Worm Breeder's Gazette 12(4): 37 (October 1, 1992)
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.
The heterochronic gene pathway controls the temporal pattern of cell divisions and differentiation in diverse cell lineages during larval development. lin-4 is required for the down regulation of lin-14 during the mid L1 stage. lin-4 is most likely activated by the food signal shortly after hatching and directly or indirectly down-regulates lin-14 protein synthesis. A possible target of lin-4 is the 3' untranslated region (UTR) of lin-14 ,which contains cis-acting negative regulatory sequences defined by lin-14 semi-dominant gain-of-function mutations.
To characterize the structure, function, and developmental regulation of the lin-4 product, we cloned lin-4 from C. elegans by RFLP mapping and transformation rescue. lin-4 is contained within an intron of an unrelated gene (of no known activity). A 693 bp Sal-Sal fragment fully rescues a lin-4 null allele ( e912 ,a >10 kb deletion of the locus). Using this rescuing piece as probe, we isolated clones from two other Caenorhabditis species, C. briggsae and C. remanei, and these clones also rescue the C. elegans lin-4 ( e912 )allele. Sequence from all three species demonstrate a high degree of homology in the rescuing region. Primers made from the highly conserved regions have allowed us to PCR amplify fragments of lin-4 homologous sequence from C. vulgaris. We have full lin-4 sequence from C. elegans and C. briggsae and partial sequence from C. remanei and C.vulgaris. In the region corresponding to the C. elegans 693 bp fragment, 50% of the bases are identical in all four species and another 20% are identical in three. All four species contain the single G/C base pair which is changed in the EMS allele, ma161 .Differences among the species consist of gaps, insertions, and deletions which seem to disrupt any putative open reading frame. These results strongly suggest that lin-4 does not encode a protein.
Nuclease protection experiments using RNA and DNA probes from a region highly conserved among the four Caenorhabditis species indicate a lin-4 transcript of approximately 60 nt. RNAse protection experiments also suggest a second transcript of approximately 20 nt from the same region. To date, the 5' and 3' ends of the 60 nt transcript have been located with sufficient precision to allow two important observations: First, the lin-4 point mutation, ma161 ,alters the transcript near it's 5' end; and second, conserved sequences within the 60 nt transcript are complementary to conserved sequences in the 3' untranslated region of lin-14 mRNA (Wightman and Ruvkun, personal communication). These observations suggest that a lin-4 regulatory RNA interacts directly with lin-14 mRNA by antisense base-pairing to somehow inhibit the translation of lin-14 protein. Experiments are under way to address the developmental regulation of lin-4 ,and to determine the precise cellular and molecular mechanism of lin-4 action.