Worm Breeder's Gazette 13(4): 86 (October 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.
Dept. of Biol. Sci., Columbia Univ, New York
mec-9 mutants are touch insensitive but their touch cells have no morphological defects. A 10 kb genomic fragment rescues the mec-9 phenotype and identifies two overlapping transcripts of 2 kb and 3 kb. The 3 kb transcript differs from the 2 kb transcript mainly in the 1 kb region at the 5' end. The 3 kb transcript level is more than 15 fold reduced in mec-3 mutants ( mec-3 is needed for touch receptor differentiation), while the 2 kb transcript level was unchanged, indicating that the long transcript is specific to the touch receptors. The two transcripts are differentially expressed. A MEC-9 -GFPfusion protein for the long transcript, produced by introducing gfp into the region encoding the 5' 1 kb portion of the 3 kb transcript, is only observed in the six touch receptor neurons and the PVD cells. Both lacZ and GFP fusions made from the short transcript showed it to be expressed in over 30 ventral cord motor neurons and 14 cells in the head. A lacZ fusion made after a common region in the two transcripts gave lacZ expression in both groups of cells. We believe that independent promoters control the production of the two mec-9 transcripts. We obtained an almost full-length cDNA from the Chris Martin library and used RACE with anchored primers to detect SL1 and SL2 splice leaders (in equal proportions). Primers from the genomic DNA and an SL1 primer also allowed us to isolate the 1 kb fragment at the 5' end of the larger transcript. Starting from the 5' end, the large protein has two Kunitz-type serine protease inhibitor domains, three EGF-like repeats (two of which are of the calcium-binding type), three additional Kunitz-type domains, three EGF-like repeats of the non-calcium-binding type, and a glutamic acid-rich domain. The short protein has the last two Kunitz domains, the downstream EGF-like repeats, and the glutamic-acid rich domain. All these domains have only been found in proteins in the extracellular space, so we think it is likely that the mec-9 proteins are secreted. We have not, however, found a signal sequence in either coding region. Agrin, a protein needed in vertebrates for the aggregation of acetylcholine receptors, has similar EGF-like repeats to those in both the long and short transcripts and also the serine protease inhibitor domains (albeit of the Kazal rather than the Kunitz type). We do not know if either of the mec-9 transcripts encode proteins with a similar function. The expression of the short transcript in the ventral cord neurons was surprising, since none of the mec-9 mutants are Unc. One possibility we are testing now is that all of our existing mec-9 mutations only affect the 3 kb transcript. We have characterized 11 mec-9 alleles with mutations in the region specific to the larger protein. In addition to two Tc1 insertions, an EMS-derived deletion, and three nonsense mutations, there are five missense mutations in the calcium-binding type of EGF-like repeats. These latter mutations suggest that the EGF-like domains are important for mec-9 function. We are sequencing five additional alleles and have yet to find mutations in the large transcript-specific region (only the most 5' and 3' parts of the region have not been sequenced). It is possible that these mutations are in common exons for the two transcripts. In addition we have looked for additional mec-9 loss-of-function alleles by screening for noncomplementing mutations. We obtained five new mec-9 alleles (26,000 haploid genomes screened). All five mutations produced only the Mec phenotype, so it is possible that the short transcript is not essential.