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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section VEST Sequencing

Although the sequence of all the genes would be expected to come from the genome sequencing project, this project was obviously going to take several years to complete. To get an early look into the genes of C. elegans, several projects have been undertaken to obtain end sequences from cDNA clones. Such partial sequences, or expressed sequence tags (ESTs), can provide quick insight into a useful fraction of genes prior to genomic sequencing. The sequences are often invaluable aids in the interpretation of the genomic sequence, once obtained.

By far, the largest project undertaken is that of Yuji Kohara (pers. comm.). The project has used as starting material size-selected, poly(A)+ RNA from total worms, and the resultant cDNA has been directionally cloned into a λ vector. More than 30,000 different clones were picked, and sequencing of clones begun. Repeated sequencing of abundantly represented clones has been minimized by periodically identifying and avoiding such clones in the full set by hybridization to the arrayed clones with pools of multiply represented clones. Both 5′and 3′ends have been sequenced from most clones. At present, there are 18,755 sequences from 11,852 clones, representing more than 3500 different genes. About half of these have significant database matches, providing clues as to their likely function in C. elegans. In the set, there are clear examples of alternative splicing (89 cases) and alternative poly(A)-addition sites (71 pairs). Of these ESTs, 1412 have been positioned on the genome map by hybridization of clones to the polytene YAC grid of the genome (>99% of these clones are represented in the currently mapped set of YACs). Placement of additional clones is now being done “in silico”by searching against the genomic sequence for matches. Clones are available to investigators by request.

Other earlier projects were much smaller in scale. In one such study, 5′-end sequences were obtained using a selected set of approximately 1500 cDNAs derived by an iterative procedure that used pools of previously picked clones as hybridization probes to avoid repicking clones with the same sequence (Waterston et al. 1992). Cluster analysis showed that these ESTs represented 1152 nonoverlapping sequences, but because only 5′-end sequences were obtained, there was no assurance that these represented that many different genes. Most of the corresponding 1152 clones were positioned on the YAC grids, and they remain available upon request. Another contemporaneous study generated 720 ESTs from 585 random clones from an unselected library, the inserts of which were not oriented (McCombie et al. 1992). Sequence from only one end was obtained from most clones. Although little map information was obtained initially, these sequences are also being positioned “in silico”and act as useful aids in sequence interpretation.

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK19995
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