In search of new mutants in cell-signaling systems of the nematodeCaenorhabditis elegans

Development of multicellular organisms is controlled mainly by cell-signaling systems. In this review I first discuss methods of genetic analysis and properties of mutants of cell-signaling systems in general and in the nematodeC. elegans. Then, I describe two of our approaches to isolating new mutants in cell-signaling ofC. elegans. The first approach is to select for mutants that have the same visible phenotype as those in known cell-signaling genes. In a survey of larval lethal mutations we found that there are quite a few mutants in which the inner surface of the body wall is detached from the outer surface of the intestine. Some of them map in genes that are known to act in cell-signaling systems in vulval induction or sex myoblast migration, which are not essential to the growth and survival ofC. elegans. Therefore, we think many of the mutations of the above phenotype disrupt cell-signaling in an unidentified essential function, and also cell-signaling in the non-essential functions. The second approach is to isolate mutants resistant to a drug expected to disturb cell-signaling. As the drug we have chosen sodium fluoride, which depletes calcium ion, activates G-proteins and inactivates some phosphatases. The mutants are grouped into two classes (three and two genes, respectively) according to degree of fluoride-resistance and growth rate of larvae. Although there is so far no direct evidence that these mutants are related to cell-signaling, they show complex epistasis that can be explained by a model consisting of a cell-signaling pathway.     

Egg-laying defective mutants of the nematode Caenorhabditis elegans

We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl. for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new components of the neural circuitry that control egg laying.     

Temperature-sensitive zygote defective mutants of Caenorhabditis elegans

Three genetically complementing temperature-sensitive mutants of Caenorhabditis elegans have been studied. Each of the three mutants has two critical times of temperature sensitivity and two distinctive corresponding phenotypes. Exposure to high temperature during gonadogenesis blocks the production of zygotes. Exposure of adults to high temperature interrupts embryogenesis of the zygotes being produced. Each of the mutants carries an autosomal mutation with a maternal effect. These mutants indicate that the individual temperature-sensitive functions are required at least twice during development and that early embryogenesis is dependent on the contribution of these functions from the maternal gonad.     

Temperature, oxygen, and salt-sensing neurons in C. elegans are carbon dioxide sensors that control avoidance behavior

Homeostatic control of body fluid CO(2) is essential in animals but is poorly understood. C.?elegans relies on diffusion for gas exchange and avoids environments with elevated CO(2). We show that C.?elegans temperature, O(2), and salt-sensing neurons are also CO(2) sensors mediating CO(2) avoidance. AFD thermosensors respond to increasing CO(2) by a fall and then rise in Ca(2+) and show a Ca(2+) spike when CO(2) decreases. BAG O(2) sensors and ASE salt sensors are both activated by CO(2) and remain tonically active while high CO(2) persists. CO(2)-evoked Ca(2+) responses in AFD and BAG neurons require cGMP-gated ion channels. Atypical soluble guanylate cyclases mediating O(2) responses also contribute to BAG CO(2) responses. AFD and BAG neurons together stimulate turning when CO(2) rises and inhibit turning when CO(2) falls. Our results show that C.?elegans senses CO(2) using functionally diverse sensory neurons acting homeostatically to minimize exposure to elevated CO(2).     

High throughput screening of mutants of oat that are defective in triterpene synthesis

The triterpenes are a large and diverse group of plant natural products that have important functions in plant protection and food quality, and a range of pharmaceutical and other applications. Like sterols, they are synthesised from mevalonate via the isoprenoid pathway, the two pathways diverging after 2,3-oxidosqualene. During triterpene synthesis 2,3-oxidosqualene is cyclised to one of a number of potential products, the most common of these being the pentacyclic triterpene β-amyrin. Plants often produce complex mixtures of conjugated triterpene glycosides which may be derived from a single triterpene skeleton. The delineation, functional analysis and exploitation of triterpene pathways in plants therefore represent a substantial challenge. Here we have carried out high throughput screening to identify mutants of diploid oat (Avena strigosa) that are blocked in the early steps of triterpene synthesis. We also show that mutants that are affected in the first committed step in synthesis of β-amyrin-derived triterpenes, and so are unable to cyclise 2,3-oxidosqualene to β-amyrin (sad1 mutants), accumulate elevated levels of primary sterols. The major differences were in Δ-7-campesterol and Δ-7-avenasterol, which both increased several fold relative to wild-type levels. This is presumably due to accumulation of squalene and 2,3-oxidosqualene and consequent feedback into the sterol pathway, and is consistent with previous reports in which specific oxidosqualene cyclase inhibitors and elicitors of triterpene biosynthesis were shown to have inverse effects on the flux through the sterol and triterpene pathways.     

Appetitive response of the nematodeCaenorhabditis elegans to oxygen

Summary Using a technique of recording the behavior of individual nematodes during exposure to various solutions, it was demonstrated thatC. elegans made more reversal behaviors after transfer to solutions of lower oxygen tension than higher. The response was stronger after the first hour in the apparatus than initially. This change was not dependent on reduced oxygen availability during the initial period. Starvation is the most likely cause of this change. A variety of mutant strains ofC. elegans that are defective in response to most known chemotactic stimuli, including two strains that have been shown to be severely abnormal in the ciliated endings of all sensory neurons of the worm's snout, all responded to changes in oxygen tension. This observation suggests that oxygen is sensed internally rather than by specialized peripheral receptor cells.I thank Deborah Higgins for technical assistance. This work was supported by a grant awarded by the Biomedical Research Support Grant Program of the National Institutes of Health.     

Thymidine-requiring mutants of Salmonella typhimurium that are defective in deoxyuridine 5''-phosphate synthesis.

In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.     

Two yeast mutants defective in endocytosis are defective in pheromone response

We have purified biosynthetically labeled alpha-factor secreted from transformed yeast alpha cells. This alpha-factor binds specifically to a cells and is internalized by a time-, temperature-, and energy-dependent process. alpha-factor is internalized in an intact form and then rapidly degraded. Two yeast mutants defective in the accumulation of an endocytotic marker, lucifer yellow CH, in the vacuole have been isolated. end1 accumulates invaginations of the plasma membrane, and end2, an internal membrane-bound organelle. One of these mutants, end1, is defective for internalization of alpha-factor. Both of these mutants are defective in pheromone response.     

The gravitropism defective 2 mutants of Arabidopsis are deficient in a protein implicated in endocytosis in Caenorhabditis elegans

The gravitropism defective 2 (grv2) mutants of Arabidopsis show reduced shoot phototropism and gravitropism. Amyloplasts in the shoot endodermal cells of grv2 do not sediment to the same degree as in wild type. The GRV2 gene encodes a 277-kD polypeptide that is 42% similar to the Caenorhabditis elegans RME-8 protein, which is required for endocytosis. We hypothesize that a defect in endocytosis may affect both the initial gravity sensing via amyloplasts sedimentation and the subsequent more general tropic growth response.     

Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.Communicated by H. Ikeda     

Responses of the nematodeCaenorhabditis elegans to controlled chemical stimulation

Summary A new method is described for studying the behavioral responses of nematodes to controlled chemical stimulation. The worm is held by the tail with a suction pipet. Behavior is recorded by an array of light sensors connected to a multichannel recorder. Several types of behavior can be detected in addition to the normal backward propagating waves of about 2 Hz that propel untethered worms forward. The most dramatic of these is the reversal bout, consisting of forward propagating waves of about 0.7 Hz, that propel untethered worms backward. The latter waves are easily distinguished from the former by the large amplitude motion caused by the fact that they contain a sharper bend at the tail. This technique was used to demonstrate that a purely temporal change in chemical stimulation can cause a large change in the probability of occurrrence of a reversal bout. These altered probabilities adapt back to the basal level in about one minute. Increased probabilities adapt faster than decreased. Stronger stimulation causes slower adaptation. Since the reversal bout is associated with changes in direction of locomotion, these observations suggest that klinokinesis with adaptation plays a role in the movement of nematodes in chemical gradients.I would like to thank Deborah Higgins for technical assistance. This work was supported by Grant RR 07024q3 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health and by the National Science Foundation under Grant GB-43561.     

Failure of sensory adaptation in bacterial mutants that are defective in a protein methylation reaction.

Chemotactic bacteria, such as E. coli, detect changes in the chemical composition of the environment. Addition of an attractant or repellent leads to an immediate response, characterized by a change in the swimming behavior of the cells--a process known as sensory excitation. However, the response gradually disappears with time, despite the continued presence of the chemical--a process known as sensory adaptation. We report here the behavior of a class of nonchemotactic mutants (cheX) that can carry out sensory excitation but are defective in the process of sensory adaptation. These mutants are also defective in the ability to carry out a protein methylation reaction which has previously been implicated in the adaptation process (Goy, Springer and Adler, 1977). The results presented here establish a firm relationship between the methylation reaction and sensory adaptation.     

Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell.

The excretory cell extends a tubular process, or canal, along the basolateral surface of the epidermis to form the nematode renal epithelium. This cell can undergo normal tubulogenesis in isolated cell culture. Mutations in 12 genes cause excretory canal cysts in Caenorhabditis elegans. Genetic interactions, and their similar phenotypes, suggest these genes may encode functionally related proteins. Depending upon genotype and individual canal, defects range from focal cysts, flanked by normal width segments, to regional cysts involving the entire tubule. Oftentimes the enlarged regions are convoluted or partially septated. In mutants with very large cysts, renal function is measurably impaired. Based on histology and ultrastructure, canal cysts likely result from defects of the apical membrane domain. These mutants provide a model of tubulocystic disease without hyperplasia or basement membrane abnormalities. Similar apical mechanisms could regulate tubular morphology of vertebrate nephrons.     

Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants

The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.     

Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis

Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups. Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS. Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix+ on alfalfa. This suggests that LPS does not play a major role in symbiosis. Mutations in lpsB, however, are Fix- in one particular genetic background, evidently because of the cumulative effect of several independent background mutations. In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix- on alfalfa.     

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.     

Lec9 CHO glycosylation mutants are defective in the synthesis of dolichol

Lec9 Chinese hamster ovary cells were found previously to be defective in the synthesis of N-linked glycans. This appeared to be the result of a defect in the synthesis of oligosaccharide lipid and lipid phosphate (Rosenwald, Stanley, and Krag. 1989. Mol. Cell. Biol. 9: 914-924). In this study we analyzed the steady state levels of long-chain polyisoprenyl lipids in Lec9 cells. We found that Lec9 cells are defective in the synthesis of dolichol. They accumulated a presumed precursor to dolichol, cis-a-unsaturated polyprenol and used this lipid in the synthesis of oligosaccharide lipid. Chain lengths of the activated polyprenols in Lec9 were the same lengths as dolichols in parental cells. Lec9 cells had increased levels of monosaccharylphosphoryl lipid and decreased levels of oligosaccharylpyrophosphoryl lipid compared to parental cells. The defect in Lec9 cells was specific for dolichol synthesis, since other aspects of [3H]mevalonate metabolism in Lec9 cells were the same as in parental cells. We hypothesize that Lec9 cells are defective in polyprenol reductase activity.