novel nematode amber suppressors

24 Pages·2003·2.7 MB·English

Copyright 0 1985 by the Genetics Society of America
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 Ill, 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 Ill 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 suppres-
sors 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 SHERMAN19 82).
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 (HUDZIAKet al. 1982), but only in one animal,
the nematode Caenorhabditis elegans, have amber suppressors been induced in
the germ line. WATERSTONA ND BRENNER(1 978) and WATERSTON(1 981) have
characterized two suppressor loci in C. elegans, sup-5 IZI and sup-7 X. The sup-
7 mutation st5 and the sup-5 mutation e1464 were shown to suppress amber
mutants via an altered tRNA (WILLS et al. 1983; KIMBLEe t 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 (BOLTENe t al. 1984). The
suf-5(e1464)m utation probably contains the same alteration in another niem-
Genetics 111: 287-310 October, 1985.
288 J. HODGKIN
ber of this tRNAl”p gene family, which has about 12 members (BOLTENe t al.
1984).
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 sup-
pressors. 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 sup-
pressors have been partly characterized with respect to the efficiency of sup-
pression and to the spectrum of amber alleles that they suppress. The 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: The following genes and alleles were used in this study. Unless otherwise noted, they
were obtained from the Cambridge collection established by BRENNER(1 974), and the genes are
listed by SWANSONE, DCLEYa nd RIDDLE( 1984). Nomenclature follows HORVITZet 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),u nc-52(e669).
LG 111: unc-93(e1500 n234) (GREENWALaDn d HORVITZ1 980), lon-I(e185),s up-5(e1464) (WA-
TERSTON and BRENNER1 978), unc-32(e189),t ra-1 (see below), dpy-I8(e364, e499).
LG IV: lin-I(e1777)( FERGUSONan d HORVITZ1 985), unc-l7(e245),d py-I3(e184), unc-5(e53)f,e m-
l(eI991, e1965) (DONIACHan d HODCKIN1 984), mor-Z(el125), unc-24(e138),d py-20(e1282, e1362,
e1415, eI422, e2017), unc-30(el9I),t ra-3 (see below), dpy-4(e1166), sDf2 (MOERMANan d BAILLIE
1981).
LG V: him-5(e1490).
LG X: sup-7(st5) (WATERSTON1 981), 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 (HODCKINa nd BRENNER1 977), 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. HODGKINS, . BRENNERP, . ANDERSON
and E. M. HEDCECOCK(f ive 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.
The 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).
Alleles of tra-1 have also been isolated as her-I suppressors (HODGKIN1 980: one allele) and by
reversion of a dominant feminizing allele of tra-1 (’HODGKIN 1983a; J. HODCKIN,u npublished
results: 26 alleles). The dominant allele used, tra-I(el575 e1816), is a double mutant derived from
tra-l(e1575)( HODCKIN1 980; J. HODGKINu, npublished results), so all of these 26 recessive derived
alleles are triple mutations of the tru-1 gene, e.g., tra-I(e1575 e1816 e1835). In the suppression
tests, only the somatic phenotype of XX animals was examined; this phenotype is affected by the
recessive masculinizing mutations (in this example e1835), but not by the dominant feminizing
mutations (e1575, e1816), which can be ignored for these purposes. Therefore, to simplify descrip-
tion, the triply mutant alleles are abbreviated by the suffix tr, e.g., e1835tr, to indicate their nature.
NEMATODE AMBER SUPPRESSORS 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. HODGKINS, . BRENNERa nd H. R. HORVITZ( e1425);f our were isolated as sup-
pressors of her-1 (HODGKIN1 980); one was obtained from the general tru screen U. HODGKIN,
unpublished results); one was a temperature-sensitive sterile (b202, KLASS, WOLFa nd HIRSH1 976);
and one was a dominant egg-laying defective (n196,T RENTT, SLJNaGnd HORVITZ1 983). The
mutation tri(f70) described by BEGUETa nd 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 ~1047e, 1093, 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. BAILLIEe,1 903 by E. M. HEDGECOCKo)n, e (e1525) by a tra-3 comple-
mentation screen U. HODGKINu, npublished results) and one (el 767) from the general tra screen
U. HODGKINu, npublished 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(1 981), one (lin-1)b y FERGUSON
and HORVITZ(1 985), one vem-I) by DONIACHa nd HODGKIN(1 984) and one (unc-93) by GREEN-
WALD and HORVITZ(1 980); 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(1 985). The marker Lon-l(e185) was often used to mark sup-5, using the
double mutant lon-I(e185) sup-5(e1464).T he 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 sup-
pressed 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
Zsolation of suppressors: The nine suppressors described in this paper were
isolated by reverting amber alleles of tra-3, a gene involved in sex determi-
nation (HODGKINan d BRENNER1 977). Three amber alleles and one nonamber
allele of tra-3 have been obtained, all of which have similar properties, causing
partial masculinization of XX animals. The tra-3 gene shows a strong maternal
rescue effect, so that tra-3 XX daughters produced by selfing tra-3/+ XX moth-
ers are completely wild-type hermaphrodites with normal self-progeny brood
sizes (average 365 viable progeny for six tra-3(e1107) hermaphrodites, as com-
290 J. HODGKIN
TABLE 1
Reversion of tra-3 mutants
Total F,
Informational suppressors Epistatic suppressors
screened
(ap~~ proximate
Allele estimate) sub-5 sub-21 sub-22 sub-23 fem-1 fem-3 tra-1 (dam)
-
el 107 25,000 e2064 e2066 e2065
e2067
e1525 100,000 e2060 e2058 e2057 e2059 e2062
e2061 e2063
2 1903 75,000 e1957 e1986
e1958
el 767 (non- 25,000 e2068 e2069
amber) e2070 e2071
pared with 330 for wild-type N2 hermaphrodites). However, almost all of these
self-progeny are transformed into abnormal sterile males, The 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. The tail phe-
notype is also variable and is never completely male. A characteristic tra-3 XX
male tail is shown in Figure la, which can be compared with the wild-type XO
male tail shown in Figure 5h. The 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 (KIMBLEe t 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) XX 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 XX her-
maphrodite 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
to 20". Only revertants will grow productively at this temperature, Several
independent revertants were obtained from each strain, as listed in Table 1.
The 18 revertants include both informational and epistatic suppressors, be-
cause tra-3 mutants can be suppressed by a variety of mutations in other sex-
determining genes (HODGKIN1 980; DONIACHa nd HODGKIN1 984). A much
larger set of reversion experiments has been carried out on the nonamber tra-
NEMATODE AMBER SUPPRESSORS 29 1
i
FIGURE I .-Tail phenotypes of tra-3 XX animals. a, tra-3(el107), unsuppressed inconiplete
male. b, sup-22@1986)t ra-3(e1903), completely suppressed fertile hermaphrodite. c, tra-3(~1107)
daughter of fra-3(eI1 07); sup-21(~2061)/+m other: 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 rever-
tants, 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.
HODGKINu,n published results).
The other nine suppressors are regarded as informational suppressors be-
cause 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 sup
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)
self-progeny. The other 223 animals were sterile or produced only Unc Tra-
3 progeny. This shows that e2060 is about 0.2 map units from unc-32 and,
292 J. HODGKIN
m
therefore, is likely to be an allele of sup-5, which is also very tightly linked to
unc-32 (SWANSONE, DCLEYa nd 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 her-
maphrodites of genotype tra-3; dpy-6 +/+ sup, and no more than I-2% pro-
duced hermaphrodite self-progeny. The 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: her-
+/+ +
maphrodites 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), 11 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.
Given these results, all five sex-linked suppressors are provisionally assigned
to the same locus, sup-21 X. All of the suppressors are dominant, so comple-
mentation testing is precluded and map position is the only criterion for re-
garding them as allelic. As shown below, the five suppressors fall into several
classes with regard to strength of suppression, and it is therefore possible that
the sup-21 locus represents several tightly linked suppressor genes.
NEMATODE AMBER SUPPRESSORS 293
d
FIGURE 3.-Phenotypes of adult hermaphrodites: the dpy-20 assay. a, Wild type N2. b, dpy-
20(e2017); (sup-21(~1957()c omplete suppression). c, sup-22(e2057) dpy-20(e2017) (weak suppres-
sion). d, dpy-20(~2017()u nsuppressed). 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, two 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.
The 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
sup-23.
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 HOSONOe t 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 (MOERMAaNn d BAILLIE1 98 1). The heterozygotes e2282/sDj2 and
e2282/e2017 have identical severe dumpy phenotypes at 20°, indicating that
294 J. HODGKIN
1
e
c) EMBRYONIC suppression of
100 x
50 X
-
0 -
sup(+) sup-22 sup-23 sup-21 sup-5 sup-7 tra(+)
FIGURE4 .-Assays for efficiency of suppression. a, Suppression of dpy-20(e2017): length mea-
surements on adult hermaphrodites (see MATERIALSA ND METHODS:L ength measurements). Vertical
axis = worm length in micrometers; error bars = f l SD. Suppressor genotypes are marked on the
bars. The leftmost bar shows the length of unsuppressed dpy-20(e2017) adults; the rightmost bar
shows the length of wild-type (N2) adults. b, Maternal suppression of tru-3. For each suppressor,
45-100 tru-3 daughters of tra-3; sup/+ hermaphrodites were picked as L4’s and were permitted
to self; the fraction producing progeny is expressed as a percentage. The suppressor alleles are
ordered as in part a. The leftmost bar indicates the negligible self-fertility(<l%) of tru-3 XX self-
progeny from tru-3 hermaphrodite parents; the rightmost bar indicates the 100% fertility of tru-
3 XX daughters of tra-3/+ parents. For sup-22 and sup-23, dumpy daughters of dpy-I3 tru-3/sup
NEMATODE AMBER SUPPRESSORS 295
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);s up 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, el 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 (WATERSTONan d 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%)o f tra-3 XX cross-progeny sired by tra-3 sup(+) XO
males; the rightmost bar indicates 100% fertility for traJ/+ 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
e1525 or eI903. All three have identical properties, so allele specifications have been omitted.
296 J. HODGKIN
high temperature (25") all 12 strains grew well, in some cases with slightly
reduced suppression of the dumpy phenotype.
The 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 sup-
pressors by this assay, which measures the amount of suppressor activity in the
maternal germ line, is shown in Figure 4b. The 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/+ XX cross-progeny are self-fertile hermaphrodites, but if the suppressor
is weak, some of these cross-progeny are sterile or intersexual. The 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. The 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: The dpy-20(e2017)a llele is well suppressed by sup-21,
as described above, and nonamber alleles are not suppressed. The amber dpy-
18 allele, e364 (WATERSTONa nd BRENNER1 978), is at least as well suppressed
by e1957 as by sup-5, whereas the nonamber allele dpy-I8(e499) is not sup-
pressed. The 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.
Muscle mutants: The paramyosin amber mutation unc-I5(e1214) is partly sup-
pressed by sup-5 and sup-7, but the double mutant e1214; e1957 was found to