Alan Coulson

The genome sequence of Caenorhabditis briggsae: A platform for comparative genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

A survey of expressed genes in Caenorhabditis elegans.

As an adjunct to the genomic sequencing of Caenorhabditis elegans, we have investigated a representative cDNA library of 1,517 clones. A single sequence read has been obtained from the 5′ end of each clone, allowing its characterization with respect to the public databases, and the clones are being localized on the genome map. The result is the identification of about 1,200 of the estimated 15,000 genes of C. elegans. More than 30% of the inferred protein sequences have significant similarity to existing sequences in the databases, providing a route towards in vivo analysis of known genes in the nematode. These clones also provide material for assessing the accuracy of predicted exons and splicing patterns and will lead to a more accurate estimate of the total number of genes in the organism than has hitherto been available.

Software for genome mapping by fingerprinting techniques

A genome mapping package has been developed for reading and assembling data from clones analysed by restriction enzyme fragmentation and polyacrylamide gel electrophoresis. The package comprises: data entry; matching; assembly; statistical analysis; modelling. Data entry can be either manual or by a semiautomatic system based on a scanning densitometer. The primary emphasis in the analytical routines is on flexibility and interactive convenience, so that the operator has full knowledge of and control over the growing map, but a variety of automatic options are included. The package continually grows to meet the needs of the Caenorhabditis project.

RNA-mediated interference as a tool for identifying drug targets.

The nematode Caenorhabditis elegans is the first multicellular organism with a fully sequenced genome. As a model organism, C. elegans is playing a special role in functional genomic analyses because it is experimentally tractable on many levels. Moreover, the lessons learned from C. elegans are often applicable across phyla because many of the key biologic processes involved in development and disease have been well conserved. Many global approaches for analysing gene activity are being pursued in C. elegans. RNA-mediated interference (RNAi) is an efficient high-throughput method to disrupt gene function. The basic technique of RNAi involves introducing sequence-specific double-stranded RNA into C. elegans in order to generate a nonheritable, epigenetic knockout of gene function that phenocopies a null mutation in the targeted gene. This technique drastically reduces the time needed to jump from the identification of an interesting gene sequence to achieving an understanding of its function. Thus, RNAi facilitates the high-throughput functional analysis of gene targets identified during drug discovery. RNAi can also help to identify the biochemical mode of action of a drug or pesticide and to identify other genes encoding products that may respond or interact with specific compounds.

Sequence of Mammalian Mitochondrial DNA

The human mitochondrial (mt) genome consists of a closed circular duplex DNA approximately 10 x 106 daltons and has been the most intensely studied animal mt genetic system. The positions of the origin of replication of H strand synthesis (Crews et al. 1979), the 12S and 16S ribosomal RNA genes (Robberson et al. 1972) and 19 tRNA genes (Angerer et al. 1976) have been located on the genetic map shown in Figure 1. A number of discrete products of mitochondrial protein synthesis have been demonstrated and three of them identified as subunits 1, 2 and 3 of the cytochrome oxidase complex (Hare et al. 1980). In comparison with other mito-systems, genes for up to four subunits of the ATPase complex, one of the cytochrome bc1 complex and possibly for a ribosomal protein would be expected to be present (see review by Borst 1977). Both strands are thought to be completely transcribed symmetrically from a point near the origin of the H strand synthesis (Aloni and Attardi 1971; Murphy et al. 1975). These transcripts are then processed to give the rRNAs, the tRNAs and a number of polyadenylated but not capped mRNAs (Attardi et al. 1979). Both the L and H strands have been shown to be coding with the L strand containing the sense sequence of the rRNA genes, most of the tRNA genes and most of the stable polyadenylated mRNAs.

2 The Genome

I. GENERAL PROPERTIES Our knowledge of the Caenorhabditis elegans genome has increased substantially since the publication of the 1988 C. elegans book (Emmons 1988); even the genome size has changed from an estimated 80 × 10 6 base pairs to 100 × 10 6 base pairs. Systematic study of the genome in the intervening years has seen the construction of a nearly complete physical map and the release of more than half the assembled sequence. Yet it is an awkward time to be writing about the genome, since our view of the genome is changing rapidly (~2 Mb of newly assembled sequence is being released per month), and as yet most of the sequence has been obtained from the gene-rich regions of the genome, with very little from the gene-poor autosomal arms. As a result, analysis of the overall sequence remains frustratingly anecdotal. Nevertheless, much has been learned, and this chapter will attempt to summarize our current understanding of the C. elegans genome. The genome is the physical basis for genetics and includes both nuclear and cytoplasmic DNAs. For C. elegans , the mitochondrial genome (13,794 bp) has been fully sequenced (Okimoto et al. 1992). The nuclear genome contains approximately 100 × 10 6 base pairs, organized into six chromosomes ranging in size from 14 × 10 6 to 22 × 10 6 base pairs (Coulson et al. 1991), which is approximately 20 times the size of Escherichia coli (the underestimate of the E. coli genome size, used as a standard, led to the underestimate of...

6 Genomics in Caenorhabditis elegans : So Many Genes, Such a Little Worm

In 1965 sydney brenner selected Caenorhabditis elegans for his studies of development and the nervous system because of its simple anatomy, its stereotyped behavior, and the ease of genetic manipulation. Even at inception, the goal of studying the worm was an understanding of how genes dictated form and behavior. This holistic view of the organism (now dubbed “systems biology”) stimulated the collection of comprehensive data sets. The anatomy was described through serial electron microscopic reconstruction with the nervous system defined at the level of the synapse (White et al. 1986). The complete cell lineage of the 959 adult somatic cells was determined (Sulston and Horvitz 1977; Kimble and Hirsh 1979; Sulston et al. 1983) and found to be remarkably consistent animal to animal. Investigators commonly sought to collect all genes affecting a certain trait through mutations (however illusory that completeness might be in retrospect). The construction of a clone-based physical map (Coulson et al. 1986Coulson et al. 1995; Sulston et al. 1988), one of the earliest genome projects, was undertaken in the early 1980s in the same spirit. The map of overlapping cosmids and later Yeast Artificial Chromosomes (YACs) (Coulson et al. 1988Coulson et al. 1991), along with efficient means of transformation, provided the community with the wherewithal to recover the DNA for any well-mapped mutant readily and rapidly. But perhaps more importantly, the existence of a nearly complete physical map in 1989 helped convince James D. Watson, head of the National Center for Human Genome Research at the time, that the worm should...