Anaerobiosis in the nematode Caenorhabditis elegans

In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C). The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia. The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures. L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products. The amounts and proportions of the end products were dependent on temperature. They did not accumulate very much in the tissues, but were mainly excreted. During anoxia, the metabolism of C. elegans was depressed to 3-4% of the aerobic value. The food-source Escherichia coli was found to be at least partly alive in the gut of the animals. To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E. coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions.     

Gastrulation in the nematode Caenorhabditis elegans

Gastrulation in Caenorhabditis elegans has been described by following the movements of individual nuclei in living embryos by Nomarski microscopy. Gastrulation starts in the 26-cell stage when the two gut precursors, Ea and Ep, move into the blastocoele. The migration of Ea and Ep does not depend on interactions with specific neighboring cells and appears to rely on the earlier fate specification of the E lineage. In particular, the long cell cycle length of Ea and Ep appears important for gastrulation. Later in embryogenesis, the precursors to the germline, muscle and pharynx join the E descendants in the interior. As in other organisms, the movement of gastrulation permit novel cell contacts that are important for the specification of certain cell fates.     

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Xenobiotic detoxification in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans is an important model organism for the study of such diverse aspects of animal physiology and behavior as embryonic development, chemoreception, and the genetic control of lifespan. Yet, even though the entire genome sequence of this organism was deposited into public databases several years ago, little is known about xenobiotic metabolism in C. elegans. In part, the paucity of detoxification information may be due to the plush life enjoyed by nematodes raised in the laboratory. In the wild, however, these animals experience a much greater array of chemical assaults. Living in the interstitial water of the soil, populations of C. elegans exhibit a boom and bust lifestyle characterized by prodigious predation of soil microbes punctuated by periods of dispersal as a non-developing alternative larval stage. During the booming periods of population expansion, these animals almost indiscriminately consume everything in their environment including any number of compounds from other animals, microorganisms, plants, and xenobiotics. Several recent studies have identified many genes encoding sensors and enzymes these nematodes may use in their xeno-coping strategies. Here, we will discuss these recent advances, as well as the efforts by our lab and others to utilize the genomic resources of the C. elegans system to elucidate this nematode's molecular defenses against toxins.     

Age-specific nuclear proteins in the nematode worm Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans is known to undergo characteristic morphological as well as physiological signs of senescence. Two-dimensional gel electrophoresis shows that alterations also occur in the pattern of the nuclear proteins as a function of age. Non-histone proteins whose level exhibits a steep fall with age are egg-specific and not involved in senescence. However, a distinct set of non-histones accumulates with age and can be considered as senescence markers. Some of these are glycoproteins, as shown by their concanavalin A-binding properties. One age-specific polypeptide, called 'protein S-28', was further characterized by peptide mapping and determination of its N-terminal amino acid sequence.     

Metabolism and aging in the nematode Caenorhabditis elegans

Research into the causes of aging has greatly increased in recent years. Much of this interest is due to the discovery of genes in a variety of model organisms that appear to modulate aging. Studies of long-lived mutants can potentially provide valuable insights into the fundamental mechanisms of aging. While there are many advantages to the use of model organisms to study aging it is also important to consider the limitations of these systems, particularly because ectothermic (poikilothermic) organisms can survive a far greater metabolic depression than humans. As such, the consideration of only chronological longevity when assaying for long-lived mutants provides a limited perspective on the mechanisms by which longevity is increased. Additional physiological processes, such as metabolic rate, must also be assayed to provide true insight into the aging process. This is especially true in the nematode Caenorhabditis elegans, which has the natural ability to enter into a metabolically reduced state in which it can survive many times longer than its normal lifetime. The extended longevity of at least some long-lived C. elegans mutants may be due to a reduction in metabolic rate, rather than an alteration of a metabolically independent genetic mechanism specific for aging.     

Trehalose extends longevity in the nematode Caenorhabditis elegans

Trehalose is a disaccharide of glucose found in diverse organisms and is suggested to act as a stress protectant against heat, cold, desiccation, anoxia, and oxidation. Here, we demonstrate that treatment of Caenorhabditis elegans with trehalose starting from the young‐adult stage extended the mean life span by over 30% without any side effects. Surprisingly, trehalose treatment starting even from the old‐adult stage shortly thereafter retarded the age‐associated decline in survivorship and extended the remaining life span by 60%. Demographic analyses of age‐specific mortality rates revealed that trehalose extended the life span by lowering age‐independent vulnerability. Moreover, trehalose increased the reproductive span and retarded the age‐associated decrease in pharyngeal‐pumping rate and the accumulation of lipofuscin autofluorescence. Trehalose also enhanced thermotolerance and reduced polyglutamine aggregation. These results suggest that trehalose suppressed aging by counteracting internal or external stresses that disrupt protein homeostasis. On the other hand, the life span‐extending effect of trehalose was abolished in long‐lived insulin/IGF‐1‐like receptor (daf‐2) mutants. RNA interference‐mediated inactivation of the trehalose‐biosynthesis genes trehalose‐6‐phosphate synthase‐1 (tps‐1) and tps‐2, which are known to be up‐regulated in daf‐2 mutants, decreased the daf‐2 life span. These findings indicate that a reduction in insulin/IGF‐1‐like signaling extends life span, at least in part, through the aging‐suppressor function of trehalose. Trehalose may be a lead compound for potential nutraceutical intervention of the aging process.     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

Mutant sensory cilia in the nematode Caenorhabditis elegans

Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory.     

Molecular genetics of cell death in the nematode Caenorhabditis elegans.

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death, which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insight into the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells.     

The avoidance of D-tryptophan by the nematode Caenorhabditis elegans.

In chemotactic studies employing countercurrent separation the nematode aenorhabditis elegans was found to avoid D-tryptophan with a threshold in the range 10(-4) to 10(-3) M. There was no response to L-tryptophan up to 10(-2) M although it appeared to partially inhibit the response to D-tryptophan.     

A uniform genetic nomenclature for the nematode Caenorhabditis elegans.

Summary A uniform system of genetic nomenclature for the nematode Caenorhabditis elegans is described. Convenient ways are specified to designate genes, mutations and strains, and to attempt to avoid name duplications.     

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.     

DNA glycosylase activities in the nematode,Caenorhabditis elegans

DNA glycosylases acting upon uracil- or 3-methyl-adenine-containing DNA have been detected in the sonic extracts of the nematode, Caenorhabditis elegans. 4 types of the asynchronously-growing worms, embryos obtained from gravid hermaphrodites, aseptically-hatched larvae, or dauer larvae. Uracil-DNA glcosylase activity was found in all 4 types of the extracts, and the activity was highest in the embryonic extract. In contrast, 3-methyladenine-DNA glycosylase activity was undetectable in the embryonic extract, while an equal level of activity was found in the other 3 types of the extracts. The results substantiate the ubiquity of base-excision repair in various organisms, and suggest that some of the repair functions may be developmentally regulated in multicellular animals.