WHAT EXACTLY IS Caenorhabditis elegans ANYWAY?

The life of a nematode

Caenorhabditis elegans is a small, free-living soil nematode that feeds on bacteria.  Nematodes belong to the animal kingdom, and to the phylum Nematoda.  This phylum is characterized by a bilaterally symmetric body (meaning that the two halves are reflections of each other) and by lack of segmentation (meaning that their body is continuous, not set off in segments like earthworms).  There are around 12,000 known species of nematodes, some of which live in soil or water, and some of which are parasites.  Parasitic nematodes you may have heard of would be Trichinella,  an organism that spends part of its life cycle in pigs, and can be transmitted to humans who eat undercooked pork.

C. elegans has a three day life cycle consisting of 4 larval molts, and has two adult forms:  self-fertilizing hermaphrodites (with both male and female sex organs) and males (which are rare, making up less than 0.05% of the population).  Under stressful conditions, the second larval stage may enter a stage known as the dauer stage.  This is a specialized third larval stage that does not feed and is resistant to starvation, desiccation and other environmental stressors!  Worms can survive for up to 70 days in this stage, allowing them to tough out bad conditions for quite a period of time. 

            The body of an adult worm is 1mm in length, and contains 959 somatic cells, the lineages of which are all known. This means that scientists have followed and mapped the developmental process from the zygote (one single cell) to the adult worm and know exactly which cells divide to produce which adult cells. 

            The body of an adult worm is relatively simple, consisting of a thick, protective exterior cuticle, a pharynx and gut, and a reproductive system.  C. elegans also has an anterior nerve ring and ventral (belly side) and smaller dorsal (back side) nerve cords.  The organism can respond to temperature, touch, taste, odor, and perhaps light.  Motion is produced by alternate contraction and relaxation of four longitudinal (lengthwise) muscle bands.  The worm is typically fed in the laboratory on E. coli., a bacterium.

C. elegans is diploid, meaning it has two copies of each of its chromosomes.  This gives it five pair of autosomal chromosomes and one pair of sex chromosomes (the hermaphrodite bears an  XX genotype, the male an  XO genotype).  Hermaphrodites can bear 300 young through self-fertilization.  The life span of C. elegans at 20^o C is 12-18 days.

Nematode strains

           Exposure to ethyl methyl sulfonate [1]; [2], ENU [3], or other mutagenic agents can be used to create mutant strains of C. elegans (for a recent review, see [4]).  The Caenorhabditis Genetics Center (CGC) at the University Of Minnesota provides a permanent storage site for the thousands of mutant strains which are available to the scientific community.

The genome size for C. elegans is 97 Megabases (Mb) (about 1/30 the size of the human genome).  This genome codes for more than 19,000 proteins.  Gene loci are named using a 3-letter italicized abbreviation for the phenotype followed by a dash and a consecutive number.  Heterozygotes are designated by adding a /+ designation, where + indicates the wild type strain copy of the gene.  Alleles appear in parentheses following the locus and identify the isolating laboratory by a letter designation followed by a number.  The chromosomal location (I, II, III, IV, V, or X) may be added following the allele identifier.   To distinguish them from the gene, protein products are capitalized and not italicized . 

Dp (“dupe”) indicates duplication; Df (“dif”) indicates a deficiency.  Transgenic strains are designated using brackets [ ] to enclose the extrachromosomal material. 

 Cloning genes of interest in C. elegans

Modern techniques in molecular biology have allowed scientists to map and sequence the C. elegans genome.  In this process, DNA fragments are prepared using enzymes called restriction endonucleases which cut the DNA (at specific sites called restriction sites) into a series of fragments,  Most restriction endonucleases produce fragments with “sticky ends”:  single stranded "tails" that are complementary to the "tails" on other fragments produced by the same endonuclease.  Because of the complementary nature of these tails, another enzyme called a DNA ligase can join the 3’ hydroxyl end of one sticky end to the 5’ phosphate end of another fragment. 

These fragments can be inserted into vectors such as plasmids, bacteriophages, or cosmids.   Plasmids occur naturally in bacteria, are circular and double-stranded, and are separate from the chromosomal DNA of the cell.  They may range in size from 1 to 100 kilobases in size.  Plasmids are duplicated during cell division just as the chromosomal DNA is, and copies are passed down to daughter cells.  Plasmids designed for cloning are usually around 3 kb in size and contain three regions:  a replication origin (necessary for replication of the plasmid), a gene for drug resistance (so that the cells that pick up the plasmid can be selected for), and the region where DNA fragments may be inserted.  This region contains a polylinker, a synthetic base sequence with several different restriction sites (so that a variety of endonucleases may be used to cut and insert DNA).  Thus, the sticky ends of the DNA fragments cut by a particular endonuclease can be ligated to the sticky ends of a plasmid cut by the same endonuclease.  The bacteriophage l virus can also be used as a vector.    Strains of this bacteriophage have been developed that can contain a 15 kb DNA insert.  Cosmid vectors are hybrids that combine some of the best characteristics of both plasmids and l phages.  A cosmid library can be produced by cutting the DNA of interest with restriction enzymes and inserting the pieces into cosmids.  Another vector is the yeast artificial chromosome.  Up to hundreds of kilobases of DNA can be carried by the YAC.

Sulston, Coulson, Waterston and colleagues have constructed a map of the C. elegans genome using the cosmid cloning method (for more information on this, see [6]).  17,000 cosmid clones of insert size 35-40 kb were ordered in overlapping contigs.    A set of 3,000 YAC clones also exists.  Cosmids were fingerprinted by digesting each with HinD III, labeling  the fragment ends, and then redigesting with the enzyme Sau3A I.  The labeled fragments for each clone were electrophoresed on an acrylamide gel, generating an average of 30 bands per clone.  Band patterns were then analyzed to find overlapping regions.  As a result of this, 98% of the genome was organized into 500 contiguous regions (contigs).  These contigs were then arranged further by hybridization to the YAC library, resulting in a map with less than 20 contigs and only a few gaps. 

 The next step was to sequence the genome, which can be carried out through two different basic methods.  In the Sanger Chain-termination method, the DNA is cloned into a vector, and heated to create single stranded DNA.  An oligonucleotide primer is hybridized next to the multiple cloning region of the vector with its 3’ end oriented towards the insert.  DNA synthesis is then carried out in 4 tubes, with each tube containing a small amount of a different dideoxy nucleotide chain terminator, so that the last base in the chain will be known.  Thus, in each tube, DNA synthesis will be terminated randomly, producing a mix of fragments of different lengths but with the same final base.  Following electrophoresis and autoradiography, the series of bands can be “read” in terms of sequence length, with each band differing by one base from the band beneath it.  Automated sequencing using fluorescent tags has also been developed.  An alternative method, the Maxim-Gilbert method,  starts with the complete DNA sequence, breaks the DNA strand at specific bases, electrophoreses the fragments and then reads the sequence.

The C. elegans genome was sequenced at two sites:  the Genome Sequencing Center in St. Louis and the Sanger Centre in Hinxton, England.  The final sequence was published in Science in December of 1998.  [5].  Sequencing was begun in the centers of the chromosomes, where cosmids were selected to form a path of overlapping clones.  Fosmids (similar to cosmids, but maintained at only one single copy per cell) were also used.  Any gaps were covered through YAC clones.  There are some remaining gaps (two internal gaps and three gaps between telomeres and outermost sequenced YACs), and the error rate is estimated to be quite low. 

As segments were completed, they were subjected to analysis in the form of comparison to known genes and other sequences (again, for details, see [5]).  EST databases consist of partial cDNA sequences (200 to 400 bp in length).  Comparison with these ESTs has identified around half of the organism's genes.  The genome data are collated into the database AceDB.    Initial analysis based on the correspondance between genomic and cDNA sequences, and on the prediction of coding genes from genomic sequence predicts 19,099 genes, each with an average of five introns.  27% of the genome is in the form of exons.   Around 42% of predicted protein products match those of other organisms.   There are several hundred genes for noncoding RNAs, including 659 tRNA genes and 29 tRNA-derived pseudogenes, as well as spliceosomal genes, ribosomal RNA genes and other noncoding RNA genes.  Gene density is high with some differences between the centers of the autosomes and the arms (local clusters of genes are more abundant on the arms), as well as between the autosomes and the X chromsome .