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[
WormBook,
2005]
The normal karyotype of Caenorhabditis elegans, with its five pairs of autosomes and single pair of X chromosomes, is described. General features of chromosomes and global differences between different chromosomal regions are discussed. Abnormal karyotypes, including duplications, deficiencies, inversions, translocations and chromosome fusions are reviewed. The effects of varying ploidy and of varying gene dosage are summarized. Dosage-sensitive genes seem to be rare in C. elegans, and the organism is able to tolerate substantial levels of aneuploidy. However, autosomal hemizygosity for more than about 3 % of the total genome may be incompatible with viability.
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[
WormBook,
2007]
Great inroads into the understanding of aging have been made using C. elegans as a model system. Several genes have been identified that, when mutated, can extend lifespan. Yet, much about aging remains a mystery, and new technologies that allow the simultaneous assay of expression levels of thousands of genes have been applied to the question of how and why aging might occur. With correct experimental design and statistical analysis, differential gene expression between two or more populations can be obtained with high confidence. The ability to survey the entire genome in an unbiased way is a great asset for the study of complex biological phenomena such as aging. Aging undoubtedly involves changes in multiple genes involved in multiple processes, some of which may not yet be known. Gene expression profiling of wild type aging, and of strains with increased life spans, has provided some insight into potential mechanisms, and more can be expected in the future.
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[
WormBook,
2006]
Physiological methods entered the world of C. elegans, a model system used for many years to study development and a plethora of biological processes mainly employing genetic, molecular and anatomical techniques. One of the methods introduced by physiologists is the use of Xenopus oocytes for expression of C. elegans ion channels. Oocytes of the South African frog Xenopus laevis are used widely for the expression of mammalian channels and transporters contributing to numerous discoveries in these fields. They now promise to aid C. elegans researchers in deciphering mechanisms of channels function and regulation with implications for mammalian patho-physiology. Heterologous cRNA can be easily injected into Xenopus oocytes and translated proteins can be studied using several techniques including electrophysiology, immunocytochemistry and protein biochemistry. This chapter will focus on techniques used for oocyte preparation and injection, and will give a brief overview of specific methods. Limitations of the use of Xenopus oocytes will be also discussed.
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[
WormBook,
2006]
Transposons are discrete segments of DNA capable of moving through the genome of their host via an RNA intermediate in the case of class I retrotransposon or via a "cut-and-paste" mechanism for class II DNA transposons. Since transposons take advantage of their host''s cellular machinery to proliferate in the genome and enter new hosts, transposable elements can be viewed as parasitic or "selfish DNA". However, transposons may have been beneficial for their hosts as genome evolution drivers, thus providing an example of molecular mutualism. Interactions between transposon and C. elegans research were undoubtedly mutualistic, leading to the advent of needed genomic tools to drive C. elegans research while providing insights into the transposition field. Tc1, the first C. elegans transposon to be identified, turned out to be the founding member of a widespread family of mobile elements: the Tc1/ mariner superfamily. The investigation into transposition regulation in C. elegans has uncovered an unforeseen link between transposition, genome surveillance and RNA interference. Conversely, transposons were utilized soon after their identification to inactivate and clone genes, providing some of the first molecular identities of C. elegans genes. Recent results suggest that transposons might provide a means to engineer site-directed mutations into the C. elegans genome. This article describes the different transposons present in the C. elegans genome with a specific emphasis on the ones that proved to be mobile under laboratory conditions. Mechanisms and control of transposition are discussed briefly. Some tools based on the use of transposons for C. elegans research are presented at the end of this review.
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[
WormBook,
2006]
Through genetic analyses, the function of genes is investigated by studying organisms where gene function is altered. In classical forward genetic screening, individuals are treated with mutagens to induce DNA lesions and mutants with a phenotype of interest are sought. After a mutant is found, the gene mutated is identified through standard molecular techniques. Detailed studies of the mutant phenotype coupled with molecular analyses of the gene allows elucidation of the gene's function. Forward genetics has been responsible for our understanding of many biological processes and is an excellent method for identifying genes that function in a particular process.In reverse genetics, the functional study of a gene starts with the gene sequence rather than a mutant phenotype. Using various techniques, a gene's function is altered and the effect on the development or behaviour of the organism is analysed. Reverse genetics is an important complement to forward genetics. For example, using reverse genetics, one can investigate the function of all genes in a gene family, something not easily done with forward genetics. Further, one can study the function of a gene found to be involved in a process of interest in another organism, but for which no forward genetic mutants have yet been identified. Finally, the vast majority of genes have not yet been mutated in most organisms and reverse genetics allows their study. The availability of complete genome sequences combined with reverse genetics can allow every gene to be studied.This chapter gives detailed protocols for the two main methods of perturbing gene function in C. elegans: RNA interference and the creation of deletion mutants. Either technique can be applied to the study of individual genes. With less than a day of actual work, RNAi creates a knockdown of gene function without altering the organism's DNA (see below). In contrast, with about a month of work, a deletion mutation permanently removes all gene function. Deciding which technique to use will depend on the nature of the experiment. The techniques can also be combined, where RNAi is used for rapid screening of loss of function phenotypes and then deletion mutants are made to study genes of particular interest. RNAi can also be carried out on a global scale, where knockdown of (nearly) every gene is tested for inducing a phenotype of interest. In this case, the reverse genetics technique of RNAi can be thought of as a forward genetic screening tool.
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[
WormBook,
2007]
Because of their free-living life cycle alternatives, Strongyloides and related nematode parasites may represent the best models for translating C. elegans science to the study of nematode parasitism. S. stercoralis, a significant pathogen of humans, can be maintained in laboratory dogs and gerbils. Biosafety precautions necessary for work with S. stercoralis, though unfamiliar to many C. elegans researchers, are straightforward and easily accomplished. Although specialized methods are necessary for large-scale culture of the free-living stages of S. stercoralis, small-scale cultures for experimental purposes may be undertaken using minor modifications of standard C. elegans methods. Similarly, the morphological similarities between C. elegans and the free-living stages of S. stercoralis allow investigational methods such as laser cell ablation and DNA transformation by gonadal microinjection to be easily adapted from C. elegans to S. stercoralis. Comparative studies employing these methods have yielded new insights into the neuronal control of the infective process in parasites and its similarity to regulation of dauer development in C. elegans. Furthermore, we have developed a practical method for transient transformation of S. stercoralis with vector constructs having various tissue- and cell-specific expression patterns and have assembled these into a modular vector kit for distribution to the community.
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WormBook,
2005]
Genetic suppression has provided a very powerful tool for analyzing C. elegans. Suppression experiments are facilitated by the ability to handle very large numbers of individuals and to apply powerful selections. Because the animal grows as a self-fertilizing diploid, both dominant and recessive suppressors can be recovered. Many different kinds of suppression have been reported. These are discussed by category, with examples, together with discussion of how suppressors can be used to interpret the underlying biology, and to enable further experimentation. Suppression phenomena can be divided into intragenic and extragenic classes, depending on whether the suppressor lies in the same gene as the starting mutation, or in a different gene. Intragenic types include same-site replacement, compensatory mutation, alteration in splicing, and reversion of dominant mutations by cis- knockout. Extragenic suppression can occur by a variety of informational mechanisms, such as alterations in splicing, translation or nonsense-mediated decay. In addition, extragenic suppression can occur by bypass, dosage effects, product interaction, or removal of toxic products. Within signaling pathways, suppression can occur by modulating the strength of signal transmission, or by epistatic interactions that can reveal the underlying regulatory hierarchies. In C. elegans biology, the processes of muscle development, vulva formation and sex determination have provided remarkably rich arenas for the investigation and exploitation of suppression.
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[
WormBook,
2006]
There are two sexes in C. elegans, hermaphrodite and male. While there are many sex-specific differences between males and hermaphrodites that affect most tissues, the basic body plan and many of its structures are identical. However, most structures required for mating or reproduction are sexually dimorphic and are generated by sex-specific cell lineages. Thus to understand cell fate specification in hermaphrodites, one must consider how the body plan, which is specified during embryogenesis, influences the fates individual cells. One possible mechanism may involve the asymmetric distribution of POP-1 /Tcf, the sole C. elegans Tcf homolog, to anterior-posterior sister cells. Another mechanism that functions to specify cell fates along the anterior-posterior body axis in both hermaphrodites and males are the Hox genes. Since most of the cell fate specifications that occur in hermaphrodites also occur in males, the focus of this chapter will be on those that only occur in hermaphrodites. This will include the cell fate decisions that affect the HSN neurons, ventral hypodermal P cells, lateral hypodermal cells V5 , V6 , and T ; as well as the mesodermal M, Z1 , and Z4 cells and the intestinal cells. Both cell lineage-based and cell-signaling mechanisms of cell fate specification will be discussed. Only two direct targets of the sex determination pathway that influence cell fate specification to produce hermaphrodite-specific cell fates have been identified. Thus a major challenge will be to learn additional mechanisms by which the sex determination pathway interacts with signaling pathways and other cell fate specification genes to generate hermaphrodite-specific cell fates.
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WormBook,
2010]
Ethanol is a widely used drug whose mechanism of action, despite intensive study, remains uncertain. Biochemical and electrophysiological experiments have identified receptors and ion channels whose functions are altered at physiological concentrations of ethanol. Yet, the contribution of these potential targets to its intoxicating or behavioral effects is unclear. Unbiased forward genetic screens for resistant or hypersensitive mutants represent an attractive means of identifying the relevant molecular targets or biochemical pathways mediating the behavioral effects of neuroactive compounds. C. elegans has proven to be a particularly useful system for such studies. The behavioral effects of ethanol occur at equivalent tissue concentrations in mammals and in C. elegans, suggesting the existence of conserved drug targets in the nervous system. This chapter reviews the results of studies directed toward determining the mechanisms of action of ethanol. Studies of the neural adaptations that occur with prolonged drug exposure are also discussed. The methods used to characterize the actions of ethanol should be applicable to the characterizations of other compounds that affect the behavior of C. elegans.
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WormBook,
2005]
Evolutionary innovation requires genetic raw materials upon which selection can act. The duplication of genes is of fundamental importance in providing such raw materials. Gene duplications are very widespread in C. elegans and appear to arise more frequently than in either Drosophila or yeast. It has been proposed that the rate of duplication of a gene is of the same order of magnitude as the rate of mutation per nucleotide site, emphasising the enormous potential that gene duplication has for generating substrates for evolutionary change. The fate of duplicated genes is discussed. Complete functional redundancy seems unstable in the long term. Most models require that equality amongst duplicated genes must be disrupted if they are to be preserved. There are various ways of achieving inequality, involving either the nonfunctionalization of one copy, or one copy acquiring some novel, beneficial function, or both copies becoming partially compromised so that both copies are required to provide the overall function that was previously provided by the single ancestral gene. Examples of C. elegans gene duplications that appear to have followed each of these pathways are considered.