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[
Methods Cell Biol,
1995]
The number of easily distinguishable mutant phenotypes in Caenorhabditis elegans is relatively small, and this constrains the number of factors that can be followed in standard genetic crosses. Consequently, a new mutation is mapped, first to a chromosome using two-factor data from one or more crosses, and then to a chromosomal subregion by successive three-factor crosses. Mapping would be more efficient if it were possible to score a large number of well-distributed markers in a single cross. The advent of the polymerase chain reaction makes this approach feasible by allowing polymorphic genomic regions to serve as genetic markers that are easily scored in DNA released from individual animals. The only "phenotype" is a band on a gel, so the segregation of many of these markers can be followed in a single cross. Following the terminology proposed by Olsen et al. (1989), we refer to polymorphisms that can be scored by appropriately designed polymerase chain reaction (PCR) assays as polymorphic seqeunce-tagged sites (STSs)...
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[
Methods Cell Biol,
1995]
Caenorhabditis elegans is in all likelihood the first metazoan animal whose entire genome will be determined. In addition, a very detailed description of the animal's morphology, development, and physiology is available (see elsewhere in this book, and Wood, 1988). Thus, the complete phenotype and genotype of an animal will be known. What is not known is how genotype determines phenotype; to study this, one needs to establish connections between genome sequence and phenotypes. Much has been done by classic or forward genetics: mutagenesis experiments have identified loci involved in a specific trait. Many of these loci have already been defined at the molecular level, and the genome sequence will certainly aid in the identification of many more. The opposite approach, reverse genetics, becomes naturally more important when more of the genome sequence is determined: Given the sequence of a gene of which nothing else is know, how can the function of that gene be determined? Reverse genetics is more than targeted inactivation. One can study a gene's function by several approaches...|
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[
Methods Cell Biol,
1995]
Sequence analysis of cosmids from C. elegans and other organisms currently is best done using the random or "shotgun" strategy (Wilson et al., 1994). After shearing by sonication, DNA is used to prepare M13 subclone libraries which provide good coverage and high-quality sequence data. The subclones are assembled and the data edited using software tools developed especially for C. elegans genomic sequencing. These same tools facilitate much of the subsequent work to complete both strands of the sequence and resolve any remaining ambiguities. Analysis of the finished sequence is then accomplished using several additional computer tools including Genefinder and ACeDB. Taken together, these methods and tools provide a powerful means for genome analysis in the nematode.
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[
1985]
Expression of the vitellogenin genes is restricted to the intestine of adult hermaphrodite C. elegans. In order to identify potential cis-acting elements involved in this developmental regualtion, we have sequenced the regions surrounding the 5' ends of five of the six members of this gene family. In addition, we have sequenced several of the promoters from the homologous genes from the related species C. briggsae. Although the various promoters are largely diverged from one another, we have discovered two potential regulatory sequences within the first 250 bp upstream of each of the genes. The first, TGTCAAT, occurs eight times as a perfect heptamer upstream of the five C. elegans genes, at least once per promoter. Allowing a 1 bp mismatch, the element is found in both orientations a total of 27 times, four to six timer per promoter. It is present preferentially at two locations: just upstream of the TATA box and, in the opposite orientation, at position -180. The second sequence, CTGATAA, is also present as a perfect heptamer in a restricted region of each promoter: near position -135. Remarkably, this sequence is also found upstream of the vitellogenin genes of vertebrates. Both sequences have been conserved in the C. briggsae promoters. We hypothesize that these two sequences are involved in the sex-, tissue-, and stage-specific expression of the vitellogenin genes.
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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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[
1989]
Transposable elements have recently been described in several species: Caenorhabditis elegans, Caenorhabditis briggsae, Ascaris lumbricoides, and Panagrellus redivivus. Because of the intense interest in C. elegans as an experimental organism for developmental genetic studies and the availability of sophisticated genetics, most is know about transposons in this species. This review focuses principally on Tc1 (Tc=transposon) of C. elegans, the best understood element in nematodes. Other elements in C. elegans and also elements in other species of nematodes will be briefly surveyed. The interested reader should also see two recent related reviews. The genome of C. elegans is 8 x 10(7) base pairs (bp) in extent, the smallest known for any metazoan. There are six chromosomes per haploid set, and about 83% of C. elegans DNA behaves as single-copy sequence in renaturation experiments. The repeated sequences are of several types, including functional genes, inverted or "foldback" sequences, and short repeated sequences of a few hundred nucleotides. The global arrangment of these short repeats is of the "short-period-interspersion" or "Xenopus" pattern. Some of the repetitive sequences consist of transposable elements, and at least five distinct families have been identified in C. elegans, Tc1 through Tc5. The sequence of one Tc1 element has been determined and shows that Tc1 resembles bacterial insertion sequence elements with terminal inverted repeats and a central open reading frame. The complete sequences for any members of the other transposon families have not been determined, but the data suggest that Tc2, Tc3, and Tc5 are also insertion sequence-like in structure and that Tc4 is foldbacklike in structure. No "retrotransposon-like" elements have been identified in C. elegans, although such elements have been described in A.
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A previous chapter in this series (1) described, primarily, the physical mapping of the 100 Mb Caenorhabditis elegans genome by fingerprinting of cosmid clones, and the linking of the contigs thus derived by YAC hybridization. At that time, the primary function of the map was to enhance the molecular genetics of the organism by facilitating the cloning of known genes, and to serve as an archive for genomic information. However, a clonal physical map - even with good alignment to the genetic map - carries only a tiny proportion of the information present in the genome. Consequently, the current objective of the C. elegans genome project (2) is to establish of the entire genomic sequence. The bacterial clone map, although incomplete by virtue of the uncloneability of regions of the genome in cosmid vectors (a factor which we shall discuss later in this chapter), has proved a sound basis for the systematic sequence analysis. The sevenfold cosmid coverage has a resolution sufficient to enable the selection of a subset of cosmids for sequencing such that, on average, each clone contributes 30 kb of unique sequence to the whole. Sequencing projects based on bacterial clone maps (3-5) of a number of other genomes of a range of sizes are also well advanced, in particular Saccharomyces cerevisiae (15 Mb; complete), Schizosaccharomyces pombe (15Mb), and Drosohpila melanogaster (150 Mb). Although it has recently been demonstrated that small bacterial genomes can be sequenced by direct shotgun sequence analysis of the entire genome with no prior mapping (6), the ability to interrelate and map clone sets, whether derived by random selection of in a directed manner, is still the most convenient route to the sequence analysis of larger genomes.
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In the next five years, molecular biology will get its first look at the complete genetic code of a multicellular animal. The Caenorhabditis elegans genome sequencing project, a collaboration between Robert Waterston's group in St. Louis and John Sulston's group in Cambridge, is currently on schedule towards its goal of obtaining the complete sequence of this organism and all its estimated 15,000 to 20,000 genes by 1998. By that time, we should also know the complete genome sequence of a few other organisms as well, including the prokaryote Escherichia coli and the single-celled eukaryote Saccharomyces
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[
Adv Exp Med Biol,
2010]
Neuropeptides are short sequences ofamino acids that function in all multicellular organisms to communicate information between cells. The first sequence ofa neuropeptide was reported in 1970' and the number of identified neuropeptides remained relatively small until the 1990s when the DNA sequence of multiple genomes revealed treasure troves ofinformation. Byblasting away at the genome, gene families, the sizes ofwhich were previously unknown, could now be determined. This information has led to an exponential increase in the number of putative neuropeptides and their respective gene families. The molecular biology age greatly benefited the neuropeptide field in the nematode Caenorhabditis elegans. Its genome was among the first to be sequenced and this allowed us the opportunity to screen the genome for neuropeptide genes. Initially, the screeningwas slow, as the Genefinder and BLAST programs had difficulty identifying small genes and peptides. However, as the bioinformatics programs improved, the extent of the neuropeptide gene families in C. elegans gradually emerged.