Worm Breeder's Gazette 16(5): 38 (February 1, 2001)
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.
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, CANADA
The monoclonal antibody MH33 was produced by Francis and Waterston (1985) and detects a structure beneath the microvilli of intestinal cells, probably the terminal web. At the comma stage of embryogenesis, staining is both apical and cytoplasmic; apical staining becomes predominant in later stages up to the adult. The MH33 staining pattern differs from that produced by MH27, which recognizes a component of the adherens junction at the basolateral side of gut cells. To identify the MH33 antigen, we screened a C. elegans expression library (kindly provided by Dr. B. Barstead) by using MH33 antibody (kindly provided by Drs. Hresko and Waterson) and isolated 24 positive clone from ~ 100,000 plaques screened. A cross hybridization experiment indicated that these positive clones all encoded the same product. Several of the largest inserts were sequenced and identified gene F10C1.7, which encodes an intermediate filament. Dodement et al., (1994) had previously identified this protein as intermediate filament b2. Two observations suggest that we have indeed cloned the correct gene. (1) Westerns could easily detect a ~60kD band in extracts from twenty worms carrying a complete transgene; (Francis and Waterston (1991) originally reported that MH33 identifies bands at 62 and 64 kD, and Dodement et al. showed that alternative splicing gives rise to two forms of b2 that differ in size by ~ 2kD). (2) A five kb fragment from the F10C1.7 5!-flanking region, fused to pPD96.04, drives gut specific expression, starting at comma stage and continuing in all later stages. The MH33-detected gut-specific intermediate filament gene should be useful as a marker of early gut development (the gene is a potential direct target of elt-2, for example, and contains the usual suspiciously located WGATAR sites) and as a probe of intestinal cell structure, in particular the generation of apical-basal polarity during gut development.