[
WormBook,
2005]
This chapter reviews analytical tools currently in use for protein classification, and gives an overview of the C. elegans proteome. Computational analysis of proteins relies heavily on hidden Markov models of protein families. Proteins can also be classified by predicted secondary or tertiary structures, hydrophobic profiles, compositional biases, or size ranges. Strictly orthologous protein families remain difficult to identify, except by skilled human labor. The InterPro and NCBI KOG classifications encompass 79% of C. elegans protein-coding genes; in both classifications, a small number of protein families account for a disproportionately large number of genes. C. elegans protein-coding genes include at least ~12,000 orthologs of C. briggsae genes, and at least ~4,400 orthologs of non-nematode eukaryotic genes. Some metazoan proteins conserved in other nematodes are absent from C. elegans. Conversely, 9% of C. elegans protein-coding genes are conserved among all metazoa or eukaryotes, yet have no known functions.
[
WormBook,
2005]
The features that differentiate the C. elegans male from the hermaphrodite arise during postembryonic development. The major male mating structures, consisting of the blunt tail with fan and rays, the hook, the spicules and proctodeum, and the thin body, form just before the last larval molt. Male and hermaphrodite embryogenesis are similar but some essential male cell fates are already established at hatching. The male mating structures arise from three important sets of male-specific blast cells. These cells generate a total of 205 male-specific somatic cells, including 89 neurons, 36 neuronal support cells, 41 muscles, 23 cells involved in differentiating the hindgut, and 16 hypodermal cells associated with mating structures. Genetic and molecular studies have identified many genes required for male development, most of which also function in the hermaphrodite. Cell-cell interactions play a role in patterning all three of the generative tissues. Male-specific neurons, including sensory neurons of the rays, hook, post-cloacal sensilla, and spicules, differentiate at the end of the last larval stage and send out axons to make connections into the existing neuropil, greatly enlarging the posterior ganglia. The hindgut is highly differentiated to accommodate the spicules and the joining of the reproductive tract to the cloaca. A complex male-specific program generates many new muscles for copulation. The cell lineage and genetic program that gives rise to the one-armed male gonad appears to be a variation on that of the hermaphrodite.
[
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.