Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans

Abstract

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.

Structural Characterization of the Human Proteome

“…We can obtain an upper estimate of the magnitude of this problem by noting that the human genome has 6% by residue of orphans. In worm, this figure is 17%, and it is considered that most genes have been identified in this genome (Reboul et al 2001). Similar figures for orphans are found in yeast and fly.…”

“…The second assumption is that all the proteins have been identified in the genome, and one has to estimate the effect of uncharacterized proteins. However, the worm, where gene prediction is more accurate than in human, and therefore even rare, and orphan protein families are more likely to be identified (Reboul et al 2001), yields a value for domain duplication of 95%, which is probably a lower estimate of the extent for humans.…”

Evidence Suggesting That a Fifth of Annotated Caenorhabditis elegans Genes May Be Pseudogenes

“…Finally, the structure of many of the annotated genes is based primarily on predictions by the computer program Genefinder. It is thought that 5.45% of all exons may be mispredicted in a way that a correct translational fusion would not be formed within the reporter gene fusions assayed (Reboul et al 2001), and splitting of one gene into two predicted genes can mean an assayed fragment would not contain the necessary promoter elements.…”

Recombinatorial Cloning Using Heterologous Lox Sites

“…This requires the phageencoded integrase (Int) and host-derived integration host factor (IHF) to catalyze recombination between a 24-bp attB site (originally in the bacterial genome) and a 243-bp attP site (originally in the phage genome) to generate the 100-bp attL and 168-bp attR sites (that originally flank the newly integrated DNA), and Int, IHF, and exisionase (Xis) to carry out the reverse reaction (for review, see Landy 1989). Whereas this system has been extensively and successfully used in cloning collections of individual genes into a nonfunctional source vector followed by recombination into functional destination vectors (Walhout et al 2000;Reboul et al 2001), it is not feasible for systems that require expression of the scFv gene, and therefore translation of each recombination site, with each vector used. For recombination to be used, either the display vector or the destination expression vector would (at some stage) have to contain the attL sites that contain stop codons in all three frames.…”

“…However, this procedure is time consuming, requiring DNA production, cleavage, purification, ligation, transformation, and screening. Recombination, which avoids the need for such manipulation, has been proposed as an alternative to rapidly transfer genes from source vectors to destination vectors, with the commercially available Gateway kit (Invitrogen; Hartley et al 2000) based on bacteriophage being most widely used to create large libraries of individual gene-specific ORFs (e.g., Caenorhabditis elegans; Reboul et al 2001). Unfortunately, this system (see Discussion) is not appropriate for selection from phage display libraries, as the length of some of the recombination signals, resulting from the asymmetric nature of the recombination process, would prevent the functional expression of scFvs in all vectors, requiring multiple recombination rounds for transfer.…”