Worm Breeder's Gazette 10(1): 28
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 have further defined the DNA sequence which encodes mec-3 by microinjection of cloned DNA into a mec-3 mutant. The entire 5.6 kb region that can rescue a mutant animal has been sequenced. Our best guess of the putative coding sequence gives the gene 8 exons. The first four are small (13-41 amino acids), and because of their small size, we consider them very tentative. The fifth is 84 amino acids. The sixth and seventh exons are 38 and 71 amino acids, and together encode a homeodomain, described below. The final exon is only 22 amino acids, of which 11 are Glu or Asp. The homeobox is usually defined as a 60 amino acid region with certain shared features: any two homeoboxes have at least 30% overall amino acid sequence identity; there are a set of 9 residues which are absolutely conserved among a set of 16 homeobox-containing genes from Drosophila; and a number of other residues are highly conserved or have a common quality, e.g. are hydrophobic, positively charged, etc. (Gehring, Science 236, 1245). The mec-3 homeobox shares 24/60 amino acids with the Drosophila gene paired, has almost as high a level of identity with other fly homeoboxes and has 8 of the 9 absolutely conserved residues. The intron between the sixth and seventh exon is between amino acids 12 and 13; none of the other known homeoboxes have an intron at this position. Considerable evidence suggests that the homeodomain functions as a DNA binding region. Thus, an appealing hypothesis is that mec-3 encodes a DNA binding protein that mediates touch-cell differentiation by regulating the transcription of genes needed for that development, some of which are presumably included in the set of other mec genes. Although there is no evidence that there is a direct interaction between mec-3 and mec-4, for example, this hypothesis is the most parsimonious at this time. Bearing this in mind, the highly acidic nature of the final exon is particularly striking, because the yeast positive regulators GAL4 and GCN4 both mediate positive control through 'acid blobs,' regions of the proteins which are not involved in DNA binding and have no particular sequence requirement except that they be acidic (Ma and Ptashne, Cell 48, 847; Hope and Struhl, Cell 46, 885). The success rate of our transformations by microinjection has increased dramatically since we switched from a standard to an inverted microscope. Recently, all injections have been done with plasmids of 8-11 kb which carry mec-3 as the only C. elegans gene. In one recent experiment, 30 transformants were found among the progeny of 22 surviving injectants. Since each parent is injected several times, the actual success rate is about 20% per injected oocyte. We make no effort to hit the nucleus of the injected oocyte. With small plasmids (as opposed to cosmids or lambda clones), most of the rescue is 'transient expression.' Only about 1/20 initially transformed animal has transformed progeny, and the transforming DNA behaves as a very unstable free duplication.