Suppressors
of the
unc-73
Gene
of Caenorhabditis ekgam
Jin-Quan Run," Robert Steven:
Ming-shiu
Hung,* Rob van Weeghel,'
Joseph G. Culottit and Jeffrey C. Way*
*Department of Biology, Nelson Laboratories, Rutgers University, Piscataway, New fersqr 08855 and tsarnuel S. Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada M5G 1x5
Manuscript received December 4, 1995 Accepted for publication January 25, 1996
ABSTRACT
The unc-7? gene of Caenorhabditis ekgans is necessary for proper axon guidance. Animals mutant in this gene are severely uncoordinated and also exhibit defects in cell migration and cell lineages. We have isolated coordinated revertants of unc-7?(e9?6). These fall into three classes: intragenic revertants, extragenic dominant suppressors (sup?9), and a single apparently intragenic mutation that is a dominant suppressor with a linked recessive lethal phenotype. sup?9 mutations cause early embryonic lethality, but escapers have a wild-type movement phenotype as larvae and adults. Gonads of sup39mutant animals show a novel defect: normal gonads have a single row of oocytes, but sup39 gonads often have two rows of oocytes. This result suggests that the mutant gonad is defective in choosing on its surface only a single site from which nuclei will emerge to form oocytes. These results are interpreted in terms of an effect of unc-73 on determination of cell polarity.
D
URING
development, cells are often reproducibly oriented with respect to neighboring cells or to the body axes. Epithelial cells are polarized into apical and basal sides, while migrating cells, neuronal growth cones, and dividing cells must orient their cytoskeletons to move or divide in a particular direction.unc-73 mutant animals move very poorly and show profound defects in axon guidance. Immunofluores- cence staining reveals that the processes of the PHC, PVN, VD,
DD,
HSN
and touch receptor neurons are misplaced in this mutant. The posterior migration of the ALM cell and anterior migration of QR and its daughters are generally shortened, and the PVM cell sometimes migrates when it should not. In addition, unc-73 animals sometimes show secondary vulval blips that are characteristic of a reversalof
polarity of the asymmetric division of the P7.p cell. Ectopic mec-3ex- pressing cells, found in unc-73 mutants, are also consis- tent with an asymmetric cell division defect (HEDGE-COCK et aZ. 1987;
SIDDIQUI
and CULOTTI 1991; MCINTIREet al. 1992;
WAY
et al. 1992). Taken together, these pleio- tropic defects suggest that the unc-73 product functions to help cells and axons orient themselves within Caeno-rhabditzs ekgans (WAY et al. 1994).
More recently,
R. STEVEN,
A.
RUE, J.
MANGILLAS, andJ.
CULOTTI (unpublished results) have shown that unc- 73 encodes a -2,000 amino acid protein with a region of similarity to Cdc24 of yeast and to the products of the dbl and bcroncogenes, which appear to encode guanine nucleotide exchange factors (HART et al. 1991; RON etal. 1991). The CBC24 gene of Saccharomyces cereuisiae is
Corresponding authm: Jeffrey C. Way, Nelson Laboratories, Bus& Campus, Biology Department, Rutgers University, Piscataway, NJ
08855. E-mail: way@biovax.rutgers.edu
Cknrtir.; 143 225-2.76 (May, 1996)
necessary for determining the polarity of the yeast cell. cdc24 mutants fail to make a bud and fail to localize newly secreted proteins to the correct site on the plasma membrane. One likely target for CDC24is CDC42, which encodes a small, membrane-anchored Gprotein. Cdc42 is a member of the rho/rac family of GTPases, which are important in controlling the actin cytoskeleton in higher eukaryotic cells (see HALL 1994 for review). cdc42 mutants have the same phenotypes as cdc24 mu- tants, and a cdc24-ts mutant can be rescued by an in- creased gene dosage of CDC42 (FIELD and SCHEKMAN 1980; ADMS et al. 1990; JOHNSON and PRINGLE 1990). To identify additional genes that might function with unc-73 in C. elegans, we have isolated suppressors of unc- 73 (e936) and characterized a C. elegans Cdc42 homo- logue. Two of these suppressors map close to the Cdc42 homologue, but are not alleles of this gene.
MATERlALS AND METHODS
General methods of C. ekguns growth and maintenance were as described by BRENNER (1974). This paper follows the standard C. ekgans nomenclature (HORVITZ et al. 1979). All mutations used in this study are described in HOCDKIN et al. (1986), except for those described below in the text. Molecu- lar biology techniques were from SAMBROOK et 01. (1989).
Since the focus of this work is on extragenic suppressors of unc-7?(e9?6), we tested whether this allele could be sup- pressed by known examples of allele-specific, gene-nonspe- cific suppressors: smgsuppressors and amber suppressors. The smg suppressors smg-3(mall7), smg-4(mal I 6 ) , and smg-6(r896) and the amber suppressor sup7(st5) do not suppress the Unc phenotype of unc-7?(e9?6).
226 J.Q. Run
Chromosome /
unc- 1 I unc-73
""
-2.2 -1.7 0
1 map unit Chromosome I /
unc-4
let-22 let-25 unc-52
" _
t
-0.4 0 1.0 1.6
I
mnDf301;5;
1mnCl
FIGURE 1.-Map positions of genetic markers used in this study. Positions of marker mutations are from HODCKIN et al. (1995). Depiction of the deficiencies mnDf?O and mnDfl05 is based on HERMAN (1978) and STARICH et al. (1995). mnCl is a crossover suppressor that is thought to be an inversion (HERMAN 1978).
with unc-73;dB hermaphrodites, and progeny males were mated with smg hermaphrodites. unc-73/+;smg
+/+
dpy her- maphrodites were identified, and from these, Unc non-Dpy progeny were picked; assuming no recombination between the dpy and smgmutations, such animals will be twethirds unc- 7?/unc-73;smg+/+ dpyand one-third unc-73/unc-7?;smg/smg. About two-thirds of these Unc animals did segregate onequar- ter Dpy Unc progeny, but never segregated non-Unc progeny, which would arise if the smg mutation had become homozy- gous and was able to suppress unc-73(e936). All unc-73;smg animals had withered tails and grew poorly; these phenotypes are seen in unc-73(e936) animals grown at 25", and in more severe alleles of unc-73 (J. WAY, unpublished results; W. FOR- RESTER and G. GARRIGA, personal communication).An unc-7?(e9?6);sup7(st5) strain was constructed and tested by a similar strategy. Presence of the sup7 mutation in this strain was demonstrated by testing for its failure to grow at 15". The c9?6 allele is likely to be a point mutation causing a partial loss of gene function. Semilethal and lethal alleles of
unc-7? have been isolated (W. FORRESTER and G. GARRIM, personal communication). The e936 mutation does not cause a DNA rearrangement that can be detected by Southern blot using the rescuing cosmid CllB5 as a probe (R. STEVEN and J. CULOTTI, unpublished results).
Isolation of revertants from unc-7?(e9?6): After treatment with 50 m M EMS for 4 hr (BRENNER 1974), unc-73(e9?6) L4 hermorphodites were put on the edge of an Escherichia coli- seeded 10-cm agar plate (five animaldplate; each animal pro- ducing -100 offspring). Plates were incubated for 7-8 days at 20°, at which point the F2 generation had reached adult-
TABLE et al.
hood and the starting side of the plate was cleared of bacteria. Most of the Unc Fe animals, even if starved, remain on this side of the plate, and non-Unc revertants are enriched on the other side where food is available. About 250,000 mutagen- ized genomes were examined (250 plates X 5 P,, animals/ plate X 100 F, animals/P, X 2 genomes/F,). Ten indepen- dent non-Unc revertants were identified and their new muta- tions assigned the allele names je1-jel0.
To test whether the reverting mutations were dominant, worms homozygous for unc-7?(e936) and heterozygous for the suppressor were constructed as follows. Each revertant was mated with unc-7?(e936)/+ males and the phenotype oftheir F, scored. All the male and hermaphrodite progeny from such crosses were non-Unc, indicating that each reverting mutation is dominant.
To map unc-73 suppressors to a chromosome, the following protocol was used. unc-73/+;sup/+ males were mated with unc-7;dpy-n hermaphrodites, where dpyn is a marker for each chromosome [chromosome I, dpy-5(~61); chromosome II, d p y IO(e128); chromosome III, d@-17(e164); chromosome W, dpy- 1?(eI84); chromosome V , dpy-l l(e224); chromosome X , l a m 2(e678)]. If the suppressor is linked to a particular dpy, then some of the non-Unc non-Dpy hermaphrodite F, progeny of such a cross will be unc-7?/unc-73;sup/~lpy and will segregate only Dpy Unc and non-Dpy non-Unc animals (excluding re- combinants; recall that all suppressors examined are dorni- nant). All suppressor mutations mapped to chromosomes I or
IZ
by these criteria, and were characterized further using markers on these chromosomes (Figure 1).In addition, to test for linkage between unw73 and sup, each suppressed strain was mated with wild-type males, and putative cross progeny were picked and examined for segrega- tion of Unc F2s. Suppressor mutations that showed linkage to dpy-5 ( l ) were inseparable from unc-73 ( l ) by this criterion.
Characterization of j e 3 The j e 3 mutation a dominant sup- pressor of unc-7?(e936), is recessive lethal, and is tightly linked to ~93'6, suggesting that je3 is an allele of unc-73 itself (Table 1). Theje3 mutation was mapped as follows. From self-progeny of
+
je? e936 +/unc-l1(e47)+ +
dpy-5 heterozygotes, 10 Dpy, non-Unc-11 and 10 Unc-11, non-Dpy recombinants were is* lated. From each recombinant F,, 10 F2s of the recombinant phenotype were picked and their F:+ progeny examined to see if the recombinant chromosome could become homozygous and therefore lacked the lethal j e 3 mutation. Of the 10 Dpy recombinants, seven were presumably+
je3 e936 dB-5/unc-l I+
+
dB-5 and segregated F2 Dpy animals that themselves all segregated Unc Dpy progeny. Similarly, one of 10 Unc non- Dpy recombinant chromosomes could not be made homozy- gous. Together, these data place the je3 mutation at about -1.8 on linkage group I, near the map position o f unc-73.The unc-73(e936) and j e 3 mutations were not separated in the above crosses: no unc-11 unc-73(e936) or unc-73(e936) dpy- 5 animals were generated. The unr-I1 unc-73(e936) double
1
The je? mutation maps to the same position as unc-7?(e9?6)
Genotype of Selected
heterozygote parent phenotypes Genotypes
+
je3 e936+/
unc-1 1+ +
dpy-5 Dpy, non-Unc 7/10+
je3 e936 dpy-5/unc-l1+ +
dpy-5 3/10+
+ +
dpy-5/unc-ll+ +
dpy-5 0/10+ +
e936 dpy-5/unc-l1+
+
dB-5 Unc-1 1, non-Dpy 1/10 unc-11 je3 e936 +/uric-I1+ +
dpy-59/10 unc-11
+
+
+ / m e - 1 1+ +
dpy-5 O/ 10 unc-11+
e936 +/uric-1 1+
+
dB-5 Hermaphrodite heterozygotes were generated and their progeny analyzed as described in MATERIALS ANDmutants have a distinctive severely Unc phenotype that was never observed among segregants from the Unc-11 recombi- nants. It is difficult to distinguish between unc-ll dpy-5 and unc-73 dpy-5, so each of the seven
+
j e 3 e936 dpy-5/unc-ll+
+
dpy-5 recombinant lines was test-crossed with unc-73(e936)/+
males to see if it was actually+
+
e936 dpy-5/unc-ll+ +
dpy-5 and would then segregate Unc-73 male progeny; none did. These results indicate that the j e 3 lethality and the e936 mutations are tightly linked.The lethality of the j e 3 mutation appears to be rescued by an unc-73(+ )bearing cosmid. evEx38 is an extrachromosomal array consisting of a cosmid, C11B5, that rescues unc-73 muta- tions. Wild-type males were crossed with an unc-11 unc-73(je3 e936)
+/uric-11
+
dpy-5 hermaphrodite, and progeny males were crossed with unc-73(rh40) Ex38 hermaphrodites. Of 55 non-Unc hermaphrodite F, progeny picked from the latter cross, nine segregated F2 Dpy Uncs and 10 putative unc-11 unr-73(je3 r936)/+ unc-73(rh40);evEx38 animals segregated F2s with an Unc-11 phenotype (thin with ratchet-like move- ment, as opposed to the Unc-73 phenotype, which is inactive, coiled, and Dpyish). The presence of j e 3 in putative unc-11 unc-73(je3 e936);euEx38 F3 segregants was inferred from the fact that -50% of their F4 progeny fail to develop beyond the L1 stage, as seen for unc-73(je3 e936) animals. Adult unc- I1 unc-73(je3 e936);evEx38 animals sometimes show ectopic pseudovulvae and are more uncoordinated than unc-lI(e47) alone, which we interpret as resulting from somatic loss of the unstable evEx38 element.It is formally possible that the lethality of je3 is due to a second mutation in an essential gene that is included within the unc-73rescuing cosmid CllB5. However, the unc-73 gene occupies 218 kb of this cosmid, and it is unclear if there are any other intact genes contained within the cosmid. It is also possible that the je3 mutation is a deletion or other rearrange- ment that simultaneously alters unc-73and a neighboring gene.
Analysis of the extragenic dominant suppressors mp39( j e 5 andje6): To test the unc-73alleles ev454::Tcl, rh40, and ev509 for suppression by sup39(je5), sup-39 homozygous males were mated with unc-73;dpy-10 hermaphrodites. Of the self-prog- eny from these unc-73/+;+ sup39/dpy-l0
+
animals, approxi- mately one-quarter (rather than 1/16) were Unc. Among these Unc animals, unc-73/unc-73;sup39/sup-39 hermaphro- dites were identified by failure to segregate Dpy progeny; these homozygotes showed an Unc phenotype indistinguish- able from the original unc-73 strain. The ev509 allele of unc-73 does not appear to be stronger than e936with respect to movement, and m454::Tcl shows more spontaneous move- ment than e936 (Figure Z ) , while rh40 animals show as little or less movement (data not shown).
The location of sup39 was determined by three- and four- factor crosses with unc-4(e120), unc-l04(e1265), dpy-l0(e128), let-22(mn22), let-23(mn23), let-25 (mn25), and cdc-42(pk57:: Tcl) as described in RESULTS, Figure 1, and Table 2.
mec-3expressing cells in sup?9(je5) animals were examined using the mec-3-lac2 fusion element jeZn2 (Table 3; WAY et al. 1991). Animals were stained with Xgal as described in FIRE et al. (1990). Secondary vulval protrusions (Table 3) in acetone- fixed adults were observed by Nomarski optics.
sup39mutants suffer from a partially penetrant embryonic lethality that decreases in severity as the mother animal ages (Table 4). Because of this variation with the age of the mother, all experiments comparing rates of embryonic lethal- ity were performed using mother animals of the same age. The embryonic lethality depends on the genotype of the mother and not the embryos (Table 5).
We tested whether a sup39(je5) mutation causes a more severe or distinctive phenotype when heterozygous with a de- ficiency. However, these experiments were complicated by the fact that sup-39 lies near or within a possible region of haplo- insufficiency in the center of chromosome ZZ (HERMAN 1978; STARICH et al. 1995). mnDf3O and mnDflO5 do not overlap,
Experiment 1
loo
I
0 2 5 15 100 780
Time (minutes) Experiment 2
100
Per cent 80
of animals moving beyond a 6o
circle 1 cm.
40
unc-73(e936)
4 unc-73(e936); sup-39(je5) o unc-73(e936); sup-39(je6) 20
0
0 15 30 60 120 300
Time (seconds)
FIGURE 2.-Movement of unc-73 mutants and suppressed strains. For each experiment, 10 animals of the indicated genotype were placed at the center of a 1-cm-diameter circle on a bacteria-covered NGM plate. Plates were examined at various intervals and worms were removed and scored as they reached the perimeter. For experiment 1, late L4 animals were used; for experiment 2, gravid adults were used. Similar results were obtained for the unc-73 mutants when a 2<m- diameter circle was used (data not shown).
and sup-39 may map between these deficiencies or under one of them. Heterozygotes of sup-39(je5) with the deficiencies mnBf?O and mnBfl05, and with lethal point mutations in let- 22 and let-23 (as controls) were constructed as follows. N2 males were mated with the following strains: sup39 unc-4, let- 22 unc-4/mnCl, let-23 unc-4/mnCl, mnBf30 unc4/mnCl, and mnBfl05/mnCI. (mnCl is a balancer for chromosome ZZ and carries the markers dpy-10 and unc-52.) Heterozygous males were then mated with unc-73 dpy-5;sup39 unc-4 hermaphro- dites, and Unc-4, non-Dpy hermaphrodite offspring were picked. (All the animals examined were thus also heterozy- gous for unc-73 and dpy-5.) For controls lacking sup39, N2 males were mated with unc-4 and male offspring were mated with let-22 unc-4/mnCI, with mnDf30 unc-4/mnCl, or with mnDflO5 unc-4/mnCl, and Unc hermaphrodite progeny were examined. Six such Unc, non-Dpy animals were picked to separate plates and their progeny examined. In one experi- ment, the first -50 eggs laid by each mother were scored; in a second experiment, the eggs laid in the first 12 hr and four subsequent 6-hr interval were picked, and hatched progeny were examined 36 hr later.
228 J.Q. Run et al.
TABLE
2Map position of sup39
Genotype of
heterozygote parent Selected phenotypes Genotypes
f unc-4 let-25/sup39 f i Unc, non-Let f let-23 unc-4//sup39 i f Unc, non-Let let-22 f unc-4/+ sup39 i Unc, non-Let
dpy-IO sup39 i unc-4/ f Unc, non-Dpy (failing to segregate Lin)
i lin-26 f
dpy-IO sup-39 i unc-4/ f f Unc4, non-Dpy unr-104 i
Dpy, non-Unc
dpy-IO sup39 f unc-4/+ f Unc, non-Dpy cdc-42:: Tcl f
Dpy, non-Unc
dpy-IO sup39cje5)
+
unc-4/ Unc, non-dpyi f sup-39(je6)fU Dpy, non-Unc
6/6 11/11
4/10 6/10
9/20 11/20
6/71 65/71 0/71 0/71 27/29 2/29 0/29 0/29
5 / 3 5 6/35 24/35
0/35
19/30 0/30 6/30
5/30
10/10 9/9
f unc-4 f/iunc-4 let-25 sup39 unc-4
+/
f let-23 unc-4f i unc-4/let-22 f unc-4 f sup-39 unc-4/let-22 i unr-4
f
+
f unc-4/d@-IO sup-39 f unc-4f sup-39 i unc-4/dpy-I0 sup-39 f unr-4
f sup-39 -t uuc-4/dpy-l0 sup39 f unc-4
f i unc-104 unc-4/dpy-l0 sup-39 f uuc-4
i
+
i unc-4/dpy-l0 sup39 f unc-4i sup-39 unc-IO4 unc-4/dpy-IO sup39 f unc-4
dpy-IO sup-39
+
f / d p y - l O sup39 f unr-4 dpy-10 f unc-104 +/dpY-lO sup39 i unr-4 dpy-10 sup39 unc-I04+/
dpy-I 0 sup-39 i unr-4dpy-IO f f i / d p ? l - l O sup-39 f unr-4
f sup-39 i uuc-4/dpy-l0 &up39 f unc-4
i i f unc-4/dpy-lO sup39 -t unr-4
f cdc-42::Tcl f unc-4/dpy-l0 sup39 f unc-4
f sup-39 cdc42::Tcl uur-4/dpy-lO sup39 f unr-4 dpy-10 sup-39 t t / d @ - I O sup39 f unr-4 dpy-IO i f + / d p y - I O sup-39 f unr-4 dpy-IO f cdc-42::Tcl +/dpY-IO sup-39 f uuc-4 dpy-10 sup-39 cdc-42::Trl t / d p y - I O sup39 i unc-4
f sup-39 i unc-4/dpy-l0 sup39 i unr-4
dpy-IO sup-39 t +/dpy-IO sup39 f unc-4
The je5 allele of sup?9 was used for all the above crosses, unless otherwise indicated. To map the sup?9(je5) mutation within chromosome I1 (Figure l ) , heterozygotes shown above were generated by standard techniques. In general, after a recombinant animal was picked, the recombinant chromosome was homozygosed and tested for the presence of sup39(je5) by checking suppression of unc-7?(e936). Unc-4 non-Dpy (sup39?) recombinants were mated with
+
+/unc-7? dpy-5(I)
males, cross-progeny segregating Dpys were identified (presumably unc-7? dpy-5/++; sup39? unc-4/+ +), and it was determined whether the Dpy segregants were always Unc. Dpy-10 non-Unc-4 (sup-?9?) recombinants were mated with +/unc-7? males, the unc-7?/+;+
+/
dpy-10 sup?9? F1 progeny were identified, and the Fs offspring from FP Dpys and F4 Uncs examined: if sup-39 is present, the UncF4s will never segregate Dpys, and the Dpy F2s will never segregate Uncs. For the cross involving lin-26, Dpy non-Unc animals were not examined because it is extremely difficult to perform test matings with vulvaless lin-26 animals; it was anticipated that sup-?9 would map to the left of lin-26 and that all the useful recombinants would thus carry lin-26. The cdc-42:: Tcl insertion causes no visible phenotype and was scored as a DNA polymorphism as described in MATERIALS AND METHODS." Results of this cross suggest that je5 and .je6 are allelic. All recombinant chromosomes carry either sup-?9(,je5) or sup?Y(je6), which cannot be distinguished.
As heterozygotes, rnnDf?O and mnDfl05 do not suppress two-thirds of the Dpy progeny would be non-Unc. Instead, unc-7?(e9?6). unc-7? dpy-5/+ males were mated with mnDf?O only rare Dpy F4 animals were identified, which presumably unc-4/mnCl, and mnDfl05/mnCl hermaphrodites, and prog- arose by recombination between unc-7? and dpy-5.
eny that segregated Unc-73 Dpy-5 but not mnCl homozygous Suppression of the Unc phenotype of unc-7?(e9?6) is com- offspring were identified. If the deficiency could suppress unc- plete in sup39 homozygotes, but unc-7?;sup?9/+ animals ex- 73(e9?6), then from the unc-73 dpy-5/
+;
mdfunc-4 F1 animals, hibit a range of phenotypes from wild type to noticeably uncoor-TABLE
3Cell migration, ectopic mec-3-expressing cells, and secondary vulval protrusions in sup39 strains
Percent of cells in abnormal
positions Percent of
duplicated mec-3 Percent of adults with
AVM ALM expressing cells secondary vulval blips
Wild type 2.6% (38) 0% (82) 0% (241) 0% (96)
unc-7?(e9?6) 61% (28) 25% (64) 3% (176) 3% (96)
sup-?9(je5) 4% (25) 0% (70) 0.4% (267) 0% (91)
unc-73; sup39
7%
(14) 15% (47) 2% (161) 2% (98)TABLE 4
Embryonic lethality as a function of the mother's age
229
Hours after mother
Percent surviving embryos Embryos examined
reaches adulthood:
Hours after mother
Percent surviving embryos Embryos examined
reaches adulthood
Experiment 1: sup-?9(je5)
0-12 12-24 24-36
7% 17% 49%
95 257 432
0-24 11% 1144
Experiment 2: sup?9(je6)
24-48 52% 653
36-48 67% 322
48 - 60 56%
223
Experiment 1: Eight sup-?9(je5) young adults were picked to separate plates and then transferred every 12 hr. Eggs laid in each 12-h period were counted, and the fraction that hatched was determined 36-48 hr later. Experiment 2: Eleven sup?9(je6) animals were picked, transferred every 24 hr, and their laid eggs examined as above. In other experiments on embryonic lethality, the age of the mother animals was made as uniform as possible within a given experiment. However, comparison of embryonic lethality rates in different experiments (e.g., Tables 4, 5, and 7) is not necessarily valid, because of variation in maternal age between experiments.
dinated, although even the most Unc of such animals always move better than unc-73;sup39(+) when prodded. This variabil- ity has made it difficult to identify possible intragenic revertants of s u p 3 9 by loss of dominant suppression (but see RESULTS).
Identification of a C. elegam Cdc42 homologue: Because gain-of-function mutations in CDC42 can suppress partial loss- of-function mutations in cdc24 of yeast, we tested whether the sup-39 mutations might be in a C. eleguns Cdc42 gene. A C. eleguns homologue of the yeast and human Cdc42 gene was identified by PCR amplification with the oligonucleotides GGNCARGARGAYT'AYGA (forward primer) and GCYTCRT- CRAANACRTIYIT (reverse primer), which correspond to the conserved sequences GlyGlnGluAspTyrAsp and LysAsnVal- PheAspGluAla (JOHNSON and PRINGLE 1990; MUNEMITSU et
ul. 1990). cDNA clones for cdc-42 were isolated and sequenced on both strands, as well as genomic coding regions, introns, and flanking sequences from wild-type C. eleguns (Figure 3). While this work was in progress, CHEN et ul. (1993) published a characterization of a C. eleguns c d c 4 2 cDNA clone. Our results
indicate that, in the region of the start codon, the genomic DNA sequence is
ttcattttttcaggcgaaAACAGACGATGAAGTG
CGTC.. . .
Several cDNA clones had the sequence gaattcg-aaatttaaggcgaaATGCAGACGATGAAGTGCGTC.
. .
; the un- derlined sequence corresponds to the splice leader SL1, indicating that the cdc-42 transcript is trunpspliced. CHEN et al. proposed a shorter N-terminus, based on a deduced amino acid sequence from a single cDNA clone in which a cloning linker directly abutted the putative start codon. It seems that our proposed N-terminus is more likely to be correct, since it is based on both the genomic sequence and a longer cDNA, the deduced N-terminal amino acid sequence is identical to the human Cdc42 homologue (MUNEMITSU et ul. 1990) and contains only an Ile to Leu substitution relative to the yeast sequence (JOHNSON and PRINGLE 1990) rather than a deletion of several amino acids, and this region was subsequently se- quenced by the C. eleguns Genome Sequencing Project, which obtained a genomic sequence identical to ours. We also cloned and sequenced cdc-42 from supjrg(je5) and s u p 3 9 ( j e 6 ) strains;TABLE 5
Maternal-effect embryonic lethal phenotype of mp39(je5)
Male parent genotype
Hermaphrodite
parent genotype n
Percent survival
of progeny n
s u p 3 9
sup-?9 Wild type
Wild type
unc-4 s u p 3 9 u n c - 4 s u p 3 9 u n c - 4 unc-4
sup-39 unc-4 unc-4
+
sup-39 unc-4/dpy-l0+
+
8 90%
9 47%
5 53%
6 92%
2 44%
2 89 %
673 1231 665 377
27 106
48% 27% Dpy 1959
47% Wild Type 26% Unc
To test whether the embryonic-lethal phenotype of sup39(je5) is due to a maternal, paternal, or zygotic defect, male and hermaphrodite parents of different genotypes were mated (first four lines) or selfed (latter three lines). For the matings, L4 hermaphrodites were mated for 1 day with excess males and then picked to separate plates. All eggs that were laid within the next 2 days were examined. Only data from hermaphrodites that produced 235% male and 75% non-Unc progeny is represented here. The fact that
+
sup?9 unc-4/dpy-l0+
+
animals produce 50% dead embryos and Dpy, wild type, and Unc viable progeny in a 1:2:1 ratio also indicates that the embryonic lethality is due to a parental, rather than zygotic, effect, and that sup39(je5) is dominant for this phenotype. n after parent genotypes is number of mothers; n after progeny is number of embryos.J.-Q. Run et al.
230
1 101 201 301 401
501
601
tgtttttctaattattgattattcatttataagagcaaaattaatcaataaacgtaggtagacgatcgaagtatcctatcagtttttaatttgataaatt ttctcgaaaggagcaatcatgttttaaattatttaaaaaatggtccgcttaagtttgtctgactgtttaacaattattgaaatcattttcgtgacatttt attgaacattgcttccaaattcccctaacataatcctgactgagatgttccagtgtttcatgtcgaattattcctatttttgtaccttttcttaaatata actagtttcaaatatatgtgcaagttgtttggtacggaaaaccgattcaaacttccgaccagttaccgatgaatcattgccaaaattcctcatctaaaga attatacgcacaagttgttttggccattttgccttatttcttcatcgtttattaaggcgtttaccgtttttttttcttgctctttcttcgataattattg
ctcccaatggtaaagaaacgctcgtggcatttcgaaagagactattgattttctgcctgatgtttcgcaattcgttttcattctctagttatctctcctt
Tcl site?
cDNA 5‘ end: aaatttaaqgcgaa. . .
tttaaaacgtgtgcgtgcacatttatgtgactcatccgtatttttcactataaaqacgtaattttaatacttttattcattttttcaaacaa~TGCAGA
MetGln
ThrIleLysCysValVa1ValGlyAspGlyAlaValGlyLysThrCysLeuLeuI1eSer~rThrThrAsnLysPheProSerGlu~ValProThrVal
ValThrAlaMetIleGlyGlyGluProTyrThrLeu
“ L
701 CGATCAAGTGCGTCGTCGTTGGAGATGGAGCTGGAGCTGTC~T~CTTGTCTCCTGATCAGCTATACCAC~CAAGTTTCCTTCTGAGTATGTGCCGACAGT
PheAspAsnTyrAla
801 CTTCGACAATTACGCCgtgtttgaaaaaaaaattattttatataaaaataatttccgaatttca~TCACA~AATGATCGGT~CGAGCCATACACATT
901 AGGATTGTTTGATACTGCTACAGGAAGATTACGATTACGATCGATTAAG~CTCTATCGTATCCACAGACCGACGTGTTTCTTGTTT~T~TCCGT~TTGCT G l y L e u P h e A s p T h r A l a G l y G l n G l u A s p T y r A s p A r g L e ~ g P r o L e u S e r T y l P h e L e u V a i C y s P h e S e r V a l V a l A l a
1001 CCAGCTTCATTCGAGAATGTCCGAGAAAAAgtaagaaataagaaatattgttctcgaatttattctatatatctgaaattgtttta~~T~CTGAAATTTC~A ProAlaSerPheGluAsnValArgGluLys TrpValProGluIleSerHis
HisCysSerLysThrProPheLeuLeuValGlyThrGlnValAsp~e~gAspAspProGlyMetLeuGluLysLeuAlaLysAsnLysGlnLysPro
1101 T C A T T G C T C A A A G A C C C C A T T C T T G T T A G T T G G T T ~ T A C T ~ G T C G A ~ T C A G G G A T G A T C C A ~ A A T ~ T C G A T G G A G C ~ C T G ~ ~ C A A ~ A ~ C C A
1201 G T G T C A A C G T A T G T T G A G A A G T ~ ~ A A A ~ ~ T T G A A A ~ A G T G A A A T A C G T T ~ T ~ T C A ~ G T T G A C G C A ~ t g a g t c a t t c t t t c c a a a t a g 1301 tcattgataatattgaaacattaaaataaaaatatcatccaatccacgggcctaatcaaataataaaatttttaaatattaaaacgaggctcttgtggaa
1401 atacgtgacactttaaaatataaatatcacttttcagttaaattgttgttgctttatatttagcaacggtcatgttttcgggaaaaacgaacaaccagaa 1501 ttctgtttttattgtgcaaaaaattatctcctgaaatgctctatattccggctgaaaataccatagttttcatattatgttgattaaaaacgcttgtact
1601 attgaagtttaaatagtttattttcagAA~GACT~TGTATTCGACGAAGCCATTCT~CCGCTCTCGACCCACCAC~CA~AGAAGAAGATTACGAAGATTACGA
1701 AGTGCAATATTCTCtaaaaacatcttccttgtctccatgttttcctctgttttgtgactgtgtttttctttctatgtccaaattttcttagcccccgact 1801 tccactcttctcatacttctaaatttccctgaaaaatgccaaacgagaagcggttgctgttatttttgacagcatcatagaagatgattttcttcaattc 1901 gtctcctgtaacttatccagtagaatctcgatgagcatgattccagaattttttaattgtttcgtatagaatttcgataagcagggcagtttatagtttc 2001 tcatctgttttcgaatttttatcgaataatcccctcgaaaatttttaaaagttcatccaaattaactatcattcttttcaatgacacctcctctttaaca 2101 ttttcgtcgtcgtttttattttcttttttatcccaaaacgtgtttatagtccggatacaccgttacgtaatgcgccttctcctttcccatggttcttttt 2201 ataattatattaataggtgttgatagcacaaccaactacatcccgagtcgcttgtttcttcatttgctgctcaaaaataccgaccagtttgtttttcttc
2301 tttccttttttttgtgcttttctgccttcacacatccatttcccattttaaactgttttctcatccactacgttttgtgtctccctcctcctttctccag
2401 aacaactctgtattttaatttttctacatattgttgttatatatttgcgatccaaaacaataaaggtgtgatataaaaaattaagatgtttataaaagat
2501 ct
ValSerThrTyrValGlyGluLysLeuAlaLysGluLeuLysAlaValLysTyrValGluCysSerAlaLeuThrGlu
LysGluLeuLysAsnValPheAspGluAlaIleLeuAl~laLeuAspProProGlnGlnGluLysLysLys
LysCysAsnIleLeu
cDNAend:
. . . .
.ttatatatttgcgatccaaaacFIGURE 3.-Sequence of the cdc-42 gene of C. ekguns. The underlined sequences are, in order: the sequence corresponding to the “upstream” oligonucleotide used in polymorphism mapping, a
TA
dinucleotide in the region of theTcl
insertion incdc-42(pk57), the remnant of the splice leader SL1 found in a cdc-42 cDNA, a putative
TATA
element, the sequence corresponding to the “downstream” oligonucleotide, and a putative poly-A addition sequence.no differences in sequence were found between cdc-42 from wild-type, sup39(je5), and s u p 3 9 ( j e 6 ) DNAs, including coding sequences, introns, and 500 bp at the 5‘ and 3‘ ends. The cdc- 42 gene lies on the cosmid M04B9, which is -50 kb to the left of lin-26 on chromosome 11
Genetic mapping of s u p 3 9 indicates that it is distinct from
cdc-42, separated by -0.25 map units (Figure 1; Table 2). T o separate cdc-42 and sup-39, we used a Tcl insertion, cdc- 4 2 : : T c l ( p k 5 7 ) -280 bp upstream of the cdc-42 start codon;
the insertion confers no overt phenotype but can be detected as a
DNA
polymorphism. The insertion was identified in anMT3126 strain background by the method of ZWAAL et al.
(1994;
R. VANWEEGHEL,
R.PLASTERK,
and J. CUI.OTTI, unpub- lished results) and outcrossed five times. A dpy-IO s u p 3 9 ( j e 5 )+
unc-4/++
cdc-42::Tcl+
strain was constructed, and fromit 30 Dpy, non-Unc and 35 Unc, non-Dpy recombinants were identified. For each recombination event, the recombinant chromosomes were homozygosed and then tested for the presence of sup-?9(je5) (legend to Table 2) and cdc-42::Tcl.
T o identify whether the
Tcl
insertion in cdc-42 was presentor absent,
DNA
from each recombinant was purified and used in a polymerase chain reaction with the upstream(GCA-
GAATTAATCAATAAACGTAGG)
and downstream(AGGAA-
GACGTTCCTAGAGAAT)
oligonucleotides (Figure 2), and with the upstream and Tcl(TCAAGTCAAATGGATGCTT-
GAG)
oligonucleotides as primers. The cdc-42(pk57)Tcl
inser- tion is oriented with its reading frame in the same direction as for cdc-42, and the Tcl oligonucleotide corresponds tothe base pairs
74
to 53 of Tcl (ROSENZWEIG et al. 1983).Amplifications were performed in a Perkin Elmer Cetus Gene- A m p PCR System 9600, using 30 cycles of 94” for 15 sec, 58” for 30 sec, and
74”
for either 50 sec (upstream and Tcloligonucleotides) or 2 min (upstream and downstream oligo- nucleotides).
Observation of gonadal defects in sup39 animals: For ob-
servation of gonadal defects, animals were stained with the nuclear fluorescent dye diaminophenylindole
(DAF’I)
usingthe Xgal staining solution without Xgal
(FIRE
et al. 1990; Table6). Gonads of sup39(je5)/let-22, sup-39/ mnDf30, and sup-J9/
m n D f l 0 5 hermaphrodites were also examined. These hetero- zygotes were constructed as described above for observations
of embryonic lethality.
A
m n D f l O 5 u n c - 4 / m n C l strain displays a gonadal defect reminiscent of sup39 mutants: emergence of cells from the gonad syncitium is delayed, and 10% of gonads ( n = 30) show two rows of oocytes.A
mnDflO5/sup-3 9 ( j e 5 ) strain shows an enhanced frequency of gonads with
two rows of oocytes (Table 6). This may result from an additive effect of two distinct defects; other more complicated explana- tions are possible. Until the location of s u p 3 9 relative to the endpoints of mnDf30 and m n D f l 0 5 is determined (which will
presumably require molecular analysis), embryonic lethality or gonad structure phenotypes in these heterozygotes cannot be strongly interpreted.
RESULTS
unc-73 Suppressors 231
TABLE 6
sup39 mutants show two rows of oocytes
Genotype
Wild type (N2) unc-73(e936) sup39(je5) sup39(je6) unc-73; sup39(je5) unc-73; sup-39(je6)
dpy-10
+
sup-?9(je5) unc-4/+ unc-104+ +
sup-39 unc-4/sup39 une-4 dpy-IO unc-4/sup39 unc-4 let-22 unc-4/sup39 unc-4 mnDf30 unc-4/sup-39 unc-4 mnDflO5 unc-4/sup39 unc-4 let-22 unc-4/+ unc-4 mnDf30 unc-4/
+-
unc-4 mnDflO5 unc-4/+
unc-4Percent of gonads showing two or more rows of oocytes
0% (72) 1.6% (122)“ 8% (50) 32% (66) 10% (48) 3% (60)
6% (50)
15% (67) 3% (32) 6% (69) 5% (60) 35% (40)
0% (50) 10% (30) 0% (57)
Gravid adults and adults large enough to contain fertilized eggs were scored. These samples may be biased by the fact that grossly abnormal animals were not picked, and some preference was shown for animals containing fertilized eggs, which might lead to an underestimate of the frequency of gonadal defects.
“For the two examples of multiple-rowed gonads in unc-
73, the gonads were severely contorted, showing several extra bends. This is not the case for gonads with multiple rows of oocytes in the other mutant strains. Strains were constructed
as described in M A T E R N S AND METHODS. Number of gonads scored are in parentheses.
After a standard
EMS
mutagenesis ofunc-73(e936),
10 independent non-Unc revertants (allelesjel-&IO)
were isolated from -250,000 independently mutagenized chromosome sets (MATERIALS AND METHODS)(BRENNER
1974). This frequency of mutant isolation is much less than that for isolation of loss-of-function mutations in a gene of average size(BRENNER
1974),so
these muta- tions may be selected to retain gene activity.unc-73(e936)
animals show a coiler phenotype and little spontaneous activity at all stages of development. Animals carrylngunc-73(e936)
and the mutationsj e l ,
je2,
j e 3 ,je5, je6, orje7
have a movement phenotype thatis indistinguishable from
unc-73(+)
animals at all stages of development (Figure2;
data not shown). Allunc-
73(e936)
animals carrying these suppressor mutations show wild-type degrees of spontaneous movement, as well as a wild-type degree of body curvature during sinu- soidal movement. Animals carryingunc-73(e936)
and the mutations je4, je8,je9,
orjelO show an intermediate movement phenotype.The position of each reverting mutation relative to
unc-73(e936)
was mapped. The allelesj e l , je2, je4,
andje7-IO
all map to chromosome Z and have proven to be inseparable fromunc-73fe936)
by recombination(MATERIALS AND METHODS). These are probably intra- genic mutations that partially or completely restore
unc-
73
function. Another mutation, je3, is a putative intra-genic dominant suppressor and recessive lethal. Two mutations,
je5
andje6,
are extragenic suppressors and both map to the center of chromosomeI1
(see below). Only je3,je5
andje6
were studied further.j e 3 is a dominant suppressor of
e936,
has a linked recessive lethal phenotype, and appears to be in theunc-73
gene itself (MATERIALS AND METHODS).unc-73/je?
e936)/unc-73(e936)
heterozygotes move like wild-type animals and segregate heterozygotes,unc-7?(e936)
ho- mozygotes, and arrested L1 larvae that are presumablyunc-73Cje3 e936)
homozygotes. (The order ofj e 3
ande936
is shown arbitrarily and has not been determined.) Linkage analysis placed j e 3 on chromosomeI
(seeMATERIALS AND METHODS). Four-factor crosses between
unc-11,
je3,e936,
anddpy-5
failed to separate the domi- nant suppressor or recessive lethal properties of je? frome936.
Also, theevEx38
element, which contains an injected cosmid that rescuesunc-73fe936)
(R.
STEVEN
andJ.
CULOTTI, unpublished results), rescues the lethal- ity of je? (MATERIALS AND METHODS). Homozygousunc-
73(je3
e936) animals hatch and crawl about for a few hours, but do not seem to develop postembryonically. Extragenic suppressors ofurn-73:
Theje5
andje6mu- tations are dominant suppressors ofunc-73(e936)
that map to chromosome/ I
and appear to define a single gene, sup-39.unc-73(e936)
larvae and adults carrying eitherje5
orje6
move in a coordinated manner and cannot reliably be distinguished from wild-type C. eleg-ans
with a dissecting microscope (Figure2).
je5
was mapped between dpy-loandlin-26,
within-0.02
cM ofunc-104
(Table2),
and has not been separated fromje6
by recombination.sup-39
mutations do not cause uncoordination when separated fromunc-73(e936),
but do induce other abnormalities described below.The suppression of
unc-73
byje5
is specific to thee936
allele.je5
does not suppress therh40, m454:: Tcl,
or
a509
alleles ofunc-73
(MATERULS AND METHODS).With respect to movement, some of these alleles are equally or less severe than
e936
(Figure2),
indicating that sup39 mutations are not simply suppressing the weakest allele of an allelic series.sup39 mutations suppress some but not all of the phenotypes caused by
unc-73(e936). unc73(e936)
males do not mate and have defective copulatory spicules(HODGKIN
1983).unc-73(e936j;sup-39Cje5)
males mate at nearly normal efficiency and have normal copulatory spicules.sup39(je5)
does not appear to suppress the appearance of secondary vulval blips or ectopicmec-3
expressing cells (Table
3 ) ,
which are presumably due to lineage alterations that may derive from an asymmet- ric cell division defect (WAY et al. 1992). Inunc-73
mu- tant animals, the posterior migration of theALM
cell is shortened so that this cell is anteriorly mispositioned, and the anterior migration ofQ R
and its daughters is often incomplete (HEDGECOCK et al. 1987).sup-39(je5)
232 J.-Q. Run et al,
Phenotypes of
sup39
mutants:sup39
mutations cause additional defects independent ofunc-73.
In an asynchronous population of animals, -66% ofje5
em- bryos( n
= 524) and 45% ofje6
embryos( n
= 876) arrest without hatching. Of the eggs that hatch, >90% develop to the L4/adult stage( n
= 1065 forje5,
1609 forjes).
Under the same conditions, 95% of wild-type eggs( n
= 257), 94% ofunc-73(e936)
eggs( n
=77),
and 46% ofunc-73(e936);sup39
(je5)
eggs hatched( n
=70).
The first embryos laid by
sup-39
mutant animals have a very poor survival rate (<15%), which increases as the mother animal ages and lays more eggs (Table4).
Embryonic lethality due to
sup-39is
a function of the mother's genotype and not that of the father or the zygote. Whensup39(ie5)
mutant fathers are mated to wild-type mothers, no embryonic lethality is observed, but when wild-type fathers are mated withsup39(je5)
mutant mothers, embryonic lethality is seen at the same frequency as from self-fertilization by mutant hermaph- rodites or when both parents are mutant (Table 5).
Rare
sup39(je5)
and(je6)
animals (<1
%)
show a Roller or Dpyish phenotype. We therefore tested whether, like other rol and dpy mutations(COX
etaL
1980),je5
can suppress the Blistered phenotype ofbli-1.
Of the 376unc-
73(e936);sup39(je5)
bli-1
animals, 55 showed a Bli p h e n e type as adults, while 22/22sup39(+) bli-1
and 74/74unc-
73;bli-1
animals showed theBli
phenotype.Phenotypes caused by
sup-39
mutations are not strongly temperature dependent. Suppression of theunc-73(e936)
movement defect is equally good at 15, 20, and 25". In addition,je5
andje6
mutant strains can be continuously propagated at 15 or 25" for several generations.Dominance
of
existingsup39
alleles:sup-?9(je5)
is dominant or semidominant for all phenotypes tested.unc-73(e936);sup-39(je5)/
+
animals show a range of movement phenotypes: some are completely wild type, while others are detectably uncoordinated but always move better thatunc-73(e936);sup-39(+).
Males of this genotype can mate.sup-39(je5)
is also dominant or semidominant for phenotypes it induces on its own.sup-39( je5)/
+
animals have progeny that suffer -50% lethality as embryos (Table 5). From a+
supXJ(je5) unc-4/dpy-l0
+ +
het- erozygote, Unc, wild-type, and Dpy offspring are pro- duced in a 1:2:1 ratio, consistent with the idea that the embryonic lethality is entirely a dominant maternal effect and is independent of the zygotic genotype.(A
dpy-10
+/+
unc-4
strain control strain showed no sig- nificant embryonic lethality.) In addition,sup39( je5)
causes a gonadal defect (see below) that is dominant and is observed in a dpy-10
+
sup-39 unc-4/+ unc-104
+
+
strain in which most of the original, mutagenizedDNA
flanking thesup-39
mutation has been crossed away (Table 6), suggesting that all the phenotypes are due to the same mutation.mnDf30
andmnDflO5
are deficiencies in the region ofsup-39.
Recombinational mapping data placessup-39
near the endpoints of m d f 3 0 and
mnDfl05
(Figure1;
Table 2). It is most likely that there is an undeletable region between these deficiencies, in which
sup-39
may lie(HERMAN
1978; STARICH et al. 1995). When sup-39(je5)
was made heterozygous withmnDf30
andmnDfl05,
no reduction or enhancement of' embryonic lethality was observed relative tosup-39(je5)/+
hetero- zygotes ( M A T E W S AND METHODS). This result is consis- tent with the idea thatsup39(je5)
is a neomorphic mu- tation whose phenotypic effects are not influenced by the presence ofsup?9(+),
or that this mutation causes an extreme gain-of-function that is not significantly en- hanced by a single copy of sup-39(+ ). Alternatively, sup- 39 may not lie under either of these deficiencies.Several attempts to isolate intragenic recessive, loss- of-function revertants of
sup-39(je5)
failed. Anunc-73
(e936);sup39 (je5)/mnCl
strain(mnC1
is a balancer for chromosomeZt
HERMAN
1978) was mutagenized with EMS(BRENNER
1974), and9000
F, animals were screened for the Unc-73 phenotype. One such animal was isolated, but when the non-mnCI chromosome was homozygosed, no lethality or other mutant phenotypes were observed. It is possible thatsup-39 (null)
mutations have a wild-type phenotype, or that in this mutant hunt, DNA damage at theje5 site was repaired by a recombina- tional repair with the balancer chromosome, but with- out affecting flanking markers(e.g.,
by the double- strand break repair mechanism of SZOSTAK et al. 1983). Alternatively, sincesup-39
maps near a region of possi- ble haplo-insufficiency, this gene may itself be haplo- insufficient, and so only wild-type revertants can be identified.Evidence that sup39 mutations
are
not amber or smg suppressors: Thesup-39(
je5) mutation is dominant and allele-specific; these properties are consistent with the idea thatje5 is an amber suppressor or some other kind of informational suppressor. However, theunc-73(e936)
mutation is not suppressible by the C.
elegans
amber suppressorsup7(st5)
or by the suppressorssmg-3,
m g -
4,
orsmg-6
(MATERIALS AND METHODS). Other data also suggest that theje5
mutation is dissimilar to known C.elegans
amber suppressors.je5
fails to suppress the amber mutationsunc-24(e1?8)
andunc-51(e369).
TheC.
elegans
amber suppressorssup-5
andsup-7
are heat sensitive for suppression and cause a cold sensitivity for growth;je5
has neither of these properties. Other properties ofje5,
such as the occasional Roller/mis- shapen body phenotypes or maternal-effect lethality, are not characteristic ofC.
elegans
amber suppressors(HODCKIN
1985).Another class of allele-specific, gene-nonspecific sup- pressors in C.
elegans
are the Smg suppressors, which stabilize unstable mRNAs(HODGKIN
et al. 1989). The smgmutations are generally recessive and cause a defect in male genitalia that prevents male mating.sup-?9@5)
males mate well, and the
je5
mutation suppresses the male mating defect ofunc-73(e936).
The existing smgunc-73
Suppressors
233tation is
dpy-5(e61),
whose phenotypic expression is un-
affected in a
dpy-5(eSl);sup39(je5)
strain. These results
argue that suppression by
sup39
is unrelated to the
smg
suppressors.
These data are
most consistent with the idea that
sup
39
is specific to
unc-73(e936)
(and possibly to the
bli
mutants). It is possible that
sup39
is a novel type of
allele-specific, gene-nonspecific informational suppres-
sor. This possibility cannot be evaluated until the gene
is cloned and the molecular mechanism of suppression
examined. However, in the course of various strain con-
structions,
sup39Cje5)
has been combined
with
unc-
73(m509), unc-73(rh40), unc-73(m454::
Tcl), d&-5(e51),
dpy-1 O(el28), dpy-11 (e224), dpy-l3(el84), dpy-l7(el64),
lon-2(e678),
lin-l7(n671),
and
unc-4(e120).
None of these
mutations were suppressed.
In addition,
as
noted above,
sup39cje5)
fails to suppress
the ALM posterior migration defect and lineage defect
that results in ectopic mc-Axpressing cells and vulval
protrusions. These observations suggest that
sup39(je5)
does not simply restore the UNG73 protein to a wild-
type state by an informational suppression mechanism.
sup39 mutations
are not alleles of cda42:unc-73
of
C.
elegans
shares a region of similarity
to
CDC24
of yeast,
and a
cdc24ts
mutation can be suppressed by increased
gene dosage of
CDC42.
We therefore cloned a
C. elegans
Cdc42 homologue
(FIGURE3;
MATERIALS AND METHODS;CHEN
et al.
1993) from wild-type and
sup39
mutant
strains to examine whether
je5
and
je6
might be gain-
of-function mutations in the
C.
elegans cdc-42
gene.
The
C. elegans cdc-42
gene lies on the cosmid M04B9,
which is 40 kb to the left of
lin-26
and
-500
kb to the
right of
unc-104
in the center of chromosome
II.
The
data of Table
2
indicate that
je5
maps to
unc-104, -0.3
map units to the left of
lin-26.
Using a Tcl insertion
upstream of
cdc-42
asa DNA polymorphism, it was possi-
ble to identify recombination events between
sup39( je5)
and
cdc-42,
such that sup39maps -0.25 units to the left
of
cdc42
(MATERIALS AND METHODS;Table
2).
Moreover,
for
sup39(+), sup39(je5),
and
sup39(je6),
we deter-
mined the sequence of the
cdc-42
coding regions, in-
trons, and 500 bp of 5’ and 3’ flanking material, and
found no alterations. Taken together, these results sug-
gest that
sup39
and
cdc42
are distinct genes.
A
gonadal defectin
mp39(je5) and sup39(je6) mu-
tants:
That the embryonic lethality in
je5
mutant ani-
mals is due to a maternal effect suggests a defect in the
germline of this mutant. We found that
sup39( je5)
and
sup39(je6)
animals show a novel gonadal defects. In
a nonmutant adult
C.
elegans
hermaphrodite, the two
gonads each consist of a tube with a proximal and distal
arm, separated by a bend (Figure 4). The germ-line
within the distal arm is a syncitium of a few thousand
nuclei within a single large plasma membrane. At the
bend, nuclei emerge from the syncitium, are encapsu-
lated by plasma membrane to form mononuclear cells,
and form a single row of oocytes (HIRSH
et al.
1976).
In
je5
and
je6
mutant animals, two rows of oocytes
within a gonad arm are often seen (Figure 4; Table 6).
The two rows appear to form as nuclei exit the gonad
syncitium; formation of two oocyte rows from a single
row within the proximal gonad is not seen. Observation
of animals stained with DAF’I indicates that nuclei in
both rows are arrested
in meiosis I with condensed chro-
mosomes (the normal arrest point for
C.
elegans
oo-cytes). Observation with Nomarski optics indicates that
nuclei in both rows are surrounded by a plasma mem-
brane; multinucleate oocytes are not seen. In addition,
in
je5
and je6mutant
animals, the gonad
syncitium often
persists around the turn
of the gonad
and nuclei
emerge in the proximal arm. However, the gonads of
je5
and
je6
animals generally appear to be slightly
shorter than in wild type. The distal tip cell at the end
of the distal gonad arm secretes a signal to nuclei in
the syncitium that represses their entry into
meiosis
(KIMBLEand WHITE 1981). It may be that the
total
linear distance from the distal tip cell to the site of
nuclear emergence is the same in
sup39
mutants and
wild-type
C.
elegans.
sup39( je5)
and the deficiency
mdflO5
show a syner-
gistic interaction with respect to gonadal abnormalities.
In
mnDflO5/sup39(+)
animals,
as
in
sup39(je5),
nuclei
emerge from the gonad syncitium in the proximal gonad
arm, and some gonads have two rows of oocytes (Table
6).
In
mdfl05/sup39(je5)animals,
these phenotypes are
pronounced (Table 6). Since it is unclear whether
sup
39
lies within the region deleted by
mdfl05,
we cannot
say whether the enhancement of the two-rowed gonadal
phenotype is an additive effect of
two distinct lesions
or an interaction between
sup39(je5)
and
sup39(null).
mdfl05,
when heterozygous,
does not suppress
unc-
73(e936)
(MATERIALS ANDMETHODS).
We tested whether the presence of two rows of oocytes
could contribute
tothe maternaleffect
embryonic lethal-
ity seen in
sup39
animals. The first-laid eggs from
sup
39
animals with normal gonads and with gonad having
two rows of oocytes were picked to a separate plate and
observed, and the parent animals were then stained with
the DNA-binding fluorescent dye DAPI and their gonads
were examined. Regardless of the gonadal morphology
of the mother animal, all embryos had a poor chance
of survival (Table
7).
Since a significant embryonic le-
thality is observed in animals with normal gonadal mor-
phology, this lethality must have some other cause.
DISCUSSION
unc-73
mutants exhibit defects in axon guidance, cell
migration, and generation of certain cell types (HEDGE-
COCKet al.
1987; SIDDIQUI
and CULOTTI 1991; MCINTIRE
et
aL1992; WAY
et aL1992), and the UNG73
protein
has
a region of similarity to Cdc24 of yeast, a cell polarity
protein that is required for bud emergence, bud place-
ment and mating process extension (R. STEVEN,
A. RUIZ,
J.
MANCILLAS, and
J. CULOTTI,
unpublished results;
FIELDand SCHEKMAN
1980; MIYAMOTO
et
al.
1987; RON
et al.234 J.Q. Run d nl.
c
I
F I C ~ I K E 4."<>ocytc formation i n sup-39(+) and s~rp-39(jr5) anilnals. Anterior is t o the left. Magnification is 2000-fold. Animals were stained with as described in MATERIALS A N D MI:.THOI)S. (A) Posterior gonad from wild-type
c.
d q y n s . Arrows indicate nuclei that have emerged from the gonad spcitium, which is located dorsally. (E) Posterior gonad of sz$~-3Y(je5). Upward arrows indicate one row of nuclei that has emerged from the syncitium, and the downward arrows indicate a second row.events, including guided migrations, segregation of de- velopmental potential, orientation of cell division, and perhaps polarized secretion (WAY et nl.
1994).
To identify additional genes that might function with unc-73, we isolated mutations that suppress the uncoor- dinated phenotype of unc-73(~936). The unc-731e936) mutation is recessive and most likely a partial loss-of- function allele: unc-73 mutations with an Unc but healthy phenotype are identified more rarely than null mutations in other genes (MCINTIRE el nl.
1992),
even though unc-73 should be a large mutational target, and semilethal allelesof
unc-73 have been isolated (W. FOR- RESTER andG.
GARRIGA, personal communication). Three classes of revertants were identified: intragenic suppressors, an intragenic dominant suppressor with a linked recessive lethal phenotype, andtwo
mutations ina
second gene, sup-39.sup39 mutations completely suppress the movement defect of unc-73(e936), as well as the defect in male mating. The low-penetrance lineage defects of unc-73, which result in ectopic vulval protrusions and extra
ma-
Axpressing cells, are not suppressed by sup-39. In addi- tion, suppression of unc-73 is completely allele-specific: phenotypes of other unc-73 mutations, even weaker
al-
leles, are not detectably improved by the presence of a sup39 mutation. These results suggest that the unc-73 and sup39products function together and that suppres- sion of uno73 by sup39is not due to an indirect bypass, e g . , an animal-wide effect suchas
changing the extracel- lular matrix to allow easier axon outgrowth.unc-73 Suppressors
TABLE 7
Embryonic lethality is not due to the presence of a two-rowed gonad in the mother animal
Percent survival of embryos from young mothers
235
With no two-rowed gonads With one two-rowed gonad
sup39Cje5) 24.3% ( n = 8 mothers, 111 embryos) 26.4% ( n = 6 mothers, 87 embryos)
sup39(je6) 1.0% ( n = 7 mothers, 212 embryos) 1.5% ( n = 5 mothers, 138 embryos)
Individual L4 animals were picked to several plates and observed until they had laid up to 30 eggs, at which point the mother was removed, fixed, and stained with DAPI, and the embryos were picked to one region of the plate where they could be monitored. From the sup39(je6) mothers with one two-rowed gonad, 16% of the embryos produced were small and misshapen; similar embryos were observed only rarely from sup39(je6)
animals with normal gonads or from sup39(je5) mothers. The survival rate of embryos shown here is particularly low because they are the first embryos produced (see Table 4).
here indicate that sup-39is not an amber or smgsuppres- sor, which are the two known types of informational suppressors in C. elegans. In addition, the sup-39 muta- tions do not suppress the lineage defects seen in an unc-73 mutant (Table
3),
suggesting that the unc-73 product is not simply being restored to a wild-type state. It is possible that the sup39mutations represent a novel type of informational suppression, but this question can only be answered when the molecular nature of the sup-39 product is known.It was not possible to address whether sup-39 muta- tions are hypermorphic, antimorphic, or haplo-insuffi- cient by examination of deficiency heterozygotes, be- cause this gene maps in or near an undeleted region in the center of chromosome
II
(Table2;
HERMAN 1978; STARICH et al. 1995). However, it seems likely that the sup39 mutations identified here are selected to retain gene function, because their frequency of isolation is only once per 125,000 mutagenized chromosome sets(RESULTS), while the frequency of loss-of-function muta- tions in a typical gene is about once per 2000 mutagen- ized chromosome sets
(BRENNER
1974). It thus seems most likely that sup-39 mutations are dominant because they either restore a protein-protein interaction, cause a gain of function in the sup-39 product, or turn this product into a poison. It may be that the e9?6mutation of unc-73 disrupts a contact between the sup-39and unc-73
products, and that the sup-?9 mutations described here restore this contact. Alternatively, it may be that the e936 mutation causes a loss of function in a particu- lar domain within the large UNC-73 protein, that otherunc-73 mutations affect different domains, and that al- tered sup-39 activity can suppress a loss of activity in only one of several unc-73 functions.
sup-39mutations have additional phenotypes that are independent of unc-73. Animals mutant for sup39 have an incompletely penetrant gonadal defect, a maternal- effect embryonic lethality, a low-penetrance Roller/ Dumpy phenotype, and show partial suppression of bli-
l(e769). In the gonad of a wild-type C. elegans, oocytes are formed as nuclei emerge from a large syncitium
( HIRSH et al. 1976). Normally the oocyte nuclei form a single row, suggesting that a single “nuclear emergence site” is identified on the syncitial surface. In sup-39mu-
tant animals, there are often two rows of oocyte nuclei that appear to come from two adjacent sites on the end of the gonad syncitium (a “two-rowed” gonad; RESULTS;
Table 6; Figure 4 ) .
One speculative interpretation of the “two-rowed’’ gonads of sup-39 animals can be based on the sequence similarity between the C. elegans UNC-73 and yeast Cdc24 proteins. Both bud formation in
S.
cerevisiae and oocyte emergence in C. elegans involve the choice of a single site on a plasma membrane. Loss-of-function mutations in the Cdc24/Cdc42 pathway will prevent bud formation because of a failure to establish a single site on the plasma membrane where secretory vesicles will fuse(FIELD
andSCHEKMAN
1980; ADAMS et al. 1990). Conversely, yeast cells with increased Cdc24/Cdc42 function will choose a bud site randomly on the cell surface, and cells with an extreme overdose of Cdc24/ Cdc42 function have multiple buds (JOHNSON and PRINGLE 1990;ZIMAN
etal.
1991). The unc-7?(e9?6) mu- tation most likely causes a decrease in the activity of an exchange factor/Gprotein pathway similar to Cdc24/ Cdc42. The sup?9 alleles described here may cause a gain of function in such a pathway. According to this idea, the “two-rowed’’ gonads could be analogous to a multiply-budded yeast cell.An unusual aspect of the maternal-effect lethality of sup-39 alleles is that its severity decreases at the mother animal ages; the older the mother is, the greater the probability that an embryo will survive (Table 4). This result could be explained by the following hypothesis. It may be that, during gonadogenesis, the sup39 product is first synthesized and used when an oocyte emergence site is chosen on the gonad syncitium. This sup39 prod- uct would then decay before fertilization, so that re- maining product does not interfere with embryogenesis. In the sup-39 mutants identified here, the amount or activity of the sup39product would be increased, so that this product would be present at fertilization and early embryogenesis, where it would be toxic. Eventually, the mutant sup39 product generated during gonadogenesis would decay, so that embryos produced by an older mother would not suffer such toxicity.