-
[
Methods Cell Biol,
1995]
Complementary DNA libraries are useful tools for uncovering genes of interest in C. elegans and finding specific homologies to genes in other organisms (Waterston et al., 1992; McCombie et al., 1992). When working with existing cDNA libraries, be sure to carefully choose which libraries would be most beneficial to the type of research being done. Some libraries may be specific for genes that are present in lower copy numbers, whereas others may be of a more general nature. It is important to fully understand the source and construction of the library you will be working with. Once an appropriate library has been chosen, work may begin to isolate a specific cDNA and sequence it completely or to survey many cDNAs by single-pass DNA sequencing. Whatever the project, it is important to develop a specific strategy for both the sequencing and the organization of the clones being characterized. The strategies and procedures we have outlined in this chapter have proven effective for rapid and comprehensive cDNA characterization.
-
[
1975]
Studies in behaviour genetics have covered a wide field: motivation, development, sensory capacities, intelligence, learning, evolution, neuromorphology and neurochemistry have all been approached using genetic techniques, and there are probably others. Whilst it is at present impossible to construct any unities one must accept that many such studies have as their common aim one of the most fundamental problems in biology: how is behavioral potential encoded in genetic terms and expressed in the course of development? The relative enormity of this problem is often matched by its inaccessibilty. It cannot be claimed that there is any agreed view of the way forward and much of the work has frankly to be opportunistic-seizing on some favourable material or a useful new analytical technique to gain a limited objective. Consequently, behaviour genetics often presents a confusing picture of numerous disjointed studies, with
-
[
1998]
The use of antibodies to visualize the distribution and subcellular localization of gene products powerfully complements genetic and molecular analysis of gene function in C. elegans. The challenge to immunolabeling C. elegans is finding the fixation and permeabilization methods that effectively make antigens accessible without destroying the tissue morphology or the antigen. Embryos are surrounded by a chitinous eggshell and larvae and adults are surrounded by a collagenous cuticle, each of which must be permeabilized to allow penetration of antibodies. In addition, antigens and antibodies are sensitive to different fixing and permeabilizing conditions. For example, some antibodies do not work well on paraformaldehyde-fixed samples, and others are sensitive to incubation in acetone. There are many protocols used in the C. elegans field; additional protocols are summarized in Miller and Shakes (1994) and on the C. elegans World
-
[
1998]
The use of antibodies to visualize the distribution and subcellular localization of gene products powerfully complements genetic and molecular analysis of gene function in C. elegans. The challenge to immunolabeling C. elegans is finding the fixation and permeabilization methods that effectively make antigens accessible without destroying the tissue morphology or the antigen. Embryos are surrounded by a chitinous eggshell and larvae and adults are surrounded by a collagenous cuticle, each of which must be permeabilized to allow penetration of antibodies. In addition, antigens and antibodies are sensitive to different fixing and permeabilizing conditions. For example, some antibodies do not work well on paraformaldehydefixed samples, and others are sensitive to incubation in acetone. There are many protocols used in the C. elegans field; additional protocols are summarized in Miller and Shakes (1994) and on the C. elegans World Wide Web page
(http://elegans.swmed.edu/). -
[
Molecular Biology of Aging,
1999]
"Gerontogenes" (genes that affect the rate of aging) can be defined operationally to refer to genes that can be altered such that a longer than normal maximum lifespan is the result. The last two decades of research in aging have demonstrated overwhelmingly that gerontogenes exist and modulate the rate of aging. The first direct demonstration that genes play a role in the aging process was carried out in the nematode Caenorhabditis elegans. Despite original prejudices that the aging process is "ineluctable" or that genes controlling longevity cannot be selected for, these results and others have shown that the process of aging, just as other biological processes, is specified by the gene. This is not to say that aging is programmed. Statements by noted developmental biologists that aging must be programmed to prevent competition with offspring are untenable for the nematode C. elegans, which has billions of descendents by the time its hypothetical "death program" kicks in to kill it. In the text below I will provide an overview, first of work primarily from my laboratory having to do with the detection and study of gerontogene variants using multigenic approaches. Subsequent work on mutants, initially from my lab but more recently from a variety of other labs as well, showing the molecular nature of these gerontogenes will be subsequently reviewed. Finally, we will close with a discussion of the role of resistance to stress in determining life-extension: a hypothesis that is gaining increasing support from a wide variety of observations in both invertebrate and vertebrate
-
[
WormBook,
2005]
A wide variety of bacterial pathogens, as well as several fungi, kill C. elegans or produce non-lethal disease symptoms. This allows the nematode to be used as a simple, tractable model host for infectious disease. Human pathogens that affect C. elegans include Gram-negative bacteria of genera Burkholderia, Pseudomonas, Salmonella, Serratia and Yersinia; Gram-positive bacteria Enterococcus, Staphylococcus and Streptococcus; and the fungus Cryptococcus neoformans. Microbes that are not pathogenic to mammals, such as the insect pathogen Bacillus thuringiensis and the nematode-specific Microbacterium nematophilum, are also studied with C. elegans. Many of the pathogens investigated colonize the C. elegans intestine, and pathology is usually quantified as decreased lifespan of the nematode. A few microbes adhere to the nematode cuticle, while others produce toxins that kill C. elegans without a requirement for whole, live pathogen cells to contact the worm. The rapid growth and short generation time of C. elegans permit extensive screens for mutant pathogens with diminished killing, and some of the factors identified in these screens have been shown to play roles in mammalian infections. Genetic screens for toxin-resistant C. elegans mutants have identified host pathways exploited by bacterial toxins.
-
[
Methods Enzymol,
2011]
The endoplasmic reticulum (ER), first compartment of the secretory pathway, is mainly involved in calcium sequestration and lipid biosynthesis and in the translation, folding, and transport of secretory proteins. Under some physiological and physiopathological situations, secretory proteins do not acquire their folded conformation and accumulate in the ER. An adaptive response named the UPR is then triggered from this compartment to restore its homeostasis. In the past few years, interconnections between the UPR and small GTPase signaling have been established. In an attempt to further investigate these novel signaling networks, we hereby provide a detailed description of experimental strategies available. We describe in detail methods to monitor both UPR and small GTPase signaling and the outcomes of such approaches in the identification of new links between those signaling pathways using pharmacological and genetic screens. In physiopathological contexts, the guidelines herein should enable researchers in the field to establish essential means for determination of functional interactions between those pathways.
-
[
1981]
This chapter is in part a review of the work of others and in part a summary of recent results from our own laboratory. It attempts to cover the currently available information on apparent neurotransmitters in the small soil nematode Caenorhabditis elegans, whose advantages of genetic manipulability and cellular simplicity have recently gained it some favor in investigations of genetic control mechanisms in neural development (for review, see Riddle, 1978). Particular attention is given to mutants that affect either the level or the action of apparent neurotransmitters, since it seems likely that such mutants may have the most to offer toward the understanding of human genetic neuropathies. The general features of C. elegans are described briefly at the outset, then each apparent neurotransmitter is considered in turn, and finally a few potential implications for other organisms
-
[
Methods Cell Biol,
1995]
One way to study cell function is to eliminate the cell and observe subsequent developmental or behavioral abnormalities in the animal. In Caenorhabditis elegans, this is usually accomplished by killing individual cells or groups of cells with a laser microbeam. Laser killing has been used to determine the functions of many mature cell types, including neurons involved in locomotion, feeding, mechanosensation, and chemosensation. These studies have been practical because only a few cell types appear to be absolutely required for viability. Laser ablation can also be sued to ask how cells interact during development. Signaling and inductive interactions between cells can be examined by removing one cell and observing the development of the remaining cells...
-
[
Methods Cell Biol,
1995]
Compared with other animals, the nematode Caenorhabditis elegans has many advantages for mutant isolation and for genetic analysis. Some of these, for example, small size and rapid growth to high density on inexpensive media, simplify the manipulation of large numbers of animals. Others, such as the lack of a muscle-driven circulatory system and the self-fertilizing hermaphroditic mode of reproduction, enable the survival of strains with genetic defects that would be lethal to more complex animals. Although a few compounds with C. elegans-specific or nematode-specific actions have been described, the vast majority appear to act on targets that are widely distributed in most or all animals, including humans (or even in most or all eukaryotes). As a result, C. elegans has been a popular organism in which to study drug action and there is a substantial body of published work. In this chapter we attempt to extract an underlying feature of this work: the methods that are used in compound-based studies of C. elegans. We present general approaches to evaluating the effects of compounds on C. elegans growth, development, metabolism, and behavior, we discuss strategies for the isolation and analysis of drug-resistant and hypersensitive mutants, and we describe the use of C. elegans for new drug discovery. We also provide, as Table I, a list of some of the compounds already studied in C. elegans, along with one or more references in which information about the detection of compound-specific effects can be found. It is hoped the table will expedite the use of compound-specific mutants as genetic markers...