Worm Breeder's Gazette 10(3): 34
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
In an attempt to identify genes that control aspects of C. elegans development, we decided to clone genes with homology to known developmental genes from other organisms, such as Drosophila. We choose to clone homeobox-containing genes for two reasons: 1) Essentially all known Drosophila homeobox-containing genes are involved in control of development, thus we assume the same for C. elegans homeobox-containing genes. 2) The availability of a large variety of sequenced homeoboxes allowed us to define a well conserved 8 amino acid region in the carboxy-terminus of the homeodomain ( Burglin, Cell 53, 339, 1988). To detect the presence of this most conserved homeodomain region in C. elegans, regardless of codon usage, we synthesized three 1024-2048 fold degenerate oligonucleotides corresponding to two homeobox consensus types in the most conserved 8 amino acids. Two of these degenerate oligonucleotides (PRD-1, PRD-2) code for the Drosophila homeobox consensus type derived from genes of the prd class. The third degenerate oligonucleotide (HB-1 probe) was designed to detect all other Drosophila homeobox-containing genes known at that time ( Antp, eve, en, bcd classes), if one mismatch is allowed in the hybridization. Control Southern blots of cloned Drosophila homeobox- containing genes (eve, Dfd, prd, en, cad) showed that under our conditions the oligonucleotides of each type specifically hybridized to the corresponding homeobox-containing fragments and not to vector or other DNA sequences. We screened the MIT cosmid library with a mixture of these probes and obtained about 100 clones which hybridized with varying intensity. DNA was isolated from these cosmids, digested with EcoRI, and analyzed by Southern blotting, independently with each type of probe. More than fifty of these cosmids showed one hybridizing band with the HB-1 probe, and several others showed specific hybridization with the PRD-1/PRD-2 probes. Some clones showed two bands, suggesting the presence of homeobox gene clusters, as found in other organisms. Clones were sorted according to hybridizing fragment size and restriction pattern into about 45 groups. Alan Coulson and John Sulston fingerprinted almost all cosmid clones detected with all three homeobox probes and mapped the true positives to 42 different contigs, with some contigs having several independent putative homeobox- containing genes. These results are not unexpected, since genomic Southern analysis using these oligonucleotides as probes revealed about 35-40 hybridizing bands. In one case the correspondence between a putative homeobox- containing cosmid and a known gene was found immediately. Several overlapping cosmids we detected had previously been shown by Gwen Freyd to contain the lin-11 gene. By hybridizing the HB-1 probe to lin-11 cDNAs (see article by Freyd, Kim and Horvitz) the positive hybridization of the cosmids was shown to be located within the lin-11 gene. Sequencing revealed that it has a true homeobox (Freyd, personal communication). So far with have obtained sequence information from 2 of the new loci and confirmed that they contain a homeobox (see Figure). We propose to name new homeobox-containing genes that cannot be assigned to any known gene 'Cehb' for C. elegans homeo-box and we would like to suggest that they be numbered sequentially. Hopefully this will prevent the confusion of nomenclature that arose in vertebrate organisms. We have obtained only partial sequence information for the two new homeobox-containing genes Cehb-2 and Cehb-3, because both contain introns in the homeobox (indicated by the 'delta') and in Cehb- 3 there is a natural EcoRI site, which prevented further sequencing of this subclone. Interestingly the intron in the homeobox of Cehb-2 is at the same position as one of the introns in JM#L1001 (=Cehb-1?; WBG, Vol.10, No. 2) and one of the introns in the Drosophila gene labial. In conclusion, with three out of the three hybridizing loci sequenced, the odds are very favorable that most of the other loci will contain homeoboxes. Can we expect to find even more homeoboxes? About 50% of the loci were only recovered as one clone, and we did not detect mab-5 (WBG, Vol.10, No. 2) and mec-3 (which we expected to detect), thus our screening appears not to have been exhaustive. In addition, new classes of homeoboxes, like the unc-86 class (see article by Finney and Ruvkun), and the cut class, are so divergent from the other classes that we wouldn't (and didn't, in the case of unc-86) detect them with our oligonucleotides. So we would expect that there are more homeobox-containing genes awaiting discovery. [See Figure 1]