NOVEL NEMATODE AMBER SUPPRESSORS

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NOVEL NEMATODE AMBER SUPPRESSORS

JONATHAN HODGKIN

MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England

Manuscript received March 18, 1985 Revised copy accepted May 25, 1985

ABSTRACT

Nine amber suppressor mutations were isolated in the nematode Caenorhab- ditis elegans by reverting amber alleles of a sexdetermining gene, tra-3. One suppressor maps to a known locus, sup-5 I l l , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5

alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 I l l and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.-Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).-Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.

ESEARCH on the molecular genetics of lower organisms has benefited enormously from the availability of nonsense suppressors, i.e., mutations that prevent polypeptide chain termination at amber (UAG), ochre (UAA) or opal (UGA) stop codons. Suppressors of this type have been identified and studied extensively in many microbial systems, in particular E. coli (reviewed by SMITH 1979) and Saccharomyces cerevisiae (reviewed by SHERMAN 1982). Nonsense suppressors are proving to be equally useful tools for the analysis of higher eukaryotes. Amber suppressors have been successfully introduced into mammalian cells in tissue culture (HUDZIAK et al. 1982), but only in one animal, the nematode Caenorhabditis elegans, have amber suppressors been induced in the germ line. WATERSTON AND BRENNER (1978) and WATERSTON (1981) have

characterized two suppressor loci in C. elegans, sup-5

IZI

and sup-7 X. T h e sup- 7 mutation st5 and the sup-5 mutation e1464 were shown to suppress amber mutants via an altered tRNA (WILLS et al. 1983; KIMBLE et al. 1982). Subse- quently, sup-7(st5) has been cloned and sequenced and has been shown to be a tryptophan tRNA gene containing an alteration at the anticodon (CCA to CTA) that would permit it to read UAG codons (BOLTEN et al. 1984). T h e

suf-5(e1464) mutation probably contains the same alteration in another niem-

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ber of this tRNAl”p gene family, which has about 12 members (BOLTEN et al. 1 9 8 4 ) .

WATERSTON ( 1 98 1 ) examined 25 suppressor mutations obtained by reverting severely uncoordinated mutants carrying amber alleles (e450, e1091) of the gene unc-13. Nineteen of these mapped to the 324-5 locus and six to the sup-

7 locus, suggesting that these loci were the only two that could mutate to yield an informational suppressor of unc-13. In the present study, amber mutants of

a sex-determining gene, tra-3, were reverted, yielding nine informational suppressors. One of these is a sup-5 allele, but the other eight map to three new amber suppressor loci, sup-21 X , sup-22 IV and sup23 IV. These amber suppressors have been partly characterized with respect to the efficiency of sup- pression and to the spectrum of amber alleles that they suppress. T h e results are relevant to the study of nonsense suppression in general and have also provided some information on dosage compensation and on maternal effects in this organism.

MATERIALS AND METHODS

Strains: T h e following genes and alleles were used in this study. Unless otherwise noted, they were obtained from the Cambridge collection established by BRENNER (1974), and the genes a r e listed by SWANSON, EDCLEY and RIDDLE (1984). Nomenclature follows HORVITZ et al. (1979).

Linkage group (LG)I: unc-I3(e312, e376, e450, e1091), unc-54(e1008, e1092, e1108, eI300, e1392).

LG 11: tra-2 (see below), unc-4(e120), unc-52(e669).

LG 111: unc-93(e1500 n234) (GREENWALD and HORVITZ 1980), lon-I(e185), sup-5(e1464) (WA-

TERSTON and BRENNER 1978), unc-32(e189), tra-1 (see below), dpy-I8(e364, e499).

LG IV: lin-I(e1777) (FERGUSON and HORVITZ 1985), unc-l7(e245), dpy-I3(e184), unc-5(e53), fem- l(eI991, e1965) (DONIACH and HODCKIN 1984), mor-Z(el125), unc-24(e138), dpy-20(e1282, e1362, e1415, eI422, e2017), unc-30(el9I), tra-3 (see below), dpy-4(e1166), sDf2 (MOERMAN and BAILLIE

1981).

LG V: him-5(e1490).

LG X: sup-7(st5) (WATERSTON 1981), unc-6(e78), dpy-7(e88), unc-I8(e81), dpy-6(e14), unc- 58(e665).

Source of tra alleles: A large number of mutations in the tra genes, which cause masculinization of XX animals (HODCKIN and BRENNER 1977), were utilized in the present study. For tra-I, 40 recessive (loss of function) alleles have been tested for suppressibility; some of these were obtained as chance segregants from general mutagenesis screens by J. HODGKIN, S. BRENNER, P. ANDERSON

and E. M. HEDCECOCK (five alleles) and some as isolates from a general screen for tru mutations (eight alleles). In this screen, hermaphrodites of genotype dpy-7

+/+

unc-18 were mutagenized and their F , , F2 and Fs progeny were examined for the appearance of non-Dpy non-Unc males. T h e two markers are tightly linked, so recombinant XO males are very rare. Results of this screen, which yielded mutations in all three tra genes, will be described in detail elsewhere U. HODCKIN,

unpublished results).

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289 Phenotypic examination of the 40 alleles permits classification into two classes, putative null or almost null (causing complete transformation of XX animals into fertile males) and non-null (causing incomplete masculinization of XX animals). The putative null alleles tested for suppressibility by sup-5 and/or sup-7 were e1099, e1516, e1728, e1729, e1730, e1774, e1781, e1783, e1822tr, e1828tr, e1829tr, e1832tr, e183?tr, e1834tr, e1835tr, el836tr, e1838t, e1844tr, e1849tr, e1856tr, e1858tr and e1860tr. The non-null alleles were e440, e1076, e1488, e17?2, e1764, e1766, e1825tr, e1830tr, el83ltr, e1837tr, e1840tr, e1843tr, e1845tr, e1846tr, e1847tr, e1850tr, e1853tr and e1857tr.

For tra-2, 19 alleles have been tested for suppressibility, 11 of which were obtained as chance segregants by J. HODGKIN, S. BRENNER and H. R . HORVITZ (e1425); four were isolated as suppressors of her-1 (HODGKIN 1980); one was obtained from the general tru screen

U.

HODGKIN,

unpublished results); one was a temperature-sensitive sterile (b202, KLASS, WOLF and HIRSH 1976); and one was a dominant egg-laying defective (n196, TRENT, TSLJNG and HORVITZ 1983). The mutation tri(f70) described by BEGUET and GIBERT (1978) was crossed into a Bristol (N2) genetic background and was found to be a weak tra-2 allele because it maps to the tra-2 locus and fails to complement tra-2(e1095). Putative null alleles tested were ~ 1 0 4 7 , e1093, e1094, e1095, e1098, e1109, e1110, e1271, e1425, e1556, e1557, e1560, e1726, e1727, e1765 and nI96; non-null alleles were e1209, b202 andj70.

For tra-3, four alleles were tested for suppressibility, two of which were obtained as chance segregants (e1107 by D. L. BAILLIE, e1903 by E. M. HEDGECOCK), one (e1525) by a tra-3 complementation screen U. HODGKIN, unpublished results) and one (el 767) from the general tra screen U. HODGKIN, unpublished results).

Ident$cation of amber alleles: Of the 22 amber alleles listed in Table 2, ten (in genes dpy-18, unc-13, unc-52 and unc-54) were identified as such by WATERSTON (1981), one (lin-1) by FERGUSON

and HORVITZ (1985), one v e m - I ) by DONIACH and HODGKIN (1984) and one (unc-93) by GREEN- WALD and HORVITZ (1980); the remainder were identified in the course of this work. Procedures for constructing double mutants with sup-5 and/or sup-7 were similar to those described by FER- GUSON and HORVITZ (1985). The marker Lon-l(e185) was often used to mark sup-5, using the double mutant lon-I(e185) sup-5(e1464). The mutation dpy-20(e2017) was also found to be useful as an indicator of suppressor genotype in some of these constructions, because it is partly suppressed by one dose of sup7 and is fully suppressed by two doses. In the case of many mutations that appeared to be incompletely suppressed, homozygous double mutants with sup-21(e1957) were constructed by crossing a heterozygous male carrying the mutation to be tested (e.g., unc-1?/+) with sup-21 hermaphrodites; the resulting males were crossed again with sup-21 hermaphrodites, yielding cross-progeny that must be homozygous or hemizygous for sup-21. Appearance of animals expressing the Unc-13 phenotype in subsequent generations indicated incomplete suppression.

Length measurements: The measurements of body length in dpy-20(e2017) animals, summarized in Figures 4a and 6, were made by picking mature hermaphrodites or males and placing them in a drop of 0.5% I-phenoxy-2-propanol on a slide under a cover slip. As soon as they had stopped thrashing, their lengths were measured using a calibrated micrometer eyepiece. There is some variability in the extent of suppression even when using a strong suppressor such as sup-7, so only strongly suppressed animals were measured. For each genotype, adult populations of comparable sizes were sampled; the 30 longest animals from a healthy plate were picked, and 20 of these were measured. Therefore, these measurements indicate maximum, rather than mean, suppression.

RESULTS

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TABLE 1

Reversion of tra-3 mutants

Total F,

screened Informational suppressors

~~

(approximate

Allele estimate) sub-5 sub-21 sub-22 sub-23

Epistatic suppressors

fem-1 fem-3 tra-1 (dam)

-

e l 107 25,000 e2064

e1525 100,000 e2060 e2058 e2057 e2059

2 1903 75,000 e1957 e1986

e l 767 (non- 25,000

e2061

e1958

amber)

e2066 e2065

e2067 e2062

e2063

e2068 e2069 e2070 e2071

pared with 330 for wild-type N2 hermaphrodites). However, almost all of these self-progeny are transformed into abnormal sterile males, T h e phenotype of these animals is variable, so that at 20" some of the animals have a her- maphroditic two-armed gonad and a rudimentary vulva. Very occasionally (<1% of animals), a few self-progeny are made in this gonad. T h e tail phenotype is also variable and is never completely male. A characteristic tra-3 X X

male tail is shown in Figure l a , which can be compared with the wild-type XO male tail shown in Figure 5h. T h e maternal rescue of tra-3 mutants, and the fact that tra-3 amber alleles are efficiently suppressed by the amber suppressors

sup-5 and sup-7 (KIMBLE et al. 1982), suggests that the tra-3 gene product is

required only in very small amounts. Reversion of amber tra-3 mutants might therefore be expected to yield a wider spectrum of informational suppressors than has been previously described.

It was found that the number of self-fertile tra-3 animals was increased at 15" (even in the case of amber alleles), so that at temperatures of 14"-15" populations of homozygous tru-3 can be propagated as self-fertile hermaphro- ditic stocks. For example, a single tra-3(eIIU7) X X hermaphrodite (daughter of a tru-3/+ mother) grown at 15" produced a total of 355 F1 progeny, of which about 20% were self-fertile and produced a total FZ brood of 1326 animals

(i.e., an average brood of 3.7 progeny). In contrast, a sibling tra-3 X X hermaphrodite grown at 20" gave 414 F1 progeny, only three of which were self- fertile, giving a total of 26 FZ progeny. Thus, homozygous tru-3 stocks can be grown at 15

"

, but not at 20

" .

Direct tra-3 reversion experiments are therefore feasible. Stocks of the four mutant strains were grown up and mutagenized with 0.025 M ethyl methanesulfonate (EMS) for 4 hr; growth at 15" was

continued for at least two generations, and then the populations were shifted

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29

1

i

FIGURE I .-Tail phenotypes o f tra-3 XX animals. a, tra-3(el107), unsuppressed inconiplete male. b, sup-22@1986) tra-3(e1903), completely suppressed fertile hermaphrodite. c, tra-3(~1107)

daughter of fra-3(eI 107); sup-21(~2061)/+ mother: partial suppression resulting from maternal inadequacy. Approximately X300.

3 allele, e2767, using a different protocol; this has yielded many mutations in the sexdetermining genes fem-1, fem-2, fem-3, tru-2 and others. These revertants, as well as the nine epistatic suppressors listed in Table 1 (one fm-2 allele, four fem-3 alleles and four tru-l(dom) alleles), will be described elsewhere

U.

HODGKIN, unpublished results).

The other nine suppressors are regarded as informational suppressors because all suppress the reference amber allele of tru-3, e2107, and have no effect on the nonamber allele, e2767. Also, all nine partly or completely s u p press an amber mutation in another gene, dpy-20(e2027).

Mupping of suppressors: Homozygous tru-3; sup stocks were established for

each of the nine suppressors. All were completely wild type with respect to sexual phenotype (e.g., Figure Ib), although some grew distinctly more slowly than the wild type

N2

hermaphrodite stock. The suppressor mutations were mapped using suppression of tru-3(e2207) as an indicator of suppressor geno-

type.

Their locations are shown in a simplified genetic map, Figure

2.

One mutation, e2060, showed tight linkage to unc-32: 224 uncoordinated hermaphrodites were picked from the self-progeny of animals of genotype

sup(e2060)/unc-32; tru-3, only one of which produced hermaphrodite (non-Tra)

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m

therefore, is likely to be an allele of sup-5, which is also very tightly linked to unc-32 (SWANSON, EDCLEY and RIDDLE 1984).

Five of the suppressors showed tight linkage to dpy-6 X for each of these five, at least 100 dumpy animals were picked from the self-progeny of hermaphrodites of genotype tra-3; dpy-6

+/+

sup, and no more than I-2% produced hermaphrodite self-progeny. T h e remaining 98-1 00% were all sterile or produced only Dpy Tra-3 progeny. This indicated that all five suppressors might have a similar genetic location, less than 2 map units from dpy-6 and distinct from the sup-7 locus, which is about 5 map units to the left of dpy-6.

Two of these five were mapped more precisely by three-factor crosses: hermaphrodites of genotype tra-3; dpy-6 sup

+/+

+

unc-58 were selfed and wild- type recombinants picked (only recombinants in the dpy-6-unc-58 interval are wild type because unc-58 has a dominant severely uncoordinated “shaker” phe- notype). For sup(e1957), 1 1 of 29 recombinants were sup/+ and 18 of 29 were sup/sup, placing the e1957 locus (designated sup-21 X ) between dpy-6 and unc- 58, which are markers 1.5 map units apart. The location of sup(e1958) is similar: from the analogous cross, four of six wild-type recombinants were sup/

+

and two of six were sup/sup.

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d

FIGURE 3.-Phenotypes of adult hermaphrodites: the dpy-20 assay. a, Wild type N2. b, dpy-

20(e2017); (sup-21(~1957) (complete suppression). c, sup-22(e2057) dpy-20(e2017) (weak suppres- sion). d, dpy-20(~2017) (unsuppressed). Approximately X80.

Initially, it appeared that a similar situation would be found with the three remaining suppressors, because all three differ in strength but are tightly linked to dpy-23 IV. However, t w o map to the left of dpy-23 (the sup-22 locus), and one maps to the right of dpy-23 (the sup-23 locus). For sup-22(e2986), three of 20 Unc recombinants and 18 of 21 Dpy recombinants from unc-I7

+

dpy-13 tra-3/+ sup-22

+

tra-3 carried sup-22. From the same cross using

sup-22(e2057), three of seven Unc recombinants and 13 of 15 Dpy recombi-

nants carried sup-22. Both e2986 and e2057 are therefore provisionally as- signed to sup-22 IV.

T h e sup-23 mutation, e2059, was found to map between dpy-23 and unc-5 by picking Dpy and Unc recombinants from dpy-23

+

unc-5 tra-3/+ sup-23

+

tra-3: 18 of 19 Dpy recombinants and two of 20 Unc recombinants carried

Eficiency of suppression: An amber allele of a gene affecting morphology,

dpy-20, has been used as an assay for comparing the strengths (efficiencies) of

the nine new suppressors, as well as sup-5(e2464) and sup-7. This allele, e2027, resembles three other dpy-20 alleles (e2362, e1415, e2422): all four mutations are recessive and cause an extreme dumpy round-nosed phenotype (see Figure 3 and also HOSONO et al. 1982). A much weaker temperature-sensitive allele,

e1282, has also been obtained, as well as a deficiency that includes the dpy-20

locus, sDj2 (MOERMAN and BAILLIE 198 1). T h e heterozygotes e2282/sDj2 and

e2282/e2017 have identical severe dumpy phenotypes at 2 0 ° , indicating that

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1

c ) EMBRYONIC suppression of

e

100 x

50 X

-

0

-sup(+) sup-22 sup-23 sup-21 sup-5 sup-7 tra(+)

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e201 7 is a null allele. All five of these alleles have been tested for suppressibility

by sup-7 and sup-21(e1957): e2017 is well suppressed by both, but the other

four showed no response. Measuring the maximum length of mature e2017 adults with various suppressor genotypes proved to be a convenient, simple and sensitive assay for strength of suppression (see Figures 4a and 6).

Homozygous dpy-20(e2017); sup strains were constructed for each of the 11 suppressors, and length measurements were carried out on these strains and on the unsuppressed

dpy

and the wild type

N2.

As can be seen from Figure 4a, the strength of suppression is variable at both sup-21 and sup-22 loci. Two

sup91 alleles, e l 957 and e2058, are good suppressors, apparently stronger than

either of the sup-5 alleles (which are probably identical with each other). A third sup-21 allele, e2064, is also reasonably strong, but the other two, e1958

and e2061, are much weaker, causing only partial suppression (Figures 3 and

4).

The suppressors at the LG IV loci are either weak (e2059) or very weak

(e2057, e1986). The e1986 suppressor is so weak that it is difficult to distinguish

from e2017 alone by this assay. However, the e1986 e2017 strain grows faster than e2017, and its larvae are distinctly less dumpy, indicating that e1986 does slightly suppress e201 7 . Suppression by the other sup-22 allele, e2057, is more pronounced (Figure 3c). Although some of these differences in length are not quantitatively significant, qualitative distinctions can reliably be made between populations of the different strains. It appears that there are two classes at the

sup22 locus and at least two (possibly four) at the sup-21 locus.

The 12 strains (the unsuppressed e201

7

and the 1 1 suppressor strains) were also compared at growth temperatures of 15" and 25". At low temperature,

e201 7 grew very slowly, as with other severe alleles of dpy-20, but the weakly

suppressed strains grew more rapidly. Suppression of the dumpy phenotype was not markedly different from that seen at 20", and the same rank order of suppression efficiencies was observed. The sup-7 strain was sterile, and both

sup-5 strains were almost sterile. Both of these suppressor loci have previously

been shown to confer a cold-sensitive sterile phenotype

(WATERSTON

and

BREN-

NER 1978, WATERSTON 1981). The strongly suppressed sup-21 strains grew well at 15", although more slowly than the weakly suppressed strains (e1959,

e2064

>

e1957

>

e2058). The slower growth may be due to a partial cold

sensitivity, as with the more extreme cold sensitivity of sup-5 and sup-7. At

tra-3 were picked; for sup-21, dumpy daughters of tra-3; dpy-b/sup-21 were picked; for sup-5,

uncoordinated daughters of unc-?Z/sup-5; tru-3 were picked; and for sup-7, uncoordinated daugh-

ters of unc-b/suf-7; tra-3 were picked. c, Embryonic suppression of tra-3. For each suppressor,

unc-32 tra-3 hermaphrodites were crossed with tra-3 males carrying the suppressor, and 40-60 non-Unc daughters were picked as L4's. These were permitted to self, and the fraction producing progeny is expressed as a percentage. Order of suppressor alleles as in parts a and b. The leftmost bar indicates the negligible self-fertility (Cl%) of tra-3 XX cross-progeny sired by tra-3 sup(+) XO males; the rightmost bar indicates 100% fertility for t r a J / + XX cross-progeny sired by wild-type males. For sup-22 and sup-23, homozygous sup tra-3 XO males were used; for sup-7 and SUP-21, hemizygous tra-3; suplo males were used; for sup-5, heterozygous unc-32/sup-5; tra-3 XO males

were used. In the experiments of b and c, all tru-3 alleles were amber, usually e1107 but sometimes

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high temperature (25") all 12 strains grew well, in some cases with slightly reduced suppression of the dumpy phenotype.

T h e heterogeneity of suppressors at the sup-21 and sup-22 loci was con- firmed by their effects on tra-3(elI07). Efficiency of suppression was measured in two ways, because tra-3 amber alleles can be suppressed by suppressor activity either in the maternal germ line or in the embryo. Animals that are tra-3 daughters of tra-3; sup/+ mothers are usually self-fertile hermaphrodites if the suppressor is strong, but they are often sterile animals with gonadal abnormalities and other signs of masculinization if the suppressor is weak, indicating that an inadequate amount of suppressed tra-3 gene product has been provided by the mother (see Figure IC). A comparison of the 11 suppressors by this assay, which measures the amount of suppressor activity in the maternal germ line, is shown in Figure 4b. T h e sup-21 alleles e2061 and e2058, which are weak by the dpy-20 assay, are also weak by this assay; but sup-23 is strong, and the sup-22 alleles are intermediate.

A different assay was made by crossing tra-3 hermaphrodites (daughters of tra-3/+ mothers) with tra-?;sup XO males: if the suppressor is strong, all tra-

3;sup/+ X X cross-progeny are self-fertile hermaphrodites, but if the suppressor is weak, some of these cross-progeny are sterile or intersexual. T h e results of this assay, which measures the level of suppressor expression in the embryo, are shown in Figure 4c. In this case, the weakest suppressors are the sup-22 alleles.

It is noticeable that the results of these three assays are not entirely concor- dant. For example, the sup-22 suppressors are very weak by the assays shown in Figure 4a and c, but are reasonably strong by the assay shown in Figure 4b, i.e., stronger than some of the sup-21 alleles. Also, sup-21(e1958) is stronger than sup-21(e2061) by the dpy-20 assay, but is weaker by the tra-3 assays. However, some of these differences might be due to background effects, i.e.,

adventitious mutations that have remained linked to the suppressor mutations. The suppression spectrum of sup-2 1 (el 957): As shown in the preceding section, sup-21(e1957) seems to be the strongest of the suppressors at the sup-21 and sup-22 loci. Its efficacy as a general amber suppressor was examined, using 22

amber alleles in 13 different genes; results are summarized in Table 2. Most of the amber mutations show some response, but in general e1957 is a weaker suppressor than sup-5(e1464) or sup-7(st5), and in a few cases it is completely ineffective. T h e results of the suppression tests are described in more detail below, as several interesting results were encountered. Also, amber alleles for some of these genes (dpy-20, unc-24, tra-2) have not been reported previously. Morphological mutants: T h e dpy-20(e2017) allele is well suppressed by sup-21, as described above, and nonamber alleles are not suppressed. The amber dpy- 1 8 allele, e364 (WATERSTON and BRENNER 1978), is at least as well suppressed by e1957 as by sup-5, whereas the nonamber allele dpy-I8(e499) is not sup- pressed. T h e weak sup-21 allele e1958 has no effect on dpy-I8(e364), nor has sup-22(e1986), but sup-23 acts as a weak suppressor of e364.

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TABLE 2

Comparison of suppression spectra

297

Amber allele

dpy-20 (e20 17)

dpy-18 (e364)

unc-I5 (e1214)

unc-54 (eZ008)

(el 108) (eIjr00)

unc-52 (e669)

unc-93 (e1500 n234)

unc-24 (el38)

unc-13 (e312) (e376) (e450) (e109Z)

lin-I (e1777)

tra-1 (e178Z) (e1825tr)b (e1828tr)b (eZ835tr)b tra-2 (eZ425) tra-3 (e1107) (e1903) (e1525)

fem- I ( e l 991)

~

sup-5 (e1464) sup-7 (stS) sup21 (e1957)

++

+++

++

+++

-

++

++a

++

+

++"

++

+

++a

++

+

+++

++

N D

++

+

N D

+++

+

+++"

+++

+

N D

+++

+

N D

+++

+

N D

+++

++

+++

+

+++

+++"

+++

++

+++

= good suppression, phenotype close to wild type;

++

= mod- erate suppression;

+

= weak suppression; f = barely detectable suppression;

-

= no detectable suppression; ND = not determined. For further description, see text.

a Inferred from other studies (see MATERIALS AND METHODS: Identij- cation of amber alleles) and not checked in this work.

For explanation of these alleles, see MATERIALS AND METHODS: Source

of tra alleles.

be no less paralyzed than e1214 alone, indicating lack of suppression. Para- myosin levels (WATERSTON 1981) in this double mutant have not been measured, so it is possible that small amounts of paramyosin are produced.

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e1108, e1300) with e1957 were constructed and compared with the unsuppressed parents and with sup-7 double mutants. In all three cases, animals carrying sup-21 moved better than the unsuppressed animals, but less well than the sup-7 doubles. Also, several ochre alleles of unc-54 have been identified by DNA sequencing (DIBB et al. 1985); double mutants of two of these alleles

(e1092, e1392) with e1957 were constructed, but they showed no response at all to e1957.

An amber allele of unc-52, e669, which causes progressive muscle degener- ation (WATERSTON, THOMSON and BRENNER 1980), is well suppressed by either one or two doses of sup-5 or sup-7 (WATERSTON 198 1); it is also well suppressed by two doses of sup-21, but not by one dose.

T h e double mutant of unc-93, e1500 n234, has no phenotype alone, but in the presence of sup-7 the amber mutation n234 is suppressed and the neo- morphic e1500 phenotype is expressed; this phenotype is a characteristic “rub- ber band” paralysis, probably resulting from a muscle defect (GREENWALD and HORVITZ 1980). Homozygous unc-93(e1500 n234); sup-21(e1957) animals do not have a paralyzed phenotype but exhibit unusual loopy uncoordinated movement, presumably reflecting low expression of e l 500.

Uncoordinated mutants: Two other unc genes, unc-24 and unc-13, were tested for suppressibility by sup-21; these genes may affect the nervous system rather than the musculature. T h e unc-24(e138) mutation results in uncoordinated forward movement, with an uneven wave form along the body; it is well suppressed by sup-7 and weakly suppressed by sup-21.

Mutations of unc-13 result in a “kinky” paralysis, with very little movement, although animals are able to lay eggs, indicating functional vulval muscles, Four amber alleles (e312, e376, e450, e1091: WATERSTON 1981) were tested for suppressibility by sup-21: two showed no response at all (e450, e1091), and

t w o showed a marginal response, only perceptible by comparing populations of suppressed and unsuppressed animals and not distinguishable on an individ- ual worm basis. This is a surprising result, because all four alleles are well suppressed by sup-5 and sup7 and are partly suppressed even by a single dose of sup-5. Reversion of unc-I3(e450) and unc-l3(el091) was used by WATERSTON (1981) to obtain most of known sup-5 and sup-7 isolates, and many of the revertants were picked as sup-5/+ heterozygotes. In order to demonstrate that these four unc-13 alleles represent four different amber lesions (rather than four isolates of the same amber, which might have unusual properties), tests for intragenic recombination were carried out (ROSE and BAILLIE 1980). Het- eroallelic unc-13 hermaphrodites were constructed and allowed to self on four to eight large (9 cm) plates; the plates were screened for wild-type animals after about three generations. T h e homozygous mutants e376, e450 and e1091

have not been observed to yield spontaneous revertants in populations of comparable size, but the heteroallelic combinations e376/e450, e376/e1091 and

e450/e1091 all yielded at least two independent wild-type animals carrying an

unc-13(+) chromosome, indicating that these three mutations are separable from one another by intragenic recombination. T h e fourth mutant, e312,

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e322/e322 and as a heterozygote e322/e450. This precluded tests for intragenic recombination, because wild-type recombinants could not be efficiently se- lected. The weaker phenotype implies that this allele may have residual unc- 2 3 function and is therefore different from the other three.

Lineage mutant: The Zin-2 amber mutation, e2777, results in a “multivulva”

phenotype in homozygotes: all P3-8.p ventral hypodermal cells undergo vulval divisions, resulting in the formation of three to four “vulval blips” along the ventral side of adult hermaphrodites (FERGUSON and HORVITZ 1985). It is completely suppressed by one dose of sup5 or su$-22(e2957). In view of this sensitivity, a double mutant Zin-l(e2 777) sup23 was constructed and found to be partly suppressed.

Sex determination mutants: Mutations in the major sex-determining genes pro-

vide sensitive indicators of normal gene function, as already demonstrated using tra-3. Numerous alleles of other sexdetermining genes (e.g., tra-2, tra-2,

fem-2: see HODGKIN 1980, DONIACH and HODGKIN 1984) have been identified, most of which have been tested for amber suppressibility, using sup-7 or sup- 5. For both tra-2 and tra-2, deficiencies have been obtained that permit classification of recessive alleles into null and non-null classes [eDfZ! for tra-2

(HODGKIN 1980), mnDf30 for tra-2 (SIGURDSON, SPANIER and HERMAN 1984)l. In the case of tra-2, the null alleles cause transformation of

XX

animals into fertile males, while ndn-null alleles cause incomplete masculinization of

XX

animals (see Figure 5a and g). Seven of 22 apparent null alleles were found to be suppressed, as was one out of 17 apparent non-null alleles. The exceptional non-null amber allele e2825tr causes more extreme masculinization than many other non-nulls, but the tail phenotype is usually incompletely male, with abnormal ray development (Figure 5g). The e2825tr

XX

animals are never fertile, nor are e2825/e2099 XX males (e2099 is the reference nonamber null allele for tra-1). It therefore seems likely that e2825tr has residual tra-2 gene activity, unlike the other seven amber alleles.

Four of the eight tra-2 amber alleles (e2782, e1825tr, e2828tr, e2835tr) were tested using sup-22(e2957). All showed partial suppression to a sterile intersexual phenotype. One, e2835tr, was slightly more suppressed than the other three but still showed signs of masculinization (Figure 5c), unlike sup-5(e2464) tra-

2(e2835tr), which is a fertile hermaphrodite. The greater sensitivity of e2835tr

is consistent with its response to the other suppressors: sup-7 acts as a dominant suppressor of this allele, but a recessive suppressor of the other seven tra-l

amber alleles. This is evident from the segregation ratios from unc-32 tra-l/+

+;

sup-7/+ hermaphrodites: for tra-I(e2835tr), the Unc-32 animals were scored

in the ratio

43

hermaphrodite: four intersexual: ten male, as compared with a ratio of 9: 1 1 : 17 for tra-Z(e1828tr).

In the case of tra-2, null alleles cause extensive but incomplete masculinization of XX animals (Figure 5d). One of 16 apparent nulls tested, e1425, was suppressed by both sup5 and sup7 to a self-fertile hermaphrodite phenotype (Figure 5e). None of three non-null alleles was suppressed. The tail phenotype of a tra-2(e2425); sup-22(e2957) XX animal is shown in Figure 5f; as with tra-

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~~

b

I

e

‘ U

I

#

C

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In the case of fem-1, null mutations cause complete feminization of X X and

X O animals. One of seven putative null alleles and zero of 14 non-null (tem-

perature-sensitive) alleles was found to be suppressed by sup-7 (DONIACH and

HODCKIN 1984). This allele, e1991, is also suppressed by sup-21(e1957): fem-

I(e1991); sup-21(e1957) X X animals are often self-fertile hermaphrodites, and

XO animals are intersexual or male (Figure Si). Successful mating by these males has not been observed, in contrast to occasional successful mating by fem- l ( e 1991); sup-7/0 males.

In summary, the sup-21 suppression spectrum is largely, but not completely, identical with the spectra for sup-5 and sup-7, and in most cases suppression is either slightly or markedly weaker with sup-21 than with the other two suppressors. T h e almost complete failure of sup-21 to affect unc-13 amber alleles is anomalous.

Dosage effects for suppressors: It was observed that the rare X O males segre- gated from dpy-20(e2017); sup-7 and dpy-20(e2017); sup-21(e1957) X X hermaphrodite stocks were much dumpier than their X X siblings, but that this was not the case for sup-5(e1464); dpy-20(e2017) stocks. This suggested that the sex-linked amber suppressors are not dosage compensated. A comparison of suppression in homozygotes, hemizygotes and heterozygotes for these three suppressors was carried out, using dpy-20 body length as an assay. T h e results are summarized in Figure 6. It can be seen that sup-7 and sup-21 are better suppressors than sup-5 in either homozygous or heterozygous hermaphrodites, b u t that suppression in hemizygous sup-7/0 and sup-21/0 males is worse than it is in homozygous sup-5 males. This shows that expression of sup-7 and sup- 21 is not compensated or only slightly compensated. T h e low expression in X O animals is not a consequence of maleness per se, because transformed (tra-1)

X X males carrying two doses of sup-7 or sup-21 show the expected high level

of suppression.

Other observations are consistent with a lack of compensation for these genes; for example, the debilitating effects of sup-7 at 20" and 15" are more marked in hermaphrodites than in males, but sup-5 hermaphrodites and males are similar in viability. At 20 O , homozygous sup-5 males mate very poorly and

sup-7 males mate well, despite the fact that the effects of sup-7 are, in general,

more severe than those of sup-5.

Tests f o r maternal message: In the case of genes, such as tra-3, that show a strong maternal effect, it is not known whether the maternal rescue is due to

FIGURE 5 .-Tail phenotypes of sex-determination mutants, unsuppressed and suppressed. a, tra-

l(e1781) X X . b, tra-l(e1781); sup-7 X X . c, tra-l(el8?5tr); sup-21 XX. d, tra-Z(e1425) XX. e, tra-

Z(e14.25); sup-5 X X . f, tra-Z(e1425); sup-21 XX. g, tra-l(I825tr) X X . h, Wild-type XO male. i , fem- l(e1991); sup-21/0 XO. For other photographs offem-l(el991), see DONIACH and HODCKIN 1984.

In parts c and f, note residual masculinization; in parts g and i, note abnormal rays. Most of these animals carried other markers to confirm genotype; complete genotypes are as follows: a, tra-

l(e1781). b, unc-32(e189) tra-l(e1781); su$-7(st5). c, unc-?2(e189) tra-I(e1575 e1816 e1835); dpy- ZO(e2017); sup-21(e1957). d, tra-Z(e1425) unc-4(e120). e, tra-2(e1425) unc-4(e120); sup-5(e1464). f ,

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a ) Hermaphrodites

*

2

-

sup f+) sup-5 sup-7 sup-21 dpy f+)

b) Males

if-

*

2

-

4 *

2

-

I

1500 p 1000

500

,xo

xx ,

sup-5 sup-7 sup-21 &Y (+)

s u p 1+)

FIGURE &--Dosage effects on dpy-20 suppression. Presentation as in Figure 4. For each suppressor (sup-5(e1464) ZZZ, sup-7(st5) X and sup-21(e1957) X), the lengths of hermaphrodites and males carrying one dose (bars marked 1) or two doses (bars marked 2) of each suppressor were measured. Heterozygous sup/+ hermaphrodites and males were generated by crossing unc-24(e138) dpy-20(e2017) hermaphrodites with dpy-20 males carrying sup. Hemizygous sup/U males were generated by him-5(e1490) for sup-21, and from a male strain for sup-7. XX males were generated using e1099, a nonamber null allele of tra-1.

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303

tra-3(nonwnber) tra-3(non-amber) dpy-4

.

sup-?

tra-3 (amber) tra-d(non-amber)

+

'

0

XX cross progeny :

1. t m - 3 h w n ~ m b e r ) dpy-4 _{' -}sup-?

tm-3(non-am6er) dpy-4 +

2. t2.a-3(non-amber) dpy-4 sup-7

tm-3(non-amber) +

'

7

3-

-

tra-3 (amber) +

.

sup-7

tra-3(non-amber) dpy-4 a

7

Phenotypes

Dumpy : Tra-3 if no rescue; hermaphrodite or intersex if rescued.

Non-dumpy : " 11

Non-dumpy : hermaphrodite

4 . t m - 3 (amber)

+

sup-7 Non-dumpy : hermaphrodite

t m - 3 ( n " b e r l +

'

7

FIGURE 7.--Scheme for detecting amber maternal mRNA from tra-3. The tru-3 amber allele used was eZZ07; the nonamber allele was eZ767. The maternal tru-3(e1767) dfy-4 chromosome also carried unc-30 in order to distinguish self-progeny dumpies from cross-progeny dumpies. The hermaphrodite parents were generated by crossing unc-30 tra-3(eZ767) dpy-4/+

+ +

hermaphro- dites with tru-3(eZ107) XO males; the male parents were generated by crossing tra-3(eZ767) dpy-4; sup7 hermaphrodites with tru-jr(e1767) XO males.

in this experiment, although tra-3(el767/e1107), sup-7/+ siblings were fully suppressed. An analogous cross, using sup-5, also gave no evidence of suppression.

Another test of this type was carried out on the gene fem-1 (DONIACH and

HODCKIN 1984), using the two apparent null alleles, e1991 (amber) and e1965

(nonamber); the results were negative (T. DONIACH, personal communication).

DISCUSSION

Amber suppressors provide information about tRNA genes and the mecha- nism of translational suppression. They also constitute useful tools for the analysis of other genes. These two aspects of the present work will be discussed in order.

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for the lower dose of EMS (0.025 M as opposed to 0.05 M) used in these experiments and assuming that all five sup-21 mutations identify the same structural gene, the mutation frequency is still much lower than the rate for loss-of-function mutations in most C. elegans genes see GREEN- WALD and HORVITZ 1980). It is notable that one sup-5 mutation, e2060, was

recovered from the tra-3 reversions, which appears to have identical properties to the original sup-5 allele, e1464. This shows that rare events, such as single- base alterations, were recoverable by the tra-3 selection. No sup-7 mutations were obtained, but it is possible that growth at 15" would have tended to select against sup-7 mutations, which are very cold sensitive. WATERSTON (1981) observed a bias against sup-7 mutations in his unc-13 reversions (19 of 25 sup-5, six of 25 sup-7), although one would expect equal numbers because both sup-5 and sup-7 mutations are likely to be identical alterations in

tRNA:zG anticodons.

If sup-21, sup-22 and sup-23 are tRNA genes, then their properties raise two

intriguing questions. First, why are different isolates at the SUP-21 and sup-22 loci so heterogeneous in strength? T h e simplest answer is that these two loci are, in fact, clusters of tRNA genes mutable to amber nonsense suppressors. T h e different isolates would then represent tRNA genes from different families or tRNA genes from the same family expressed at different levels and therefore with different efficiencies. Ample precedent for this kind of heterogeneity can be found in microbial systems. For example, yeast amber suppressors have been shown to vary in strength both between and within families: the tyrosine- inserting amber suppressors (six loci) are all high efficiency suppressors, whereas the leucine-inserting amber suppressors (six loci) are all low efficiency

(SHERMAN 1982). Also, the three serine-inserting ochre suppressors differ in strength (ONO et al. 1981), although their coding sequences are identical

(BROACH, FRIEDMAN and SHERMAN 1981). In C. elegans, it has already been established that different members of a tRNA gene family are not identical in

coding sequence. BOLTEN et al. (1984) found that two wild-type t R N A z z G

genes differed at position 18, a site likely to affect transcription, so that one of these genes may be dysfunctional or expressed only at a low level. Also, it is clear from WATERSTON (1981) and from the present work that sup-5 and sup-7 differ in efficiency, although both are almost certainly members of the same tRNAT'p family.

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305

compared with the sup-5 and sup-7 spectra. Many of the differences can be ascribed to generally inefficient suppression by sup-21, sup-22 and sup-23 mu- tations, but some of the differences are less easily explained. A conspicuous contrast can be made between dpy-20, for which sup-21 is at least as strong as sup-5, and unc-13, for which sup-5 is a strong suppressor and sup-21 is almost ineffective. Two explanations can be considered: first, that sup-5 and sup-21 cause insertion of different amino acids; and second, that sup-5 and sup-21 have different tissue specificities. T h e first explanation seems rather unlikely, in that it requires the assumption that the amino acid inserted by sup-21 is unacceptable o r almost unacceptable at any one of four positions in the unc- 13 gene, while the amino acid inserted by sup-5 is highly acceptable at all of these positions. T h e second possibility, tissue specificity, is more attractive, particularly in view of the apparent differences between expression in the germ line and expression in the embryo that are indicated by the tra-3 suppression assays (Figure 4b and c). T h e fact that sup-21(e1957) is a good suppressor of

dpy amber mutations (dpy-18, dpy-20), a poor suppressor of ambers in muscle genes (unc-15, unc-52, unc-54, unc-93) and a very poor suppressor of ambers in unc-13, which may be a neuronal gene, could be explained by a high level of sup-21 expression in hypodermal cells, a low level in muscle cells and a very low level in neurons. Tissue-specific tRNA species have been described in other systems (e.g., SPRAGUE, HACENBUCHLE and ZUNIGA 1977). If this explanation

is correct, then it should be possible to create changes in the tissue specificity of sup-21 by selecting for wild-type movement in an unc-I3(e450); sup- 2I(e1957) strain. It would also increase the utility of these suppressors, because sensitivity to sup-21 as compared with sup-5 would provide information about the sites of expression of any gene for which an amber allele is available.

Molecular analysis of the suppressor strains may rapidly clarify the nature of sup-21, sup-22 and sup-23. As shown by BOLTEN et al. (1984), alteration of a tRNAT'p gene around the anticodon from the wild-type sequence TCCAGA to the suppressor TCTAGA results in the creation of a new XbaI site. Probing XbaI digests of genomic DNA from sup-5 and sup-7 strains with a tRNATrp probe revealed the existence of two new bands on Southern blots, as predicted. If the new suppressors are also tRNATrp genes, then comparable Southern analysis of these strains should reveal yet more new bands. It would not be surprising if the new suppressors were members of the same family as sup-5 and sup-7, because there appear to be at least ten genes in this family (BOLTEN

et al. 1984). If they are not tRNATrp genes, then the most likely candidates are tRNAG'" genes. This is because the majority of EMS-induced mutations in

C . elegans are GC to A T transitions (BOLTEN et al. 1984; DIBB et al. 1985), and the only two mutations of this type that yield a conventional amber suppressor are TGG(Trp) to TAG and CAG(Gln) to TAG. Unfortunately, pre- dictions of this type are not very reliable: in yeast, amber suppressors inserting tyrosine, serine, leucine, glutamine, glutamic acid, lysine and tryptophan should all be possible, but in practice only the first three classes have been found (SHERMAN 1982). T h e failure to find the other classes remains unexplained.

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remain to be discovered, because seven of the eight new suppressors that I have analyzed seem to have distinctive properties. Two sup-21 isolates, e1957 and e2058, may be identical, but the other three sup-21 isolates, as well as the sup-22 and sup-23 suppressors, are distinguishable from each other and from sup-21(e1957). Probably several more suppressors could be identified by tra-3 amber reversion, particularly if a different mutagen were used in addition to EMS. It is possible that this approach might permit characterization of an entire tRNA gene family in a higher organism.

An incidental observation made during this work is the absence of dosage compensation for sex-linked amber suppressors. T h e measurements of dpy-20 suppression summarized in Figure 6 show that there is little or no compensation of the levels of sup-7 X and sup-21 X in hemizygous (XO) males compared with homozygous ( X X ) hermaphrodites or homozygous tra-1 X X males. How- ever, it is believed that many sex-linked genes in C. ehgans are dosage compensated (HODGKIN 1983b), either by increasing expression of genes on the single X chromosome of the male (as in Drosophila, reviewed by BAKER and BELOTE 1983) or by decreasing expression of genes on the two X chromosomes of the hermaphrodite. T h e sup-7 and sup-21 genes therefore appear to be exceptional. Conceivably, the lack of compensation is an effect of the suppressor mutations themselves, but this is not likely (for sup-7 at least) because the anticodon loop does not form part of the promoter in other tRNA genes (GALLI, HOFSTETTER and BIRNSTIEL 1981; FOLK and HOFSTETTER 1983). It could be that sex-linked tRNA genes in C. elegans are, in general, uncompensated, in contrast to those of Drosophila, some of which are known to be compensated (BIRCHLER, OWENBY and JACOBSON 1982). Perhaps the compensation machinery does not work with genes transcribed by RNA polymerase 111. Alternatively, these tRNA genes may have remained uncompensated because they are members of multigene families distributed over both sex chromosomes and autosomes, and therefore, the difference in gene dosage between male and hermaphrodite would be much less than with single-copy genes.

T h e second part of this discussion is concerned with the use of amber suppressors as tools for the analysis of other genes. Many of the points raised here have been made elsewhere (e.g., WATERSTON 1981; BOLTEN et al. 1984; FER- GUSON and HORVITZ 1985), but they are repeated here for the sake of com- pleteness and also to illustrate the fact that the same principles apply to amber suppression in a higher eukaryote as apply in microbial systems.

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quency, examination of allelic series, analysis of temperature-sensitive mutants, complementation testing and the use of genetic deficiencies. T h e availability of amber suppressors is a powerful additional tool, but it is clear from the present work that it is not wholly infallible, because not all amber alleles are necessarily null or amorphic. For the gene tra-I, one of eight amber alleles is not a null, and for unc-13, one of four amber alleles. In both of these cases, it is conceivable that the apparent residual activity of the gene is due to a dominant tightly linked extragenic suppressor, but this seems very unlikely. More probably, there is a low level of gene activity for both of these alleles,

tra-l(e1835tr) and unc-Z3(e312), resulting either from low-level endogenous read-through of the amber codon or from low-level activity of the incomplete amber polypeptide fragment. There are well-known precedents for both of these phenomena (e.g., PELHAM 1978; LEHMAN and CHIEN 1973). Non-null amber mutations of the second type are likely to be located close to the 3’ end of the coding sequence [as in the case of unc-54(e1300) (WILLS et al. 1983)], and this may turn o u t to be true of e1835tr and e312. Several other cases of apparent non-null amber alleles in C. elegans have been described by FERGUSON and HORVITZ (1985).

On the whole, however, non-null amber alleles seem to be rare in C. elegans,

so the identification of an allele suppressible by sup-7 is a strong indication that this is, indeed, a null allele. T h e frequency of amber alleles among putative nulls seems to vary from gene to gene in the limited sample so far examined: for example, six of

22

for unc-54 (WATERSTON 1981), seven of 22 for tra-1,

one of 16 for tra-2, three of four for tra-3 (this work) and one of seven for

fem-1 (DONIACH and HODGKIN 1984). It is encouraging that ambers have been found in almost all genes for which more than 15 mutations are available, and amber alleles have now been obtained in more than 40 C. elegans genes [M. EDGLEY and D. L. RIDDLE (Caenorhabditis Genetics Center), personal communication].

T h e identification of an amber allele also shows that a given gene has a protein product. This is not a trivial conclusion in the case of a gene whose biochemical function is unknown; indeed, some theories of development have proposed that untranslated R N A sequences play important regulatory roles

(e.g., BRITTEN and DAVIDSON 1969). T h e fact that so many C. elegans genes

have already been shown to have amber alleles argues against the relevance of such theories to these genes.

An amber allele must lie within a portion of the gene that codes for a polypeptide and must also result in a TAG triplet in the mutant gene. Both of these properties are likely to be important in the molecular analysis of a gene. For example, S. BRENNER has pointed out that about one-fourth of amber mutations in C. elegans should create a CTAG sequence and, therefore, result in a new restriction site for the restriction enzyme Mae1 (SCHMID et al.

1984).

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suppressed, while those in the second class should be poorly suppressed, because the maximum amount of protein product restored by the strongest suppressor, sup-7, is less than 40% of wild-type levels (WATERSTON 1981). So far, the analysis of C . elegans amber alleles has provided some support for this approach: for example, the nuc-1 (endonuclease) amber mutation is very well suppressed, while amber mutations in muscle protein genes (unc-15, unc-54) are poorly suppressed (WATERSTON 1981). However, it appears that not all amber alleles of a given gene show the same sensitivity: as shown here, one amber allele of tra-1, e1835tr, is more efficiently suppressed than the other seven tra-1 amber alleles. If only this allele had been available, one might have incorrectly concluded that the tra-1 product is needed only in catalytic amounts. Even so, varying the dosage of suppressors does provide a means of changing the level of wild-type activity for a given gene in a fairly controlled manner. T h e availability of the new suppressors permits further variation, because most of these suppressors are weaker than sup-5 and sup-7.

A related use of amber suppressors, as a means of testing for maternal mRNA in the embryo, is described in this paper. Neither of the two maternal- effect genes tested, tra-3 and fem-1, showed any response in these experiments. Unfortunately a negative result is inconclusive because the amount of maternal amber message could simply have been insufficient to give a positive result. Also, the amber mRNA may be less stable than wild-type mRNA and, therefore, may not be detected. Nevertheless, the method may be used for other maternal-effect genes and could provide information about such genes and about translational control during early development.

Finally, if the proposed tissue specificity of the new suppressor loci is sub- stantiated by further work, then they will provide useful tools for examining where particular genes are expressed. Otherwise, the utility of these suppressors is somewhat limited because most of them are so weak. No case has been found of an allele suppressed by sup-21 that is not better suppressed by sup-7, and for many genes, sup-7 is a much more powerful suppressor. One minor advantage of sup-21 is that homozygotes are viable at 15 O , unlike sup-7 or sup- 5 homozygotes, but for almost all purposes the most useful C . elegans amber suppressor remains sup-7.

I am grateful to the workers cited in MATERIALS AND METHODS for providing strains and information, and to ANDREW FIRE, IVA GREENWALD, TARITHA DONIACH and MICHAEL SHEN for their critical reading of the manuscript.

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