Worm Breeder's Gazette 13(2): 96 (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.
We are interested in understanding the role basic/helix-loop-helix (bHLH) transcription factors have in cell fate specification. Members of this family of transcription factors found in both vertebrates and invertebrates include the achaete-scute complex, myoD, twist, and atonal. We employed a reverse genetics approach to identify new bHLH genes. Since bHLH proteins form homo- and heterodimers in solution, genes of this family can be identified by screening for dimerization with a labeled protein probe.
We probed an embryonic C. elegans lambda- gt11 cDNA expression library (courtesy of P. Okkema and A. Fire, WBG Vol. 12, #2) with [32P]-labeled E12 protein, a human transcription factor known to dimerize with several other bHLH proteins. Three identical clones were isolated in an initial screen of 6 x10 +E5plaques. From the predicted protein sequence, the cDNA encodes a 191 amino acid polypeptide with a calculated molecular weight of 21.5 kD. This sequence has similarity to genes in the Drosophila and Mouse achaete-scute complex, a family of bHLH proteins required for neurogenic cell fate determination (see diagram below). The achaete-scute genes have been shown to specify proneural equivalence groups in Drosophila.
This gene, hlh-3 ,has been mapped to the interval between the let-23 and rol-6 on chromosome II. Reverse transcriptase PCR experiments have indicated that hlh-3 has both a SL1 and a SL2 trans-spliced leader sequence. Currently, we are sequencing the genomic DNA flanking hlh-3 to determine if it might be part of an operon. Temporal and spatial localization studies of hlh-3 are currently underway. We are also determining if our gene can rescue any known mutation in the region, and analyzing how it may interact with other genes.
Finally, we have also begun studying T05G5 .2,a sequence identified in the C. elegans genome project and brought to our attention by Mike Krause and Dave Turner (thanks!) that has significant similarity to hlh-3 and members of the achaete-scute complex.
The two nematode genes were scored as being most similar to the achaete-scute complex genes based on the sequence of their basic domain. An interesting observation is that the only region of conservation among these b/HLH proteins is in the b/HLH domain; there is little to no similarity outside this domain. The expression pattern and regulation of hlh-3 and T05G5 .2may differ from each other given that their b/HLH domains are not identical and that the flanking sequences of these two genes are quite different Further experiments will clarify what differences may exist between these two genes and members of the achaete-scute family.