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.

Some precious hermaphrodites have gone missing: Transgenic arrays of her-1 V promoter fragments induce dominant sdc phenocopy

Marc Perry, Nimerta Rajwans, Richard Ikegami

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
     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.