Worm Breeder's Gazette 12(3): 50 (June 15, 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.

Characterization of the Axonal Guidance and Outgrowth Gene unc-33

W. Li[1], J. E. Shaw[2]

[1]R K. Herman
Department of Genetics and Cell Biology, University of Minnesota, St Paul, MN 55108

[2]Department of Genetics and Cell Biology, University of Minnesota, St Paul, MN 55108

unc-33 mutants have been shown by Hedgecock et al. (1985, Dev. Biol. 111:158170) to have abnormalities in axonal outgrowth and a superabundance of microtubules in neuronal processes. As reported earlier, we have cloned unc-33 by tagging the gene with the transposable element Tc4 .Northern blot analysis of poly(A) RNA prepared from N2 worms showed three unc-33 transcripts: 2.8, 3.3 and 3.8 kb in size. In a gamma-ray-induced unc-33 deletion mutant, all three transcripts were smaller by 0.5 nucleotides, the size of DNA missing from the mutant genome. The abundance of each of the three wildtype transcripts appeared to be approximately constant throughout C. elegans development. By using different segments of cDNA sequence as probes in Northern analysis, the unc-33 transcripts were found to differ only at their 5'-ends. This conclusion was confirmed by RACE-PCR and RNAase protection experiments. By sequencing the PCR products, it was shown that the two larger wild-type transcripts acquire the trans-spliced leader SL1 and that the 3.8-kb message contains four exons at its 5' end that are not contained in the 3.3-kb message. The smallest transcript (2.8 kb) does not appear to have a spliced leader, and its transcriptional initiation site was determined both by RACE-PCR and RNAase protection experiments. In sum, the 3.8-kb message consists of SL1 and ten exons (I-X); the 3.3-kb message begins with SL1 spliced to the 5' end of Exon V and includes exons VX; and the 2.8-kb message begins within exon VII and includes exons vm-x. The 3' UTR of the messages is 1100 nucleotides long. The three putative polypeptides encoded by the three messages consist of 854, 679 and 523 amino acid residues; they overlap in C-terminal sequence but differ by the positions at which their N-terminal begin; none has significant similarity to any other known protein.

Two TR679 -induced unc-33 mutants (one kindly supplied by E. Hedgecock) were found to have distinct Tc4 elements inserted at the same site, within exon VII about 13 bp upstream of the 5' end of the 2.8-kb message. On northern blots, both Tc4 mutants showed three approximately wild-type-size messages, in addition to small amounts of at least two larger transcripts, corresponding to Tc4 insertions. The wild-type-size messages were analyzed by PCR and RNAase protection for one of the Tc4 mutants. It was found that the Tc4 sequence and 28 additional upstream nucleotides were spliced out of the two larger messages and that the Tc4 sequence was trans-spliced off the smallest message such that SL1 was added 13 nucleotides upstream of the normal 5' end of the 2.8-kb message. In these altered patterns of splicing, the terminus of Tc4 was used as a 3' splice acceptor.

A 2.1-kb ORF cDNA fragment covering all three messages was cloned into an E. coli trpE expression vector. Rabbit polyclonal antibodies were generated against the fusion protein produced from the trpE- unc-33 fusion gene in E. coli. On Western blots, the antibodies recognized three proteins in wild-type animals throughout development. The three proteins were either missing or altered in several unc-33 mutants. Indirect immunofluorescence staining of larvae and adults by the antibodies showed that they appeared to stain all neurons almost exclusively in their processes and only stain a few cell bodies. During embryogenesis the antibody staining was very strong in the bodies of cells that we presume are neurons as the embryo reached about 400 500 cells. As development advanced, the neuronal processes started to stain brightly and many fewer cell bodies showed staining. The antibodies to unc-33 product were also used to stain several uncoordinated mutants, including unc-44 ,which has neuronal defects very similar to those seen in unc-33 .It was found that the majority of the staining in unc-44 mutants was located in the neuronal cell bodies with very faint staining in the processes throughout development, implying that unc-44 product plays a functional role in the distribution of the unc-33 proteins.

Literature Cited:

Hedgecock et al. ,1985, Dev. Biol. 111:158170