Worm Breeder's Gazette 13(4): 89 (October 1, 1994)

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.

An HLH-1 Reversion Screen.

Lihsia Chen1, Michael Krause2, Andrew Fire3

1 Carnegie Institute of Washington, Baltimore, MD 21210, The Johns Hopkins University, Dept of Biology, Baltimore, MD 21218.
2 NIH-Laboratory of Molecular Biology, NIDDK, Bethesda, MD 20890.
3 Carnegie Institute of Washington, Baltimore, MD 21210.

  The MyoD family of transcription factors has been implicated in the
determination and differentiation of vertebrate skeletal muscles. A single homologue
of the MyoD family, designated hlh-1 ,was identified in C. elegans; this gene is
expressed in body wall muscle cells and their clonal precursors(1). Animals
homozygous for an hlh-1 null mutation ( cc450 )die as larvae or young adults, suffering
muscle contraction and morphogenesis defects. Despite these defects, the hlh-1 ( cc450
)mutants appear to contain normal numbers of body wall muscles with surprisingly
well-organised myofilament structure(2).
  Elucidating the role of HLH-1 will entail the identification of its regulatory targets
and of the factors that promote myogenesis in its absence. We have taken both
biochemical (cDNA subtraction screening) and genetic approaches to this question.
This article describes a genetic reversion screen undertaken to identify compensating
regulatory mutations, affecting either a definitive Hlh-1 target or a parallel myogenic
pathway. The starting strain used in the screen was homozygous for hlh-1 ( cc450
),and was rescued by an extrachromosomal array carrying a handicapped hlh-1 gene
with the last two exons deleted (the array also carries the dominant visible marker
rol-6 ( su1006 ).While the array allows viability and fertility in the cc450 background,
the rescue is incomplete, with resulting animals exhibiting poor movement and slow
growth. Potential revertants can be easily identified in this screen as animals that
exhibit improved movement and growth. There are five types of mutations that might
give the above phenotype:
  1) intragenic reversion at the hlh-1 locus;
  2) extragenic suppressors which bypass the requirement for hlh-1 ;
  3) amber suppressors ( cc450 is an amber mutation) and potentially other
informational suppressors, which might allow HLH-1 protein expression by the cc450
mutant allele;
  4) mutations in the transgene array which allow improved expression or function
of the handicapped hlh-1 gene; and
  5) chromosomal mutations which improve the rescued phenotype of array
containing animals. We were most interested in whether mutations of class 2 could be
isolated (it should also be noted that class 5 might also include loci with direct roles in
myogenic regulation). To bias ourselves against mutations in the transgene array, we
chose animals that no longer express a strong Rol-6 phenotype.
  Following EMS mutagenesis, we isolated l0 strains that displayed improved
movement (from 30,660 chromosomes). Three of these strains proved to be amber
suppressors; this gives an indication of the completeness of the screen, since these are
likely to represent anticodon transitions in one of the four known amber suppressors
previously shown to suppress cc450 .Of the remaining seven strains identified in our
screen, four still contained the extrachromosomal array, suggesting that the array was
still required for viability of the strains. In one other strain, the array has integrated
into a chromosome. We don't know for these five strains whether the array or an
endogenous locus has mutated to allow the improved rescue.
  Two revertants which do not require the transgene DNA were recovered. One
contained a semi-dominant suppressing mutation that is tightly linked to hlh-1 ;this
strain may be a second site intragenic revertant (the cc450 mutation is still present).
While rescue appears to be complete in this strain, the animals have longer bodies than
wild type. The second strain contains an unlinked semi-dominant suppressor.
Rescue in this strain is only partial. Both strains stain strongly with antibodies
against the Hlh-1 protein. We are currently trying to determine in these two strains if
the HLH-1 antibody recognizes Hlh-1 protein or a (as yet hypothetical) cross-reacting
Hlh-1 homologue which has been overexpressed.
  The screen described above was an extensive search for mutations that can
produce healthy animals capable of fairly good movement in the cc450 background.
The strain used, however, did not allow for the identification of mutations that might
less completely suppress defects in cc450 mutants. We are currently carrying out
additional genetic screens that should enable us to identify such mutations.
 (1) Krause et al. (1990). Cell 63, 907-919.
 (2) Chen et al. (1994). Development 120, 1631-1641.