Worm Breeder's Gazette 13(2): 68 (February 1, 1994)
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.
Because it has been recently shown that the gene unc-7 shares sequence similarity to the Drosophila genes ogre and passover, we were interested in making a systematic search for other members of this family in C. elegans. From sequence comparisons of the three genes, we designed a pair of degenerate oligo primers which could be used in PCR reactions to amplify a short stretch (approx. 180 bp) derived from a single exon in unc-7 .Our initial experiments utilized genomic DNA as a template in order to avoid bias against the representation of particular transcripts in cDNA libraries. From this search four candidates were found which could encode polypeptides related to unc-7 .The sequences of three of these candidates, pcr-32 ,-49, and -55, have not been previously reported in databases. The fourth candidate was found to be identical to a portion of the cDNA clone cm10a 8,identified by the sequencing consortium. We noted that the sequence for pcr-55 suggested the presence of an intron internal to the PCR primer sequences, indicating that we could be missing candidates which might have large introns internal to these sequences.
To address this question and extend our search, we used a cDNA library (provided by B. Barstead) as the source of template for PCR reactions. In this case we used a third degenerate oligo primer (designed from a conserved region approx. 570 nt downstream of primer 2) in conjunction with primer 1 in an initial PCR amplification. The products from this reaction were diluted and reamplified using primers 1 and 2. Among 34 clones showing sequence relationship to unc-7 ,we identified the same four candidates, as well as a fifth that is identical to a portion of the expressed sequence tag EST02207 ,submitted to Genbank by McCombie and colleagues. Further computer searches showed that two other sequences have been reported which share similarity in this region, the cDNA cm9 d9 and the expressed sequence tag EST01007 is predicted to have one mismatch in the second primer site. It is doubtful that we have identified all of the unc-7 related genes--for example, the unc-7 sequence itself did not appear in our screen of the cDNA library and the sequence for EST02207 appeared only once in our collection. In addition, the apparent absence of the primer 1 site in cm9 d9 raises the possibility that more divergent members of the family will be completely missed by our screen.
Comparison among the group of unc-7 -relatedsequences reveals a few invariant positions internal to the primer sites; a pair of cysteines and nearby amino acids(in bold) are very highly conserved (with the exception of pcr-32 ,which lacks the first cysteine). Because of the strategy used for generating products from the cDNA library, it is likely that most of these family members share similarity in regions further 3' as well. We are currently mapping the unc-7 -relatedgenes with the goal of determining whether they might correspond to Unc or Let genes, and we are further screening to determine whether this gene family is represented in vertebrates.