Worm Breeder's Gazette 14(4): 60 (October 1, 1996)
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 Molecular and Medical Genetics, University of Toronto, Toronto, ON, CANADA
The her-1 locus, which specifies male sexual fates, contains two promoters that drive the synthesis of two male-specific transcripts in response to the X:A ratio. Our original characterization of her-1 revealed the stronger P2 promoter that regulates the smaller abundant mRNA is activated ubiquitously, early in embryogenesis. However, this transcript (and its accompanying promoter) is not necessary for her-1 rescuing function. Fusing fragments from the weaker, upstream, P1 promoter to lacZ reporters did not reveal reproducible patterns of embryonic gene expression. In contrast, this P1 promoter drives enough synthesis of her-1 to rescue XO her-1 null animals--demonstrating that biological assay for her-1 is more sensitive than beta-gal activity in fixed animals (Perry et al., (1993) G&D 7: 216). The discovery that reporter constructs containing multiple introns increase the steady-state levels of fusion proteins (Fire et al., (1995) 10th International C. elegans meeting, p. 213) encouraged us to re-evaluate the sex and tissue specificity of the her-1 P1 promoter. The same three fragments, from P1 (1.5 kb and 1.8 kb), and P2 (3.54 kb), were fused to five intron variants of GFP. Each construct was injected at a concentration of approx. 100 ug/mL (along with an equivalent amount of pRF4 (rol-6(d))). Analyses of reporter gene expression in initial lines carrying extrachromosomal arrays were reminiscent of the published data described above and not particularly illuminating. The her-1-P1::GFP levels approach the limit of detection; and her-1-P2::GFP expression is relatively easy to detect. Some GFP-positive eggs had unusual morphologies when viewed with DIC, as if the arrays disrupt or arrest embryonic development. Since further data collection was impeded by technical problems (see below) we are reluctant to interpret any intriguing expression patterns we saw. him-8 strains were assayed for sex-specific expression in these experiments; because more than one-third of the resulting F1 Rol animals are males this makes generating hermaphrodite Rol lines slightly more labour-intensive. Plasmids carrying either the her-1 P1 or P2 promoter can significantly alter this sex-ratio such that most--sometimes all--of the F1 Rol animals are males. Exceptional F1 Rol hermaphrodites rarely transmit F2 Rol hermaphrodites, adding to the labour-intensive nature of generating stable Rol lines. Closer inspection of the F1 progeny revealed two rare classes of small sickly animals: (1) Dpy, dead and dying hermaphrodites (some of these could be coaxed into rolling); and (2) variably Rol Tra (masculinized) intersexes. Infrequently the weakly Rol Dpy animals were partially masculinized. Establishing Rol lines with these plasmids in a non-Him genetic background is more challenging, although we discovered that rare Rol, Egl or Dpy animals sometimes segregate Rol, Egl or Dpy animals in non-Mendelian ratios, as if they carry an extrachromosomal array, and these animals contain GFP-positive embryos. Our interpretation of these observations is not that her-1 promoter arrays transform the missing XX hermaphrodites into healthy Rol XX males; rather, we believe both the glowing dead embryos, and the sick semi-Rol and Tra animals are the missing XX hermaphrodites. Since the her-1 gene is repressed in XX hermaphrodites the observation that increased dosage of the P1 or P2 promoter masculinizes suggests a model where a factor (or factors) that regulates her-1 binds instead to sites on the extrachromosomal arrays, resulting in expression of the endogenous her-1 gene (masculinization). We speculate that these factors are also involved in an essential process such as X-chromosome dosage compensation, which could explain the observed Dpy-ness and lethality. Three genes mutate to lf phenotypes of disrupted X-linked dosage compensation (with an attendant reduction in viability), and masculinization due to ectopic expression of her-1: sdc-1 X, sdc-2 X, and sdc-3 V (De Long et al., (1993) Genetics 133: 875, and references therein). We propose that one or more of these proteins binds directly to control elements in the her-1 gene, effectively reducing the amount of available sdc activity. Since the SDC-2 (Dawes et al., (1996) West Coast Worm Mtg. no. 36) and SDC-3 (Davis et al., (1995) 10th International C. elegans meeting, p. 54) proteins associate with X-chromosomes in XX hermaphrodites and form complexes with other dosage compensation proteins (Albrecht et al., (1996) West Coast Worm Mtg. no. 2), we cannot distinguish if we are titrating SDC proteins alone or proteins that bind to SDC proteins. Patience and perseverance have resulted in a few transmitting Rol lines carrying these plasmids. Perhaps continuous passaging of self-fertile animals selects arrays containing lower copy numbers of the toxic sequences. It is also likely that some partially Rol, partially Tra, self-fertile animals may be mosaic (i.e., some cells are less sensitive to sdc titration). We will vary the concentration of the injected DNA and test our hypothesis that the dominant sdc effect is dose-sensitive. A second prediction is that masculinization depends on her-1(+) so we will assay her-1(lf) strains for sexual transformation. The current assay, lack of Rol hermaphrodites, depends on a negative result. Since dying feminized xol-1 XO animals are rescued by sdc-2(lf) mutations we could assay dominant sdc phenocopy by scoring xol-1 XO animals for viability, a positive result (Miller et al., (1988) Cell 55: 167). Finally, it should prove possible to map finely the sequence elements conferring this sdc phenocopy by generating arrays with sub-fragments of DNA from the P1 and P2 promoters.