagenesis (BRENNER 1974). Two schemes were employed. In both, 24-well microtiter dishes (Costar) were filled with 1.5 in1 NGM agar (BRENNER 1974) and the wells were seeded with E. coli OP50 about 24 hr before irradiation. To insure that animals could not crawl out of the wells in which they had been placed, the dishes were allowed to dry for at least 48 hr before use. In the first scheme, adult secondgeneration (R) hermaphrodites from EMS-mutagenized N2 animals were placed in individual microtiter wells. After 2 hr, each adult was removed to a “rescue” well; the eggs laid in the first well were counted and irradiated with either 1.0 kr X-irradiation or 5 Jm-z UV radiation. Both doses are sublethal to wild type, permitting the identification of hypersensitive broods upon later examination (usually after 3 days). An egg-laying period of greater than 2 hr gave a heterogeneous population with respect to radiation sensitivity. The second scheme differed from the first as follows: the Fz adults were incubated overnight in the first microtiter well, which served as the “rescue” well. Each adult was then placed in a second well, where it received immediately a dose of 60 Jm-’ UV radiation. Irradiated adults were removed after 24 hr, and the dishes were scored for surviving progeny 24 to 48 hr later. The second scheme was a n improvement on the first because it eliminated the need to count individual eggs, a step required in the first scheme by the variation in the number of eggs laid from well to well. Genetic mapping and complementation: With the exception of rad-6, the mutants were indistinguishable in appearance from wild type. The rad-6 mutant has a slightly dumpy appearance, more pronounced in hermaphrodites, with variable expressivity. In assessing the results of genetic crosses, radiation sensitivity itself was used to assign the Rad phenotype. This was usually determined by allowing 5-6 adult hermaphrodites to lay eggs for up to 2 hr. Following removal of adults, eggs were counted and either incubated in the dark as controls or administered a dose of either 1.0 kr X-or 5 Jm-’ UV radiation. The plates were scored for survivors 2-3 days later. Since recognition of the Rad phenotype requires progeny testing, genetic positioning was accomplished

C. ELEGANS RADIATION-SENSITIVE MUTANTS

161

mainly by a series of three-factor crosses. Results, which are presented using the nomenclature of HERMAN and HORVITZ (1980), were as follows: rad-1 I: dpy-5(7/7)(unc-13 rad-1); dpy-5(6/9)rad-1(3/9)unc-75. In a two-factor cross, 11/12 Dpy segregants from a + +/dpy-5 rad-1 heterozygote were radiation sensitive. rad-2 V: unc-60(3/3)(dpy-11 rad-2); dpy-11(2/11)rad-2(9/ll)unc-42. rad-3 I: dpy-5(1/9)rad-3(8/9) unc-54; dpy-5(5/7)rad-3(2/7)unc-13. rad-4 V: (rad-4 dpy-11)(4/4)unc-42; unc-60(5/10)rad-4(5/10)dpy-l1. rad-5 III: dpy-1(10/10)(unc-36 rad-5); (rad-5 unc-36)(13/13)dpy-18. rad-6 III: unc-36(7/16)rad-6(9/16)dpy-l8.In a two-factor cross, 0/12 Unc segregants from a rad-6/ unc-32 heterozygote were Rad. rad-7 IV: unc-5(5/13)rad-7(8/13)dpy-4.In a two-factor cross, 0/12 Unc segregants from a rad-7/unc5 heterozygote were Rad. rad-8 I: (rad-8 dpy-5)(8/8)unc-54; dpy-5(2/8)rad-8(6/8)unc-13.In a two-factor cross, 10/10 Unc segregants from a + +/rad-8 unc-13 heterozygote were Rad. rad-9 111: (rad-9 unc-36)(6/6)dpy-18; dpy-1(10/11)rad-9(1/11) unc-36. A simplified genetic map, indicating the positions of the rad mutants and the visible mutants used in mapping, is shown in Figure 1. As a consequence of the above mapping data, complementation analyses were required for only three pairs of mutations: rad-3 and rad-8 on LG I, rad-5 and rad-9 on LG 111, and rad-5 and rad-6 on LG 111. All three pairs were found to complement with respect to brood size, temperature sensitivity and radiation sensitivity. The mutations rad-4 and him-7 both affect meiotic X-chromosome nondisjunction and map to similar locations. The following data indicate that they are not allelic: (1) rad-4, but not him-7, animals were hypersensitive to UV radiation; (2) the brood sizes of rad-4, but not him-7, mutants were greatly reduced at 15'; and (3) rad-4/him-7 heterozygotes showed wild-type levels of radiation sensitivity and X-chromosome nondisjunction (6 self-progeny broods of rad-4/him-7 animals yielded 4 males out of 2029 total progeny). es and hermaphrodite self-progeny Measuring male and hermaphrodite fertilities: Male fert broods were scored according to the methods described by HODGKIN, HORVITZ and BRENNER (1979). Sick animals were defined as those that hatched but either failed to reach adulthood or displayed grossly abnormal features. The percent sick animals was calculated relative to total viable zygotes. Brood data were derived from the analysis of six complete broods at 20" and at 25". The percentage of male self-progeny was obtained by scoring a minimum of IO00 animals of each genotype. The 25O/2Oo brood-size ratios were normalized by assigning N2 a value of 1.0. The 25' progeny testing was performed by shifting adults from 20' to 25' and analyzing the broods of their progeny. Quantitation of radiation and MMS sensitivities: Eggs were isolated and purified by the method of EMMONS, KLASSand HIRSH(1979) except that the NaOH and NaOCl concentrations were 0.2 N and 170,respectively, and the eggs were washed three times with phosphate-buffered saline (PBS), consisting of 8 g NaCI, 0.2 g KCl, 2.25 g NaZHP04 and 0.2 g KHzPOI per liter. The purified eggs, typically at concentrations of I O 4 per ml, ranged in age from roughly 30 min to 180 min (where zero age corresponds to time of fertilization) and could be diluted and plated accurately with a 1-ml pipet. To test UV- and X-radiation sensitivities, eggs were diluted into PBS to give between 25 and 300 survivors per plate and 0.1-ml aliquots were pipetted onto a series of agar-filled Petri plates, which were then irradiated a t different doses. Survival was scored as the percentage of animals reaching adulthood 4 to 6 days after irradiation relative to unirradiated controls. X-radiation was supplied by a Mueller RTlOO irradiator at a dose rate of 470 roentgens (r) per minute. UV radiation was provided by a single Sylvania germicidal 15-W bulb (G15T8) at a distance of 53 cm. Irradiated animals were protected from ambient light. The UV dose rate was 1 Jm-'s-' as determined by ferrioxalate actinometry (JAGGER 1967). Potassium ferrioxalate was purchased from Duke Standards (Palo Alto, CA). MMS sensitivities were determined both on plates and in liquid. Although MMS can be incorporated directly into the medium before pouring plates, it was more convenient to spread the mutagen on solid agar. The response to MMS using this spreading method was found to be influenced by the moisture content of individual plates as well as the time between MMS application and addition of worms. The method was standardized by flooding plates with sterile HzO,

162


I

It

m

m P

X

I

I

FIGURE1.-A simplified genetic map of C. elegans showing loci used in this work. immediately shaking off excess H20, and incubating overnight at 37'. To each plate 0.1 ml of the appropriate MMS stock was added and spread evenly across the agar surface. After 30 min, the plates were seeded with E. coli OP50, and eggs, prepared by the hypochlorite method, were added. Final MMS concentrations were calculated on the assumption that the MMS was uniformly incorporated into the medium. Terminal developmental stages were determined by size estimates, ~ MMS sensitivities in liquid were determined by using a dissecting microscope at 2 0 magnification. suspending eggs in PBS containing 50 mM MMS. Aliquots of 25 pl were withdrawn at various intervals and plated. RESULTS

Mutant isolation, mapping and complementation: Two schemes, detailed in were used to screen 6434 Fz broods of EMS-mutagenized animals for hypersensitivity to either UV- or X-radiation. Of the 485 candidates produced by this initial screening process, 9 proved to be consistently hypersensitive, a yield of 0.14%. From the same 6434 broods, 14 him mutants were detected on the basis of the high incidence of male self-progeny. Thirteen were recessive and may be similar to the meiotic nondisjunction mutants described by HODGKIN, HORVITZand BRENNER(1979). The remaining him mutant was dominant and has been studied as one member of a collection of dominant X-chromosome nondisjunction MATERIALS AND METHODS,

C. ELEGANS RADIATION-SENSITIVE

163

MUTANTS

C. K. KARIand P. S. HARTMAN, unpublished results). mutants (R. K. HERMAN, None of the him mutants showed significant departures from the wild-type pattern of radiation resistance (see below). The nine rad mutants (for abnormal radiation sensitivity) were outcrossed twice to wild type and bFhaved as single, recessive Mendelian factors. Linkage testing, mapping by three-factor crosses, and complementation testing led to the assignments shown in Figure 1.The nine mutations defined nine genes, rad2 through rad-9. Viabilities and fertilities of rad mutants: Self-progeny broods of the rad mutants showed a variety of departures from the wild-type pattern (Table 1). Although some (e.g., rad-2, rad-2, rad-3) gave brood sizes similar to that of wild type, the brood sizes of others (rad-5, rad-8 and rad-9) were reduced by factors of greater than ten. Four of the mutants (rad-5, rad-6, rad-7 and rad-9) gave significantly reduced broods when reared at 25O (Table 1). Temperature shift experiments on rad-5 produced asymmetrical curves, with the slope of the shiftdown curve displaying a gradual slope relative to the shift-up curve (Figure 2). Similar responses have been noted with other temperature-sensitive mutants of C. elegans (HIRSHand VANDERSLICE 1976).When rad-5 fourth stage larvae (L4's) or young adults were shifted from 15' to 25O, many unfertilized oocytes were subsequently laid at the higher temperature. The rad-5 temperature-sensitive period extended over most of the life cycle. Meiotic X-chromosome nondisjunction, as measured by the frequency of male self-progeny, was not significantly increased in any of the rad mutants (Table 1);however, the rad-4 mutant showed reduced X-chromosome nondisjunction, which will be discussed in more detail in a later section. A number of mutants produced large numbers of inviable zygotes or sick animals or both. Male rad mutants were generally less fertile than wild-type males (Table 2). The him-5 mutation-which by itself reduced male fertility less than 50%-was used to generate rad-8 and rad-9 males, because repeated attempts at generating males by heat shock were unsuccessful. Mutants with low male fertilities (Table 2) also showed small hermaphrodite self-progeny brood sizes (Table 1).All mutant males tested produced equal numbers of X- and nullo-X-bearing sperm (Table 2). Relative sensitivities to radiation: Radiation sensitivity decreases markedly TABLE 1 Self-progeny broods of rad mutants At 20' Inviable zygotes

Yo Sick

250/20° brood size ratio

0.4 8.3 5.1 0.4 1.7 4.3 32.1 5.4 12.6 16.0

1.0 0.6 0.9 1.0 1.4 0 0.1 0.4 1.9 0.2

%

rad genotype

Average brood size

90Males

Wild type

262 k 33 155 f 86 238 k 23 152 f 53 89 f 29 19 f 13 138 f 30 65 f 14 19 f 16 24 k 12

0.2 0.2 0.1 0.2 t0.1 0.2 0.3

rad-1 rad-2 rad-3 rad-4 rad-5 rad-6 rad-7 rad-8 rad-9

0.3

0.3 0.4

0.2 1.4 0.7 1.6 10.4 17.7 23.3 3.5 70.2 62.2


164

wU

EQUIVALENT TIME AT 2 5 O (days)

FIGURE2.-Temperature-shift experiments with r a d 4 Procedures were identical to those of HIRSHand VANDERSLICE (1976), with plates shifted from 25' to 15' (0) and from 15' to 25' (0). except zygotes were isolated as eggs by the hypochlorite method (see MATERIALS AND METHODS). Egg hatching occurred at about 1 2 hr; animals reached adulthood at about 48 hr and began egg laying at about 57 hr (WOODet al. 1980).

TABLE 2 Sizes and sex ratios of broods sired b y rad moles Cross progeny Male genotype

Hermaphrodite

Male

'3 Wild-type fertility

Wild type rad-1 rad-2 rad-3 rad-4 rad-5 rad-6 rad-7 rad-8; him-5 rod-9; him-5 him-5

789 399 696 730 128 69 272 680 0

815

100 49 87

9

11

0 1

531

524

66

394 701 723 125 73 277 674 0

90

16 9 34 85

throughout the nematode life cycle, particulariy during embryogenesis (KLASS 1977; SAMOILOFF 1980; P. S. HARTMAN and R. K. HERMAN, unpublished). The procedure of collecting young embryos, encased in relatively impermeable egg shells, by dissolving gravid hermaphrodites with a hypochlorite solution (EMMONS, KLASS and HIRSH1979) yielded fairly homogeneous and reproducible populations of zygotes with respect to response to UV- or X-radiation, as illustrated in Figures 3 and 4.The kinetics displayed by wild type are typical of most organisms; that is, a marked shoulder is apparent at low radiation doses, followed by exponential inactivation. All nine rad mutants are significantly hypersensitive to UV radiation (Figure 3), but the response to X rays is more varied (Figure 4). The nine rad mutants show three basic patterns of radiation sensitivity. Two mutants (rad-2 and rad-2) are extremely hypersensitive to both UV- and X-radiation. One mutant (rad-3) is extremely UV hypersensitive but exhibits near normal X-ray resistance, and the remaining six mutants are moderately UV hypersensitive and show less, if any, X-ray hypersensitivity. For every case of hypersensitivity, the shoulder of the inactivation curve was reduced or abolished.


0 20 40 20 40 DOSE(Jm-

165

a

0 40

FIGUREa.-UV-radiation sensitivities of wild-type (N2) and rad mutant eggs. The wild-type inactivation curve is presented in each panel for comparison.

1234 1234 1234 DOSE (kr) FIGUREI.-X-ray sensitivities of wild-type (N2) and rad mutant curve is presented in each panel for comparison.

The wild-type inactivation

Other mutants that might be expected to be radiation hypersensitive were quantitatively assayed for their UV- and X-radiation sensitivities. These included him-2 through him-10, unc-86 (a weak Him), the nuclease mutant nuc-2 (SULSTON 1976),flu-2 (BABU1974),rec-2 (ROSEand BAILLIE1979) and the 14 him mutants isolated in this study. Of these 28 mutants only him-2 proved significantly hypersensitive to either UV or X-radiation (Figure 5). It is worth noting that eggs derived from unc-86 hermaphrodites by hypochlorite treatment were actually considerably more resistant to UV- and Xradiation than eggs from wild type. But because unc-86 hermaphrodites are defective in egg laying (HORVITZ and SULSTON 1980; SULSTON and HORVITZ 1981),

166

P. S. HARTMAN, A N D R . K. HERMAN

B o

\

10 \

z w 0 K w

o \ \\

0

-1

1

0

20

40

1

2

3

1

.

4

DOSE(Jm-2) DOSE (kr) FIGURE5.-Sensitivities of wild-type (-----; data taken from Figures 3 and 4 ) and him-1 (0)eggs to: (A) UV, (B) X rays.

the embryos obtained by hypochlorite treatment are older on average than wild-type embryos. The unc-86 patterns of radiation response were obtained from wild-type eggs by allowing them to develop 5-10 hr before irradiation. MMS sensitivities: We used three methods to assay MMS sensitivities. Two involved chronic exposure to low levels of MMS, and the third involved relatively brief acute exposures. In the first chronic exposure method, eggs collected by hypochlorite treatment of gravid hermaphrodites were placed on plates containing various concentrations of MMS and allowed to develop. Even at the highest MMS concentration used (0.36 mM), virtually all eggs hatched for every strain tested, but development became arrested at progressively earlier larval stages with increasing MMS concentration. Arrest was fairly uniform, with over 70% of each population arresting at a particular developmental stage for a given MMS concentration; development before arrest was slower than for untreated controls. Figure 6 shows the response of wild type and the rad-3 mutant in this assay. Wild-type development was not arrested by concentrations of 0.1 mM and below, although at 0.1 mM, development was significantly slower than for untreated controls. A concentration of 0.36 mM was necessary to arrest development of the majority of the population at the first larval stage (Ll).For rad-3, development of most animals was arrested at L1 by 0.1 mM MMS. None of the other eight rad mutants showed significant MMS hypersensitivity by this assay. In the second chronic exposure method, L4 larvae were placed on plates containing various concentrations of MMS and removed after 24 hr; progeny remaining on the MMS plates were then allowed to develop and scored for predominant stage of developmental arrest. By this assay, rad-3 was again hypersensitive compared to wild type, but in addition, rad-2, which showed no hypersensitivity in the first chronic exposure method, was even more hypersensitive than rad-3; see Figure 7. None of the other rad mutants showed MMS hypersensitivity by this assay. In the acute exposure assay, eggs were incubated in 50 mM MMS for various times before plating. The effect of acute MMS exposure was similar to that of UV- and X-irradiation in that longer exposure times decreased egg-hatching frequencies, but most of the survivors, albeit sterile, achieved adulthood. Among the nine rad mutants, only rad-4 showed hypersensitivity to acute MMS

C. ELEGANS RADIATION-SENSITIVE

MUTANTS

0.1

0.4

0

0.2

0.3

167

MMS CONCENTRATION (mM) FIGURE6.-Terminal developmental stage attained by wild-type (0)and rad-3 (0)animals after chronic exposure to MMS. Zygotes were placed on MMS-containing agar medium as eggs and subsequently scored for stage of arrest.

MMS Concentration (mM) FIGURE7.-Terminal developmental stage attained by wild-type (O),rad-2 (A), and rad-3 (0) animals after chronic exposure to MMS. L4 larvae were placed on MMS-containing agar medium and removed after 24 hr; progeny left on MMS plates were subsequently scored for stage of arrest.

exposure, as shown in Figure 8. Although both rad-2 and rad-3, which showed MMS hypersensitivity in one or both of the chronic exposure assays, gave wildtype survivals in the acute exposure assay, they were exceptional in that if the MMS-treated eggs were not washed by centrifugation before plating, there was sufficient MMS carryover to render rad-2 survivors severely uncoordinated (similar to the phenotype of unc-36) and to terminate development of rad-3 survivors at the L1 stage. Both effects were eliminated by the washing step, which was unnecessary for wild-type or the other rad mutants. Effects on recombination: Seven of the nine rad mutations were examined for their effects on genetic recombination (Table 3). Intervals on linkage groups I1 and X were selected for testing; none of the rad mutants map on these linkage groups, hence construction of the appropriate tester stains, homozygous for a particular rad mutation and heterozygous for two cis-linked visible markers, was straightforward. Recombination was not tested in rad-8 or rad-9 mutants because the large number of sick animals made it difficult to score phenotypes reliably. The seven rad mutations appear to have little if any effect on genetic recombination over the intervals tested. Both rad-4 and rad-6 show slight but statistically significant deviations from the wild-type recombination frequency between dpy-7 and unc-3. The deviations are small, however, particularly in comparison with those found for some meiotic mutants (BAKERet al. 1976), including C. elegans (HODGKIN, HORVITZ and BRENNER1979; ROSEand BAILLIE

168

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HARTMAN, AND

a

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K. HERMAN

' " " 7

3

cn

* :

0 30 60 90 120

TIME ( m i d FIGURE8.-Sensitivity

of wild-type (0)and r a d 4 (e)eggs to 50 mM MMS. TABLE 3 Recombination in rad mutants

Segregation from dpy-10 unc-4/++11 rad genotype

Wild type rad-1 rad9 rad-3 rad-4 rad-5 rad-6 rad-7

Frequency of recombinants

22/1172 16/1036 34/1749 26/1142 13/782 9/556 9/895 23/1347

%

Recombination

1.9 1.6 1.9 2.3 1.7 1.6 1.0 1.7

Segregation from dpy-7 unc9/++X Frequency of recombinants

% Recombina-

207/1231 165/1041 135/922 182/1058 169/842 105/ 598 68/579 140/816

18.5 17.4 15.9 19.0 23.0 19.5 32.5 19.0

tion

From 4 to 16 hermaphrodites were picked and their total self-progeny scored. Scores are given as Dpy non-Unc progeny plus Unc non-Dpy progeny/total progeny.

1979). It is possible that the slight deviations were caused by differences in genetic background unrelated to the rad mutations. Effects on spontaneous mutation: Spontaneous mutability was assessed for seven of the nine rad mutants by comparing the frequencies of reversion of unc-58 (HODGKIN, HORVITZ and BRENNER 1979) in rad+ versus rad genetic backgrounds. For rad+ and all rad strains tested except rad-5, the large majority of plates screened were devoid of revertants (Table 4). In the case of rad-5, however, revertants were found on 31 of 34 plates. These are the combined results of two separate experiments. In the second experiment, special care was taken to avoid the possibility of introducing revertants in the inocula: nine plates were each inoculated with five hermaphrodites, all of which were taken from a small plate containing no revertants and initiated by a single rad-5; unc58 hermaphrodite; spontaneous revertants were generated on all nine plates. As in the first experiment and as expected for spontaneous mutations (LURIAand DELBRUCK 1943), the numbers of revertants per plate fluctuated widely, from a few animals to hundreds. The fluctuation presumably resulted from differences in the time of occurrence of the first reversion; the time differences could have involved either different times in germ line development or different generations of growth, or both. The unc-58 mutation was extracted by recombination from


169

TABLE 4 Effect of rad mutations on spontaneous reversion of unc-58 Genotype

unc-58 rad-1; unc-58 rad-2; unc-58 rad-3; unc-58 rad-4; unc-58 rad-5; unc-58 rad-6; unc-58 rad-7; unc-58

Plates containing revertants"/total plates screened

0/35 2/22 5/18 3/20 0/24

31/34 0/24 0/25

As described by HODGKIN, HORVITZ and BRENNER (1979), each strain was grown to starvation on 9-cm Petri plates: the plates were then screened for non-Unc revertants.

the rad-5; unc-58 stock; ten plates of the resulting strain yielded no revertants. The generality of the effect of rad-5 on spontaneous mutability was tested by looking for spontaneous levamisole-resistant mutants, according to the methods of LEWIS et al. (1980). Spontaneous levamisole-resistant mutants were found on 4 of 15 rad-5 plates, as compared with 0 of 15 for N2. It thus appears that rad-5 is a general spontaneous mutator. The rad-4 mutant shows reduced X-chromosome nondisjunction: The incidence of male self-progeny of rad-4 hermaphrodites was about one-tenth that of wild type, as shown in Table 5. In addition, rad-4 appeared to suppress Xchromosome nondisjunction in him-I, him-3, him-6, him-9 and unc-86 mutants by factors of between three and five (Table 5). Crosses between rad-4 males and rad-4 dpy-12 hermaphrodites gave equal proportions of male and hermaphrodite cross-progeny; we conclude that the low incidence of rad-4 male self-progeny is due to reduced X nondisjunction and not to inviability of males. We cannot be certain that the reduced X nondisjunction found in the rad-4 strains is caused by the rad-4 mutation; the original rad-4 stock was established after two outcrosses to wild type, and the construction of the him rad-4 mutants involved additional outcrosses, but it is possible that the effect on X nondisjunction is caused by a closely-linked mutation. If such were the case, the absence of suppression for some him rad-4 mutants might then result from the absence of the him-suppressing mutation. We have tested this idea in the case of him-8 by reextracting rad-4 from the him-8; dpy-11 rad-4 strain, which showed little suppression of him-8 expression, and using it in the construction of a him-2; dpy-11 rad-4 strain; the latter strain gave the expected suppression of him-2 expression. The hatching of rad-4 eggs was cold sensitive. Over 90% of the eggs laid at 25' hatched, compared with less than 25% at 1 5 O . The egg-hatching frequencies were the same for parents shifted from 25' to 15' at various larval stages, up to L4, as well as for parents always kept at 15' and taken from a stock maintained for a number of generations at 1 5 O . The egg-hatching frequencies at 15' decreased as a function of parental age; when measured over Z-day intervals, they were 44.7% (268/600), 14.1% (148/1051), 7.3% (48/657), and 3.2% (4/122) from parents in the 1st and 2nd, 3rd and 4th, 5th and 6th, and 7th and 8th days of sexual maturity, respectively.


170

TABLE 5 Self-progeny broods of him rad-4 mutants

Genotype"

No. of broods analyzed

Males/total

% Male

0.32 0.03

Wild type rad-4

20 90

18/5542 3/9689

him-2; dpy-22 him-2; rad-4 dpy-11 him-2; rad-4 dpy-lZ/+

16 48 10

426/1454 52/1049 276/954

him-2; dpy-22 him-2; rad-4 dpy-11

8 20


Relative to control

0.1

-

29.2 5.0 28.9

0.2 1.0

15/956 6/493

1.6 1.2

0.8

4 32

27/1028 5/897

2.6 0.6

0.2

dpy-11; him-4 rad-4 dpy-11; him-4

16 90

9/223 9/165

4.1 5.5

1.3

dpy-12 him-5 unc-76 rad-4 dpy-11 him-5 unc-76

9 20

276/1162 42/299

23.8 14.0

0.6

him-6; d p y - l i him-6; rad-4 dpy-12

20 76

213/1260 20/427

16.9 4.7

0.3


a 20

271/895 132/596

30.3 22.1

0.7


8 32

63/1497 4/354

4.2 1.1

0.3


8 26

153/1270 11/67

12.0 16.4

1.4

unc-86; dpy-11 unc-86; rad-4 dpy-12

8 36

26/1114 6/1040

2.3 0.6

0.3

-

-

-

-

-

-

a The marker dpy-11 V simplified strain construction because of its close linkage to rad-4. The rad-4 dpy-22 him-5 unc-76 strain was recovered after picking Dpy Unc segregants from rad-4 dpyll/him-5 unc-76 animals.

Parental-effect tests, analogous to those used to analyze temperature-sensitive and HIRSH1976; MIWAet al. 1980; WOODet mutants of C. elegans (VANDERSLICE al. 1980) were performed at 1 5 O . In the selfing test, approximately 25% (205/921) of the progeny from rad-4 dpy-ll/++ hermaphrodites were Dpy, with less than 1%(9/921) inviable zygotes. A maternal rad-4" allele was therefore sufficient to permit survival of rad-4 homozygotes at 15". The mating of rad-4 dpy-11 hermaphrodites with wild-type males reduced the number of inviable zygotes from about 75% to about 30%. Thus, a rad-4+-bearing sperm was capable of rescuing a rad-4 oocyte. On the other hand, rad-4 sperm made in a heterozygous male did not rescue rad-4 oocytes. A sex-specific lethal effect in the rad-6 mutant: When rad-6 unc-36 hermaphrodites were crossed with rad-6 males at 1 5 O , nearly equal numbers of male and hermaphrodite cross-progeny were produced, as shown in Table 6.

171


TABLE 6 Some crosses involving rad-6 Cross

Temoerature

?& Male cross-prog-

env

Total cross-progeny scored

rad-6 unc-36 x rad-6 rad-6 unc-36 x rad-6 unc-36 X r a d 4 rad-6 unc-36 X N2

15' 25 25" 25'

53.5 71.2 49.1 51.4

1323 812 1368 1795

When the same cross was done at 25", however, over two-thirds of the crossprogeny were male. All of the rad-6 unc-36 self-progeny at 25' (and 15") were hermaphrodites; this means first, that the hermaphrodite parent does not produce nullo-X ova at 25", and second, that the rad-6 mutant does not transform XX animals into pseudomales (KLASS,WOLFand HIRSH1976; HODGKIN and BRENNER 1977; HODGKIN 1980) at 25'. The cross between rad-6 males and unc-36 hermaphrodites at 25" gave a normal cross-progeny sex ratio; this means that rad-6 males do not produce nullo-X sperm at a frequency much greater than half. Finally, the last cross in Table 5 shows that oogenesis of rad6 hermaphrodites at 25" is not sufficient to produce the aberrant sex ratio; the rad-6+ allele contributed by the male parent led to a normal sex ratio. We are therefore left with the conclusion that rad-6 hermaphrodites are less than half as viable as rad-6 males at 25". The two sexes are about equally viable at 15". Egg-hatching frequencies as a function of parental age in rad-8 mutants: Less than 5% of the eggs laid by rad-8 hermaphrodites on the 1st day of egg laying hatched. The frequency of egg hatching increased with parental age so that by the 5th day of egg laying, 75% of the eggs hatched (Figure 9). The overall egghatching frequency for complete broods was about 30%.This phenomenon was not temperature dependent. Virtually all zygotes that hatched developed to adulthood, and all viable progeny, whether derived from the early or the late portion of a brood, laid eggs with the same pattern of hatching shown in Figure 9. Inviable zygotes were examined by Nomarski microscopy and found to be arrested only after a large number of cleavages had occurred. Parental-effect tests were conducted on rad-8. Among 1434 eggs laid by rad8 unc-13/++ hermaphrodites, only 0.7%did not hatch, with Unc hermaphrodites comprising 25% of the viable progeny. Mating young rad-8 unc-23 hermaphrodites with wild-type males reduced the percentage of unhatched eggs to 3.2% (27/850); some of the unhatched eggs may not have represented cross-progeny since 0.6% of the viable progeny were (Unc) self-progeny. When young rad-8 unc-23 hermaphrodites were crossed with rad-8 unc-13/++ males, only 5.0% (28/558) of the eggs laid did not hatch. Among the viable progeny, 48% were Unc, and the vast majority of these were (rad-8) cross-progeny because about half were male. We conclude that rad-8 sperm made in a heterozygous male can rescue the early oocytes made by a rad-8 hermaphrodite. It follows that rad-8 hermaphrodite sperm tend to be defective. This is not the only defect apparent in rad-8 animals, however, which take nearly twice as long as wildtype animals to reach adulthood. This slow development is maternally influenced: rad-8 self-progeny of heterozygous hermaphrodites develop at the wildtype rate, but rad-8 cross-progeny from a mating between rad-8 hermaphrodites and heterozygous males develop at the slow rate.

172

P. S. HARTMAN, AND

R.

K. HERMAN

00 80 60 40

20

TIME ( d a y s ) FIGURE%-Viability of self-progeny of rad-8 hermaphrodites as a function of parental age. Wildtype egg-hatching frequencies were in excess of 95% regardless of parental age. DISCUSSION

W e have described nine mutants of C. elegans that are hypersensitive to ultraviolet radiation. The mutations are all recessive to their wild-type alleles and define nine genes, named rad-1 through rad-9. The mutations seem to be widely distributed on the C. elegans genetic map, mapping to four of the six linkage groups, Some of the properties of these mutants are summarized in Table 7. The absence of alleles among our set of nine mutants indicates that we are far from saturating the class of genes capable of giving UV-sensitive mutants. On the other hand, our frequency of recovery of rad mutants, 9 per 6434 F2 broods examined or 1.4 x is only about five times greater than the frequency at which recessive mutations, including null mutations, are recovered in an average C. elegans gene after standard EMS mutagenesis, about 2.5 X lop4 (BRENNER 1974; GREENWALD and HORVITZ 1980). We therefore suggest that many mutations occurring in genes that are potentially capable of yielding UVsensitive alleles are not recoverable in fertile UV-sensitive mutants by our procedures. One reason for this may be that only certain alleles give a UVsensitive phenotype. Another, perhaps better, reason may be that many of the rad genes encode indispensable functions; for such genes, the only alleles that we would recover would be those that retain enough residual gene expression to permit the animals to survive and reproduce. That we have indeed selected for such hypomorphic or leaky alleles of essential genes is suggested by the fact that the reproduction of four rad mutants was sensitive to high temperature (to varying degrees; see Table 1) and the reproduction of another mutant, rad-4, was cold sensitive; our mutant isolation procedure was not specifically designed to isolate conditional lethal mutants. It has been estimated that greater than 50 loci control DNA repair and related phenomena in yeast (LEMONT 1980) and in Drosophila (SMITH, SNYDER and DUSENBERY 1980). It also appears that a significant proportion of mutations affecting radiation sensitivity, mutagen sensitivity and meiotic chromosome behavior in Drosophila may define genes that specify essential functions (BAKER, CARPENTER and RIPOLL1978; BAKERand SMITH 1979). Radiation-sensitive mutants of C. elegans have been identified in two other laboratories. Several alleles of flu-2, originally isolated on the basis of altered


173

TABLE 7 Summary of properties of rad mutants ?& Wild-type fertility

MMS Gene

rad-1 rad-:! rad-3 rad-4

Hermaphrodite

Male

59

49

90

87 90

58 34

UV hypersensitivity"

X-ray hypersensitivity"

hypersensitivity"

++

-

16

++ ++ ++ +

++ -

Other properties

+ + +

rad-5

7

9

+

+

-

rad-6

52

34

+

-

rad-7 rod-8

25

85

-

7

0

-

-

rad-9

9

1

+ + + +

-

-

Decreased X-chromosome nondisjunction; cold-sensitive embryogenesis Increased spontaneous mutability; ts sterile Hermaphrodites less viable than males at 25' Young hermaphrodites lay many inviable eggs

Symbols: ++ = exhibits this characteristic strongly; + = exhibits this characteristic moderately; - = exhibits this characteristic weakly if at all.

intestinal autofluorescence (BABU1974), confer hypersensitivity to gamma radiation and EMS but not UV (BHATand BABU1980). Extracts from flu-2 animals contain reduced levels of kynureninase, a tryptophan-degradative enzyme. None of our rad mutations, all autosomal, is an allele of flu-2, an X-linked gene. We have examined the intestinal autofluorescence of all nine rad mutants and found no abnormalities. Although we found that the most severe allele of flu-2 was not gamma-radiation hypersensitive (data not shown), we irradiated zygotes that were less than 2 hr old, whereas BHAT and BABUirradiated animals that were up to 26 hr old. N. ISHII(personal communication) has isolated two mutants that are hypersensitive to UV radiation. Their relationship to flu-2 and the rad mutants is unknown, as they have not been characterized genetically. It seems likely that at least some of the rad mutants have altered DNA repair capacities, although biochemical analysis of the mutants, which we have not done, will be necessary to prove this point. Specific DNA repair defects have been ascertained in radiation-sensitive and mutagen-sensitive mutants of other organisms, including eukaryotes such as yeast (e.g., LEMONTT1980; PRAKASH and FRIEDBERG 1980) and Drosophila (BOYDet al. and PRAKASH 1980; REYNOLDS 1980; BOYD and HARRIS1981). Sensitivity to more than one DNA-damaging agent is characteristic of many DNA-repair defective mutants (for reviews, see 1980). The findings that two C. elegans rad GENEROSO, SHELBY and DE SERRES mutants-rad-2 and rad-2-are very hypersensitive to X rays and that threerad-2, rad-3 and rad-4-are hypersensitive to MMS therefore suggests that these mutants may be abnormal in DNA repair. The effect of MMS on C. elegans embryos depended on whether the exposure was acute (in liquid for short periods at high MMS concentrations) or chronic (on agar medium at low concentrations). The response to acute exposure was similar to the responses to UV- and X-radiation: with increasing dose, fewer

174

P. S . HARTMAN, AND

R. K.

HERMAN

eggs hatched, but those animals that survived embryogenesis generally reached adulthood. Chronic MMS exposure, on the other hand, did not block embryogenesis but led to the arrest of development at a larval stage, the particular stage of arrest depending on the MMS concentration. The manifestation of MMS hypersensitivity by each of the three hypersensitive rad mutants depended on the conditions of exposure: the rad-2 mutant was hypersensitive when fourth stage larvae were placed on MMS-containing agar but not when eggs were given either acute or chronic doses; rad-3 hypersensitivity was apparent when either eggs or L4 animals were given chronic doses; and rad-4 hypersensitivity was seen only after acute exposures. The MMS hypersensitivities of mutants of Saccharomyces cerevisiae (PRAKASH and PRAKASH 1977), Neurospora crassa (DELANGE and MISHRA1981) and Escherichia coli (S. LINN,personal communication) have also been shown to vary depending on the mode of exposure to MMS: chronic exposure on solid medium versus acute exposure in liquid. Certain DNA repair-defective mutants of yeast (for review, see VON BORSTEL and HASTINGS 1980) and Drosophila (for review, see BAKERet al. 1980) show altered spontaneous mutability. Among our C. elegans mutants, rad-5 showed enhanced spontaneous mutation in two independent tests: reversion of unc58(e655), which appears to occur by any mutation within the unc-58 gene that inactivates mutant expression (HODGKIN, HORVITZ and BRENNER1979; GREENWALD and HORVITZ 1980), and mutation to levamisole resistance, which can occur at several loci (LEWISet al. 1980). The rad-5+ function, which may be essential because the mutant does not reproduce at 2 5 O , could possibly involve DNA repair or replication. We have not yet measured mutagen-induced mutation frequencies in rad-5 animals. Our finding that rad-4 reduces spontaneous meiotic X-chromosome nondisjunction to about one-tenth the wild-type frequency indicates that the wild-type frequency of X-chromosome nondisjunction is not simply the lowest possible but is subject to natural selection. This should not be surprising because the Xchromosome nondisjunction frequency is clearly important in controlling the incidence of male animals. Since crossing over seems to be necessary for normal disjunction in many organisms (BAKERet al. 1976), we were interested to see whether rad-4 would show increased X-chromosome recombination. In fact, it does, but the increase is small, only about 25%. The rad-4 mutation partially suppresses certain him mutations, which by themselves have the effect of enhancing X-chromosome nondisjunction: X-chromosome nondisjunction in five him mutants was reduced to one-fifth to one-third the usual levels by rad4; five others were affected little or not at all. This suppression pattern may eventually assist in delineating various classes of meiotic nondisjunction mutant, but there is no clear pattern of suppression apparent. For example, among the class of well suppressed him mutants are him-2, in which nondisjunction is probably largely restricted to the X chromosome and occurs at one of the highest frequencies; unc-86, in which X-chromosome nondisjunction occurs at a relatively low frequency; and him-6, in which both autosomal and X-chromosome nondisjunction frequencies are elevated (HODGKIN, HORVITZ and BRENNER 1979). B. MEYER(personal communication) has identified a mutant that greatly suppresses him-5; it maps between dpy-12 and unc-76 and is clearly not an allele of rad-4. The rad-4 mutant shows another abnormality: embryogenesis is cold semi-


175

tive. The cold sensitivity is maternally influenced in two senses: 1)rad-4 zygotes generated by a heterozygous hermaphrodite parent are not cold sensitive: and 2) for homozygous rad-4 hermaphrodites maintained at low temperature, the older the parent at the time of fertilization (i.e., the older the gonad), the more likely is the failure of embryogenesis of the zygote progeny. The relations among the various properties of rad-4 are not clear. Indeed, we emphasize the possibility that despite repeated outcrossing more than one mutation may be involved in producing the different phenotypic abnormalities. This statement obviously applies to other rad mutants as well, particularly rad-6 and rad-8, discussed below, which were found to exhibit phenotypes quite unexpected on the basis of their UV hypersensitivities. The reproduction of the rad-6 mutant is partially temperature sensitive; moreover, in crosses between rad-6 hermaphrodites and rad-6 males, it was found that the viability of hermaphrodite cross-progeny is less than half that of male cross-progeny. The two sexes are about equally viable at 15'. It is possible that an alteration in dosage compensation is involved in producing this temperature-sensitive, sex-specific semilethal phenotype. Many sex-specific lethal loci are known in Drosophila-see BELOTEand LUCCHESI(1980a) for a summaryand at least some affect dosage compensation (BELOTEand LUCCHESI1980b; CLINE1979). There are other examples in C. elegans of sex-specific lethalsand BRENNER 1977;J. HODGKIN, both XO-specific lethals or semilethals (HODGKIN personal communication; B. MEYER, personal communication), and XX-specific lethals (B. MEYER,personal communication), but the bases of the lethalities of these mutants are also not as yet known. Most of the self-progeny of young (but not old) adult rad-8 hermaphrodites do not survive embryogenesis. The defect appears to be in the rad-8 hermaphrodite sperm or seminal fluid because rad-8 sperm made by a heterozygous male are capable of rescuing the early oocytes made by rad-8 hermaphrodites. Among a set of 35 embryonic lethal mutants subjected to this same test ( W O O D et al. 1980; MIWAet al. 1980),only one, B235, behaved similarly. It was suggested that B235 might be defective in blockage of polyspermy (WOODet al. 1980). The same could be said for rad-8. Indeed, since sperm are gradually depleted during the period of egg laying (WARDand CARREL1979), the problem of polyspermy in rad-8 might decrease with parental age; this would account for the observed increase in zygote survival with parental age (but would not account for the slow development of rad-8 progeny). We thank CLAIRE KARI for technical assistance, ANNROSEfor providing the rec-1 mutant, and American Cyanamid for a gift of levamisole. This work was supported by Public Health Service and by postdoctoral fellowships from the National Grants GM22387 and AGO2845 to R. K. HERMAN, Science Foundation (SPI-7914828) and the American Cancer Society (PF-1880) to P. S. HARTMAN. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is supported by Contract N01-AG-9-2113 between NIH and the Curators of the University of Missouri. LITERATURE CITED

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Corresponding editor: A. CHOVNICK