The sex determination gene
her-1 is required to specify male development. Mosaic and sequence analyses have suggested that
her-1 encodes a secreted ligand involved in a cell signaling event, which might ensure that all cells in an individual undergo the same sexual differentiation (Hunter and Wood, 1992; Perry et al., 1993). However, the structure of the active protein and its mode of action are unknown. In order to characterize the structure and subcellular localization of Her-1 we have raised an antibody against a Her-1/MBP fusion protein. Even though this antibody recognizes Her-1 produced in bacteria and Xenopus oocytes we have not been able to detect endogenous Her-1 in whole mounts or on Western blots. We tried to overexpress HA-and myc-tagged Her-1 under the control of the heat shock promoter in vivo. Several extrachromosomal arrays which rescue the
her-1 null mutant have been obtained but we have not been able to detect Her-1 protein in any of these lines with either our anti-Her-1 antibody or with the corresponding anti-tag antibody. We are currently attempting to concentrate the protein by immunoprecipitation prior to Western blot analysis. As an alternative approach to learn what parts of Her-1 could be important for function we are attempting to clone
her-1 from C. remanei. Since earlier attempts using
her-1 as probe for low stringency hybridization have failed we are trying to clone by synteny (Kuwabara and Shah, 1994). Trent et al. (1991) have described two non-sex-specific transcripts flanking
her-1 in C. elegans. Probes that recognize these transcripts were used to screen Chris Link's C. remanei ssp. vulgaris genomic library. In the primary screen two plaques which cross-hybridize with the upstream
her-1 probe have been isolated. In addition to the 1.2kb mRNA, which is necessary and sufficient for all known functions,
her-1 encodes a second, 0.8kb, RNA which consists of the last two exons (exons 3 and 4) of the 1.2kb transcript transspliced to SL1. The 0.8kb RNA is transcribed from its own promoter (P2) which is located in the second intron of the 1.2kb transcript. This RNA potentially codes for the last 64 amino acids of Her-1 if the translational machinery can overcome an upstream out-of-frame AUG which is followed immediately by a stop codon. In order to test whether this small transcript is translated in vivo we injected a construct which contains an HA-tagged version of the 0.8kb transcript under the control of P2 into
him-8 worms. So far we have not been able to detect any protein from this construct in whole mounts. However, we have observed an unexpected under-representation of hermaphrodites in four independent transmitting lines which we are currently investigating further. References: Trent, C. et al., (1991) Mech. Dev. 34, 43-56; Hunter, C. P. and Wood, W. B., (1992) Nature 355, 551-555; Perry, M. D. et al. (1993) Genes Dev. 7, 216-228; Kuwabara, P. E. and Shah, S., (1994) Nucleic Acids Res. 22, 4414-4418.