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[
WormBook,
2005]
Mutations in many genes can result in a similar phenotype. Finding a number of mutants with the same phenotype tells you little about how many genes you are dealing with, and how mutable those genes are until you can assign those mutations to genetic loci. The genetic assay for gene assignment is called the complementation test. The simplicity and robustness of this test makes it a fundamental genetic tool for gene assignment. However, there are occasional unexpected outcomes from this test that bear explanation. This chapter reviews the complementation test and its various outcomes, highlighting relatively rare but nonetheless interesting exceptions such as intragenic complementation and non-allelic non-complementation.
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[
Methods Cell Biol,
1995]
This chapter is devoted to providing information on techniques applicable to studying transcription and translation in Caenorhabditis elegans. These techniques are constantly evolving and being passed among workers, each making improvements or adaptations. None of the techniques discussed below are original, but, rather, have emerged from a variety of sources over the years, making it difficult to trace their origin or give credit to the originators. Although each technique has been used successfully, for each there are alternative methods available in the literature that work equally well. In fact, depending on the available resources, you might find that an alternative technique suits your needs and facilities better than the one described below. For this reason, the procedures discussed below are usually accompanied by one or more references that will allow you to look at other, related methods. Where appropriate, there will also be a discussion of factors to consider when
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Sorrentino V, Deplancke B, Ouhmad T, Cornaglia M, Gijs MA, Auwerx J, Williams EG, Krishnamani G, Frochaux MV, Nicolet-Dit-Felix AA, Lin T, Mouchiroud L
[
Curr Protoc Neurosci,
2016]
Phenotyping strategies in simple model organisms such as D. melanogaster and C. elegans are often broadly limited to growth, aging, and fitness. Recently, a number of physical setups and video tracking software suites have been developed to allow for accurate, quantitative, and high-throughput analysis of movement in flies and worms. However, many of these systems require precise experimental setups and/or fixed recording formats. We report here an update to the Parallel Worm Tracker software, which we termed the Movement Tracker. The Movement Tracker allows variable experimental setups to provide cross-platform automated processing of a variety of movement characteristics in both worms and flies and permits the use of simple physical setups that can be readily implemented in any laboratory. This software allows high-throughput processing capabilities and high levels of flexibility in video analysis, providing quantitative movement data on C. elegans and D. melanogaster in a variety of different conditions. 2016 by John Wiley and Sons, Inc.
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[
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.
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[
2010]
Over 30 years ago, Nobel laureate Sydney Brenner recognized that an intellectually straightforward strategy to delineate the basic principles in neurobiology is to utilize a model organism with a nervous system that is simple enough to lend itself to anatomical, cellular, genetic, and molecular analysis, yet be complex enough that lessons learned in that organism would give us insight into general principles of neural function. The humble organism he chose, the nematode Caenorhabditis elegans, is now one of the most thoroughly characterized metazoans, particularly in terms of its nervous system. One of Brenner's motivations in adapting C. elegans as a model organism was to understand the totality of the molecular and cellular basis for the control of animal behavior (Brener 1988). In this chapter, we review what is arguably the best-studied aspect of C. elegans behavior: response to chemical stimuli. The C. elegans neurobiology literature can be intimidating for the uninitiated; we attempt to limit the use of "worm jargon" in this review. For a more C. elegans-centric review, we refer you to other excellent sources (Bargmann 2006).
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[
Methods Cell Biol,
1995]
In studying embryos of many species, methods of fragmenting and culturing embryonic tissues or cells have been useful for addressing questions of blastomere autonomy in early and later embryogenesis, for exposure to drugs or other agents that perturb specific processes, and for direct labeling of DNA or RNA. For Caenorhabditis elegans workers, the small size of the embryo and the impermeability of the chitinous eggshell and inner vitelline membrane have made such experiments difficult. A method of permeabilization and blastomere isolation, a culture system that will support further cellular development and differentiation, and assay methods for assaying the degree of development and its relative normality after experimental manipulation are minimal requirements for a satisfactory C. elegans embryonic culture system. Methods of isolating early blastomeres have included crushing of the eggshell and extrusion, laser ablation of neighboring blastomeres within an itact eggshell, laser puncturing of the eggshell producing extrusion, and digestion of the eggshell followed by shearing or manual stripping of the vitelline membrane. This last method is described in detail below. Permeabilization of complete embryos can be achieved by the same methods; in addition, one-cell embryos within the shell can be permeabilized to certain drugs such as cytochalasin D by gentle pressure on an overlying
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[
1987]
Vitellogenins of many insects, vertebrates, nematodes and sea urchins are very similar in size and amino acid composition. We have determined the nucleotide sequences of the genes that encode vitellogenins in nematodes (C. elegans) and sea urchins (S. purpuratus), and compared the deduced amino acid sequences to the published sequences of two vertebrate vitellogenins (X. laevis and G. gallus). This comparison demonstrated unequivocally that the nematode and vertebrate proteins are encoded by distant members of a single gene family. The less extensive sequence data available for the sea urchin gene indicates that this, too, may be a member of this family of genes, as may the vitellogenin genes of locust. On the other hand, we were unable to detect any similarity between these genes and the D. melanogaster yolk protein genes. Thus it appears that while nematodes, vertebrates, sea urchins and at least some insects utilize the same family of genes to encode vitellogenins, Drosophila uses a different gene family. All of the vitellogenin genes are regulated in a tissue-specific manner. They are expressed in the intestine in nematodes, in the liver in vertebrates, in the fat body in insects, and in the intestine and gonad in sea urchins. Their production is limited to adult females in all species except sea urchins, in which they are expressed by adults of both sexes. In nematodes we have identified two heptameric sequence elements repeated multiple times in all eleven of the vitellogenin genes sequenced. One of these elements is also present in the vertebrate promoters and has recently been shown to be required for transcriptional activation. All of the 5' ends of the vitellogenin mRNAs of nematodes, vertebrates and locust can be folded into potentially-stable secondary structures. We present evidence that these structures have been strongly selected for and presumably perform some function in regulation of vitellogenin production.