Worm Breeder's Gazette 14(5): 59 (February 1, 1997)
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.
Eotvos Lorand University, Department of Genetics, Budapest H-1088, Hungary
The body size in the animal kingdom is thought to be determined quantitatively, but there are some examples when a giant phenotype determined by a simple Mendelian allele. We are interested in molecular mechanisms leading to giant phenotype. In C. elegans, any recessive loss-of-function allele of the gene lon-2(X), such as the reference allele e678, determines an elongated phenotype. The lon-2 gene seems to be expressed throughout the postembryonic development. The epistatic relationships of body size determining genes (such as daf-4, coding for a serine/threonine kinase receptor binding a TGF-beta-like ligand) have already been characterised. The pleitropic phenotype of any daf-4 allele is small and the phenotype of the daf-4 lon-2 double mutant is also small. However, the lon-2(+) exerts a maternal effect rescuing of the Daf(c) phenotype of daf-4 mutant indicating, that lon-2 is a negative regulator of daf-4 activity, and there might be a link between lon-2 and the TGF-beta-like signal transduction pathway. This is supported by the fact, that genomic clones rescuing sma-4 often confer a Lon phenotype. Product of sma-4 is a cytoplasmic signal transducer for the cell surface receptor encoded by daf-4, therefore Lon phenotype can be caused by over-activation of the TGF-beta signalling pathway in C. elegans (Savage, WBG 13). The transposon induced allele of lon-2, g309, appeared in an unusual mutator strain was used for molecular cloning. Both the mutator (RC301) and the mutant derivative (RC309) strains were kindly provided by R. Cassada. As for the mutator transposon, RC301 is a low-copy number strain; it has even less copy of Tc1 than N2. The frequency of (germline) transposition, as well as that of the reversion in RC309 is rather rare. When Tc1 was used as a probe, the Southern hybridization pattern of RC301 and g309 differed at 5 places. According to ACeDb, the lon-2 can approximately be localized to a 100 kb large segment of the physical map and potentially be covered by some of the 24 overlapping cosmid clones between C13C10 and C08A11. Using the Tc1 transposon as a probe, we identified a fragment, that could also show a Tc1 pattern difference between RC301 and RC309 genomes. This fragment was purified, circularized and then amplified by a reverse PCR method using Tc1 specific primers directed in the opposite direction outward from the Tc1 core region, and then it was hybridizated to the set of cosmids. As a result, two overlapping cosmids, C37H11 and R13G1, proved positive. Both of them were able to rescue the wild type when injected them into lon-2 homozygote recessive mutants. The overlapping genomic DNA region, which was subcloned from these cosmids, was about a 8 kb large EcoRI fragment. It was cloned into a vector and was still able to recsue the wild type phenotype when injected into Lon hermaphrodites. The transgenic animals were wild type in size and shape, however, they were little abnormal in movement and were either sterile or produced only Lon progeny. The sequence of this fragment was determined by using the data of C. elegans sequencing project, that consists a predicted gene, F43C9.3. The predicted product of this gene seems to be a novel protein. Search for sequence homologies indicated both myosin-like and a signal transduction pathway's regulator protein-like similarities. Regulatory proteins of two similar, independent functions (namely, regulating the cytoskeleton and a transduction signal) have been known.