Dietary restriction in the nematode Caenorhabditis elegans

The first observation of the positive effect of reduced food intake on mammalian life span was made 70 years ago. In the decades that followed, researchers successfully applied this method to increase the life span of a very wide range of animals. The nematode Caenorhabditis elegans is an excellent model organism for studying the aging process. However, relatively little effort has been made to study the effects of dietary restriction in C. elegans. In this review we discuss the difficulties of subjecting C. elegans to dietary restriction, the effects of dietary restriction on metabolism and stress defense, and the potential role of different signaling pathways in DR-induced life extension. Recent experiments suggest that the TOR (target of rapamycin) pathway, rather than insulin-like signaling, might be involved in mediating the life-extending effect of dietary restriction.     

Transposition of Tc1 in the nematode Caenorhabditis elegans.

We have identified a strain of Caenorhabditis elegans in which the transposable element Tc1 is genetically active. Most spontaneous mutations affecting the unc-54 myosin heavy chain gene of C. elegans variety Bergerac are due to insertions of Tc1 within unc-54. The Bergerac genome contains an unusually high number of Tc1 elements, but this is not responsible for transpositional activity. Another variety of C. elegans, strain DH424, contains an equally high number of Tc1 elements, but transpositions are not detected. Tc1 insertion mutations are genetically unstable. They revert to unc-54+ in both germ-line and somatic cells. Germ-line revertants are wild type and contain precise or nearly precise excisions of Tc1. Somatic revertants are genetic mosaics; they contain small patches of revertant muscle tissue in otherwise mutant animals. The pattern of mosaicism often allows us to know when and where during muscle development the excisions occur. Somatic reversion can be over 1000-fold more frequent than germ-line reversion.     

Nutritional alteration of life span in the nematode Caenorhabditis elegans

The longevity of the free-living nematode Caenorhabditis elegans was studied under two different nutritional regimes, one axenic and the other monoxenic. Axenic nematodes showed typical sigmoidal survival curves with exceptionally long tailing. Monoxenic worms died off much faster and the maximum life-span in bacterial culture was generally three to four times shorter than that obtained in axenic culture. When nematodes were transferred from axenic to monoxenic culture and vice versa at near adulthood the survival patterns observed were reminiscent of the final medium. These results are in agreement with the hypothesis that worms may die off prematurely in bacterial culture by toxins given off by the bacteria.     

Effects of vitamin E on the nematode Caenorhabditis elegans

Vitamin E at 200 μg/ml significantly extended the mean lifespan and extended maximum lifespan of the nematode Caenorhabditis elegans when supplied early in the prereproductive stage. At this concentration, vitamin E increased growth, but did not affect fecundity or the length of the reproductive period. The vitamin E effect was not passed from the parents to the progeny. Evaluations of the effects of vitamin E on lipofuscin accumulation were inconclusive. The results are compared to previous studies on C. briggsae and Turbatrix aceti.     

Spectrofluorometric analysis of the effect of centrophenoxine on lipofuscin accumulation in the nematode Caenorhabditis elegans

A 41.3% mean decrease in lipofuscin was found in the nematode Caenorhabditis elegans following treatment of 6.8 mM centrophenoxine for 21 days. It is proposed that the spectrofluorometric technique is a convenient and more accurate method for determining cellular content of lipofuscin than planimetric and histochemical methodologies. This study further demonstrates the similarity of nematode lipofuscin to mammalian age pigment and provides a rapid, inexpensive method for evaluating the effects of pharmaceuticals on age-related lipofuscin accumulations.     

Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.

When grown at a temperature from 16 degrees to 25 degrees and placed on a thermal gradient, the nematode Caenorhabditis elegans migrates to its growth temperature and then moves isothermally. Behavioral adaptation to a new temperature takes several hours. Starved animals, in contrast, disperse from the growth temperature. Several mutants selected for chemotaxis defects have thermotaxis defects as well; these behaviors depend on some common gene products. New mutants selected directly for thermotaxis defects have unusual phenotypes which suggest mechanisms for thermotaxis.     

Evidence of a mate-finding cue in the hermaphrodite nematode Caenorhabditis elegans

When males of the roundworm Caenorhabditis elegans come into association with their hermaphroditic counterparts they cease foraging behavior and begin to mate. Here we detail several assays used to demonstrate that a diffusible cue is correlated with this process. This cue is sexually dimorphic, given off only by the hermaphrodite and eliciting a response only in the male. Males are attracted to, reverse direction of movement frequently, and remain in regions of agar conditioned with hermaphrodites. From our studies we suggest a form of kinesis that works by attracting males to their mating partners from a distance and functions, once males arrive, in holding attracted males in close proximity. The hermaphrodite vulva is not required for the cue. Males from general sensory mutants osm-5 and osm-6 fail to respond to the cue, whereas male-specific mutants lov-1 and pkd-2 respond. Finally, that males from multiple isolates of C. elegans also respond similarly to this cue indicates that this cue is robust and has been maintained during recent evolution.     

Cell lineages of the embryo of the nematode Caenorhabditis elegans.

Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.     

Extrachromosomal copies of transposon Tc1 in the nematode Caenorhabditis elegans.

Extrachromosomal copies of the 1.6-kilobase transposable element Tc1 are present at the level of between 0.1 and 1.0 copy per cell in Caenorhabditis elegans strain Bergerac. Extrachromosomal elements were detected and studied using Southern hybridizations employing a Tc1-specific probe. The amount of extrachromosomal Tc1 DNA was roughly constant during development in Bergerac, which has approximately 300 integrated chromosomal copies of Tc1 in its haploid genome. Extrachromosomal Tc1 DNA was not detected in strain Bristol, which has 30 chromosomal copies of Tc1. Three forms of extrachromosomal DNA were detected. The predominant form was a 1.6-kilobase linear molecule with ends corresponding to the ends of an integrated Tc1 element. The other two forms were, respectively, relaxed and supercoiled circular copies of the element. Structural assignments were based on electrophoretic mobility, the results of sedimentation velocity and equilibrium density gradient experiments, and on the sizes of the products produced by treatment of purified extrachromosomal DNA with restriction endonucleases. The suggestion is made that these extrachromosomal transposable elements are the products of excision events known to be occurring at high frequency in somatic cells in Bergerac.     

Toward a physical map of the genome of the nematode Caenorhabditis elegans

A technique for digital characterization and comparison of DNA fragments, using restriction enzymes, is described. The technique is being applied to fragments from the nematode Caenorhabditis elegans (i) to facilitate cross-indexing of clones emanating from different laboratories and (ii) to construct a physical map of the genome. Eight hundred sixty clusters of clones, from 35 to 350 kilobases long and totaling about 60% of the genome, have been characterized.     

A second trans-spliced RNA leader sequence in the nematode Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the 22-nucleotide RNA sequence called the spliced leader (SL) is trans-spliced from the 100-nucleotide-long SL RNA to some mRNAs. We have identified a trans-spliced leader (SL2) whose sequence differs from that of the original spliced leader (SL1), although both are 22 nucleotides long. By primer-extension sequencing, SL2 but not SL1 was shown to be present at the 5' end of the mRNA encoded by one of the four glyceraldehyde-3-phosphate dehydrogenase genes. The other three glyceraldehyde-3-phosphate dehydrogenase genes encode mRNAs that have the SL1 but not the SL2 sequence at their 5' ends. Therefore, the trans-splicing process can discriminate the transfer of SL1 from that of SL2 in a gene-specific manner.     

Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions

The nematode Caenorhabditis elegans was cultured monoxenically with E. coli as a food source and the influence of the bacterial growth conditions on the life span was studied. When bacterial growth was restricted by reducing the concentration of bactopeptone, which was supplied as the energy source in nematode growth medium (NGM), the nematode's life span tended to be prolonged without a marked effect on postembryonic development. The effect of bactopeptone on the life span was clearly observed during the postreproductive period (that is, after the egg-laying stage of the wild-type C. elegans) rather than during the larval to young adult stage. Evidence is presented that this alteration of the life span was not brought about by any factor in the bactopeptone but by the concentration of bacteria.     

Cell-specific proteomic analysis in Caenorhabditis elegans

Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-l-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.In a complex eukaryote like Caenorhabditis elegans, cell heterogeneity restricts the usefulness of large-scale, mass spectrometry-based proteomic analysis. Enriching for specific cells is challenging, and researchers cannot systematically identify low-abundance proteins expressed in specific cells from whole-organism lysates. Cell-selective bioorthogonal noncanonical amino acid tagging (cell-selective BONCAT) offers a way to overcome these limitations (1, 2). We have previously engineered a family of mutant Escherichia coli methionyl-tRNA synthetases (MetRSs) capable of appending the azide-bearing l-methionine (Met) analog l-azidonorleucine (Anl) to its cognate tRNA in competition with Met (3, 4). Because Anl is a poor substrate for any of the natural aminoacyl-tRNA synthetases, it is excluded from proteins made in wild-type cells but is incorporated readily into proteins made in cells that express an appropriately engineered MetRS. Controlling expression of mutant MetRSs by expression only in specific cells restricts Anl labeling to proteins produced in those cells. Tagged proteins can be distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to alkynyl or cyclooctynyl probes that permit facile detection, isolation, and visualization of labeled proteins. This strategy has been used to selectively enrich microbial proteins from mixtures of bacterial and mammalian cells. For example, Ngo et al. (5) found that proteins made in an E. coli strain outfitted with a mutant MetRS could be labeled with Anl in coculture with murine alveolar macrophages, which were not labeled. Using similar approaches, Grammel et al. (6) identified virulence factors from Salmonella typhimurium that were expressed in the course of infection of murine macrophages, and Mahdavi et al. (7) profiled Yersinia enterocolitica proteins that were injected into HeLa cells. In a complementary approach, Chin and coworkers (8) recently reengineered orthogonal Methanosarcina barkeri and Methanosarcina mazei pyrrolysyl-tRNA synthetase/tRNA pairs for codon-selective incorporation of a cyclopropene lysine derivative into proteins made in E. coli, Drosophila melanogaster ovaries, and HEK293 cells; however, this technique requires the expression of both exogenous aminoacyl-tRNA synthetases and tRNAs. Here, we configure cell-selective BONCAT for cell-specific proteomic analysis in the nematode C. elegans (Fig. 1A). We first demonstrate that restricted expression of a mutant C. elegans phenylalanyl-tRNA synthetase (CePheRS) can label proteins with p-azido-l-phenylalanine (Azf; Fig. 1B) with spatiotemporal selectivity in the live worm. We then show that cell-selective BONCAT combined with stable-isotope labeling of amino acids in cell culture (SILAC) provides a convenient and effective approach for unbiased discovery of proteins uniquely expressed in a subset of cells.Open in a separate windowFig. 1.Cell-selective BONCAT analysis in C. elegans. (A) A mutant C. elegans PheRS is capable of tagging proteins with the reactive noncanonical amino acid Azf. Spatiotemporal selectivity is achieved by controlling expression of the mutant synthetase using cell-selective promoters in transgenic lines. Proteins synthesized in cells that do not express the synthetase are neither labeled nor detected. (B) Structures of amino acids and probes used in this study: l-phenylalanine (Phe), p-azido-l-phenylalanine (Azf), dibenzocyclooctyne-functionalized tetramethylrhodamine (TAMRA-DBCO), and sodium dithionite-cleavable dibenzocyclooctyne-functionalized biotin (Diazo Biotin-DBCO).     

A selection for myosin heavy chain mutants in the nematode Caenorhabditis elegans.

The unc-54 gene of Caenorhabditis elegans encodes an abundant myosin heavy chain protein expressed in body-wall muscle cells. We have designed genetic techniques that select directly for unc-54 mutants. This selection is based upon properties of the unc-54 dominant allele e1152. Mutations that eliminate dominance of e1152 are null alleles of unc-54. Deletions have been identified by their genetic properties. We have defined mutationally a number of essential genes near unc-54, and we have described the genetic fine structure of this region of linkage group I. As much as 27% of the unc-54 mutations induced by the bifunctional alkylating agent 1,2,7,8-diepoxyoctane are multisite deletions. Extrachromosomal free duplications that include unc-54 are also described.     

Testing the rate-of-living/oxidative damage theory of aging in the nematode model Caenorhabditis elegans

The integration of the rate-of-living and oxidative damage theory of aging predicts that lifespan extension is linked to low energy metabolism, low ROS production rates, low molecular damage and a slow aging rate. In the long-lived Caenorhabditis elegans Ins/IGF-1 mutant daf-2(e1370), low carbonylation levels and postponed morphological decline comply with the latter two of these predictions. However, metabolic rates in daf-2(e1370) refute the rate-of-living theory. The apparent contradiction between increased ROS generation and long lifespan in daf-2(e1370) is reconciled by an enhanced stress defense, acknowledging oxidative damage as a probable cause of aging.     

Goalpha regulates olfactory adaptation by antagonizing Gqalpha-DAG signaling in Caenorhabditis elegans

The heterotrimeric G protein G(o) is abundantly expressed in the mammalian nervous system and modulates neural activities in response to various ligands. However, G(o)'s functions in living animals are less well understood. Here, we demonstrate that GOA-1 G(o)alpha has a fundamental role in olfactory adaptation in Caenorhabditis elegans. Impairment of GOA-1 G(o)alpha function and excessive activation of EGL-30 G(q)alpha cause a defect in adaptation to AWC-sensed odorants. These pathways antagonistically modulate olfactory adaptation in AWC chemosensory neurons. Wild-type animals treated with phorbol esters and double-mutant animals of diacylglycerol (DAG) kinases, dgk-3; dgk-1, also have a defect in adaptation, suggesting that elevated DAG signals disrupt normal adaptation. Constitutively active GOA-1 can suppress the adaptation defect of dgk-3; dgk-1 double mutants, whereas it fails to suppress the adaptation defect of animals with constitutively active EGL-30, implying that GOA-1 acts upstream of EGL-30 in olfactory adaptation. Our results suggest that down-regulation of EGL-30-DAG signaling by GOA-1 underlies olfactory adaptation and plasticity of chemotaxis.