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We investigated the cis-acting sequences regulating expression of the Caenorhabditis elegansgene hlh-1, a homolog of the MyoD family of myogenic regulatory factors. The hlh-1gene is expressed in mature body wall muscle, in clonal muscle precursors, in a set of early embryonic blastomeres (the MS-granddaughters), and in six glial-like cells called GLRs. The entire natural hlh-1expression pattern is recapitulated in transgenic animals containing an hlh-1:: lacZfusion with 5.1 kb of hlh-1sequences beginning upstream of the coding region and extending into the second exon. Deletions and rearrangements in this 5.1-kb sequence were assayed for their effects on reporter gene expression in transgenic animals. Deletions removing a segment 434-550 bp upstream of the coding region resulted in a loss of all aspects of the expression pattern, suggesting that at least one common regulatory factor is required for all expression. Deletions of other regulatory elements affected distinct aspects of the expression pattern; hence, each expression subpattern exhibits a different set of sequence requirements. Interspecies sequence comparison and lacZexpression constructs with the related nematode Caenorhabditis briggsaeindicated that the C. briggsaeand C. elegansgenes contain equivalent sets of control signals. The results from this work implicate the hlh-1promoter as an integration point for diverse temporal and spatial control signals.

We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than ‘sense’ RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.