of unc-54. The mutants have no visible phenotype, and their viabilities and ... The following genes and alleles were used: dpy-5 (e61) I, unc-13 (e51) I, unc-54 ...
Copyright 0 1986 by the Genetics Society of America
@-GLUCURONIDASE MUTANTS OF THE NEMATODE CAENORHABDITIS ELEGANS MARISA SEBASTIANO, MARINA D’ALESSIO AND PAOLO BAZZICALUPO
International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, via G . Marconi 10, 80125 Napoli, Italy Manuscript received June 17, 1985 Revised copy accepted October 28, 1985
ABSTRACT Using a screening procedure that is based on a histochemical stain for the enzyme @-glucuronidase,we have isolated several mutants of t h e nematode Caenorhabditis elegans affected in @-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped o n the right arm of linkage group I (LG I), 1.1 map units to t h e left of unc-54. T h e mutants have n o visible phenotype, and their viabilities a n d fertilities a r e unaffected. Linked revertants of two of the mutations have been isolated. T h e y restore enzyme activity to almost wild-type levels; the @-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for @-glucuronidase.
T
HE free-living nematode Caenorhabditis elegans is an important model
organism for the study of complex biological phenomena such as metazoan development and behavior. T h e knowledge of the development of this organism at the cellular level, the possibility of mutational and microsurgical manipulation of its development and the application of recombinant DNA technology to its study have made C. elegans attractive to biologists who hope to get an integrated picture of its development. However, the description of the biochemical mechanisms involved in cell and tissue differentiation is, in general, more primitive than that available for model organisms, such as Drosophila o r mammals. Although a few biochemical mutants have already been identified in C. elegans (reviewed by SIDDIQUIand VON EHRENSTEIN1980; see also SANFORD,GOLOMBand RIDDLE1983; RANDand RUSSELL1984), more such mutants would clearly be useful. In a preliminary search, we investigated the presence and tissue distribution of several enzymes in C. elegans using histochemical techniques. We then focused on ,&glucuronidase, which cleaves the glycosidic bond between D-glucuronic acid and a variety of moieties. During C. elegans development, p-glucuronidase seems to be subject to various types of regulation (M. SEBASTIANO, M. D’ALESSIOand P. BAZZICALUPO, unpublished results; N. WOLF and D. HIRSH,personal communication). T h e enzyme is not detectable in early emGenetics 112: 459-468 March, 1986.
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bryos, first appearing around the 250-cell stage in the cells destined to become the embryonic gut. Somewhat later, pharyngeal cells also start to produce it. During postembryonic life, intestinal cells show very little o r no enzyme activity, and a limited number of cells of the pharynx seem to become the main site of enzyme production. When sexual maturity is reached, the hermaphrodite vulva and male tail also contain @-glucuronidase. T h e study of the factors leading to this spatial and temporal pattern of synthesis is interesting and may help us to understand cell-specific regulation of gene expression. @-Glucuronidase is also attractive because it has been studied biochemically in a great variety of organisms, ranging from bacteria to mammals (TOMINO et al. 1975; NOVELand NOVEL1976; LANGLEYet al. 1983), so that many technical approaches to its study have been worked out. Finally, the study of naturally occurring genetic variants of this enzyme in mice has led to the formulation of interesting models of regulation of gene expression in eukaryotes (PAIGEN 1979). In this paper, we report the isolation and genetic characterization of C. elegans mutants affected in ,&glucuronidase activity. T h e enzymes produced by wild-type and mutant strains have been biochemically analyzed. T h e mutants we have isolated have already been used as recipients in experiments attempting the transformation of C. elegans with the E. coli /3-glucuronidase gene, by itself o r fused to the regulatory regions of nematode genes (R. JEFFERSON, S. BURGESS, N. WOLF and D. HIRSH, personal communication; M. KLASS, personal communication). T h e approach and the procedures used for the study of &glucuronidase should be applicable to the study of several other enzymes in C. eleguns. MATERIALS AND METHODS Chemicals: 4-Methylumbelliferil-@-bglucuronide,naphthol AS-BI P-D-glucuronide, pararosanilin, glucaro-l,4-lactone and glucuronic acid lactone were from Sigma Chemical Company, St. Louis, Missouri. Nematodes: A11 nematodes were grown at 20 It 1.5" on NGM agar plates seeded with E. coli according to BRENNER (1974); chicken-egg plates were prepared by dispersing the yolk of one chicken egg in 50 ml of Ty broth (tryptone 10 g, yeast extract 5 g, NaCl 8 g/liter). The suspension was brought to a boil for 3-4 min, homogenized with a Sorvall Omnimixer to a fine slurry and poured onto 12 90-mm Petri plates personal communicontaining NGM solid medium (D. BAILLIEand R. ROSENBLUTH, cation). After drying overnight, the plates were used for mass growth of worms to purify enzymes. The following genes and alleles were used: dpy-5 ( e 6 1 ) I , unc-13 ( e 5 1 ) I , unc-54 ( e 1 9 0 ) I , let (7202) I, lev-11 ( x 1 2 ) I , let-49 (st44) I , dpy-IO ( e 1 2 8 ) 11, unc-32 ( e 1 8 9 ) 11, d p y - I 3 ( e I 8 4 ) IV, unc-42 ( e 2 7 0 ) V , him-5 ( e 1 4 9 0 ) V and dpy-3 ( e 2 7 ) X. In addition, the following deficiencies, all located on LG I, were used: eDf3, eDf4, eDflO, e D f l 1 , e D f l 3 and e D f l 5 . All have been described by SWANSON, EDCLEYand RIDDLE(1384). The wild-type progenitor strain is N2. The mutations b 4 0 5 , 6 4 0 6 , b 4 0 7 , b408, b409 and b410 (isolated in D. HIRSH'Slaboratory in Boulder, Colorado) as well as g b 2 5 , gb29, gb32, gb44 and gb85 (isolated in Naples) reduce @-glucuronidaseactivity. gb94 and g b l 7 3 , which are revertants of 6 4 1 0 , and g b 8 5 1 , which is a revertant of gb85, were isolated in Naples. Mutagenesis and screening: Worms to be mutagenized were synchronized, and ethyl methanesulfonate (EMS) was used as mutagen according to BRENNER (1974). T o look
C. ELECANS GLUCURONIDASE MUTANTS
46 1
for /?-glucuronidase minus mutants, 25-50 mutagenized worms were placed on each of a series of 90-mm Petri plates. After enough progeny (-1000/plate) had reached adulthood, gravid worms were counted, and F2 eggs were prepared from them by treating KLASSand HIRSH1979). These F2 the worms with Na hypoclorite and KOH (EMMONS, animals were screened when they grew and became gravid. The mutation frequency was calculated from the number of gravid F1 animals. An average of 15-20 viable embryos per parent can be recovered by the hypoclorite method if the F1 animals are starved slightly. Not more than one mutant from each plate was kept. The frequency of reversion was based on a rougher estimate of the number of F1 animals analyzed, since more worms had to be screened. The screening procedure was based on the coupled reaction described by HAYASHI, NAKAJIMA and FISHMAN (1964). Naphthol AS-BI /?-D-gIucuronideis a substrate for /?glucuronidase; the enzyme liberates naphthol from the substrate, and the free naphthol reacts with p-rosanilin hexazonium salt, forming a bright red precipitate at the reaction site. Worms were collected from the plates, rinsed from bacteria and other debris by centrifugation and, then, resuspended in 4-5 vol of 0.6% SDS in H 2 0 for 5 min at room temperature. After washing thoroughly from the detergent, the worms were incubated at 20" for 2-3 hr in a freshly prepared solution containing 0.1 M Na acetate, pH 4.9, 0.25 mM naphthol AS-BI /?-D-glucuronideand 1.8 mM p-rosanilin hexazonium salt. Then, worms were screened under a stereomicroscope after diluting the reaction mixture in M9 buffer and pouring it onto a 60-mm agar plate. Wild-type worms show a cherry red stain mostly at the pharynx and at the vulva, whereas mutants remain colorless. Mutant candidates were picked with a platinum wire and were deposited on a new plate seeded with bacteria. The embryos that they contained hatched, crawled out of the mother and grew to adults. This procedure was used also in scoring genetic crosses. When few worms were to be checked, a wire or a micropipette was used with the aid of a microscope to transfer worms from one solution to the next. In looking for revertants, worms were screened much more rapidly because the red worms stood out more clearly among the mass of colorless ones. Enzyme assay: &Glucuronidase was assayed using 4-methylumbelliferil /?-D-glUCUrOnide as fluorogenic substrate. The 200 PI reaction mixture contained 0.4 mM substrate, 0.1 M Na acetate, pH 4.9, and various amounts of enzyme. Incubation was usually for 1 hr at 20". After incubation the mixture was brought to 1 ml with 0.2 M Na&Os, and the amount of 4-methylumbelliferil liberated was calculated by reading the mixture in a Turner Fluorimeter with excitation light of 365 nm and emission at 445 nm. Activity units were arbitrary; one unit corresponds to the amount of enzyme that in 1 hr at 20", pH 4.9. hydrolyzes 225 nmol of 4-methylumbelliferil-/?-~-glucuronide Extracts: T o measure the specific activity of /?-glucuronidase in various strains, 15,000-20,000 worms were synchronized as dauer larvae and were then fed for 2430 hr. Worms were harvested by washing them from plates and were cleaned by flotation on 35% sucrose. After removal of sucrose, they were resuspended in 0.5 ml of 10 mM Tris-HCI, pH 7.5, and disrupted by sonication. The suspension was brought to 0.2% Triton, allowed to stand for 30 min at 4" and then centrifuged at 12,000 X g for 10 min. The supernatant was assayed as described. Protein concentration was determined using the Bio-Rad protein assay with bovine serum albumin as standard. Enzyme purification: Approximately 5 g of worms were cleaned by flotation on sucrose, were disrupted by passing them three times through a French pressure cell at 12,000 p.s.i. and all the forms of the enzyme were solubilized by incubation with 0.2% unpublished results). Debris and deoxycholate (M. D'ALESSIOand P. BAZZICALUPO, insoluble material were separated by centrifugation at 100,000 X g for 3 hr. From the supernatant, which is the crude extract, the enzyme was purified by (1) acid precipitation in sodium acetate, pH 4.5, (2) ammonium sulphate (45-80%) fractionation, (3) size fractionation on a Sephadex G200 column and (4) ion exchange chromatography on a DEAE cellulose column. The specific activities of the peak fractions from the DEAE
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M. D’ALESSIO
AND P. BAZZICALUPO
column were between 300- and 500-fold higher than that of the crude extract, depending on the preparation. These fractions were used for the characterization of the enzyme from the different strains.
RESULTS
Mutant isolation: T h e histochemical method of HAYASHI,NAKAJIMAand FISHMAN(1964) uses the combination of a naphthol-@-D-glucuronide as substrate and $-rosanilin as a stain to localize @-glucuronidaseactivity in cells and tissues. We have found conditions that allow the use of this reaction to screen for C . elegans mutants with very low o r no @-glucuronidaseactivity. Animals are killed by the cuticle permeabilization step of our staining procedure. Therefore, we developed conditions for staining the worms which allowed us to recover viable progeny of stained animals. Our method takes advantage of the fact that C. elegans hermaphrodites retain their fertilized eggs in utero before laying them. T h e eggs are protected from the environment by a vitelline membrane and a chitinous shell; we have found that a short treatment with the ionic detergent sodium dodecyl sulfate (SDS), although killing the adult, permeabilizes worms without inactivating the enzyme and without killing the embryos. Worms to be screened are permeabilized with SDS, incubated in the presence of a mixture of substrate and stain and then observed under a stereomicroscope. Wild-type worms display red stain at the pharynx, vulva and tail. Carcasses of nonstaining hermaphrodites are picked and placed on a new plate; the embryos in utero develop and grow to adults that can be tested again. This method has proved very efficient. In reconstruction experiments, two unstained but otherwise equally treated worms were added to about 1000 stained ones. We always were able to find the unstained worms after 30-45 min. In addition, the number of false positives after the first screen was quite low and was not a problem in this work. Using EMS as a mutagen, we isolated 11 independent mutants that showed no red staining. T h e mutations have been designated b405 through b410 and gb25, gb29, gb32, gb44 and gb85. In principle, the lack of red color could be explained by a lack of enzyme activity or by some trivial reason; for instance, a decreased or increased sensitivity to SDS of the cuticle o r enzyme. However, in extracts from all the mutants, @-glucuronidaseactivity was extremely reduced or undetectable, being less than 0.2% that of wild type (Table 1). Other enzymes such as a- and pglucosidase, a- and P-galactosidase and a-fucosidase were fully active in all the extracts (data not shown). It is clear, therefore, that the screening procedure has led to the identification of mutants affecting @-glucuronidaseactivity and that this is the reason for the lack of stain. Mapping and complementation: T h e mutants were outcrossed twice to eliminate unwanted mutations; they do not show any visible phenotype except for the lack of red stain in the assay described above. Life cycle and fertility of the mutants d o not differ appreciably from those of wild type. T h e mutations are all recessive, since heterozygotes stain red. Six mutants were crossed with mapping strains, each carrying a visible marker on one chromosome. In each
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TABLE 1 @-Glucuronidaseactivity of wild-type and mutant strains Units of 0-glucu-
ronidase per mg of protein
Allele
gus- I b405, b406, b407, 6408, b409, gb25, gb32, gb44, gb85 gb29 b410 gb94 b410" gbl73 b410" gb851 gb85" +
11.3 f 2.1
