[
Worm Breeder's Gazette,
1993]
Fluoroacetic acid which inhibits aconitase, an enzyme in both the Krebs and glyoxylate cycles, was discovered to be a potent and specific inhibitor of reproduction in a toxicity test using the nematode Caenorhabditis elegans. Fluoroacetic acid reduced reproduction in the second generation by 50% at concentrations 3000 times lower than the LC(50) of 76 mM. Four concentrations (1.7, 4.2,8.5, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations the survival rates were unaffected, and first generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. To determine whether fluoroacetic acid inhibited reproduction because it interfered with both the Krebs cycle and the glyoxylate cycle, malonic acid which inhibits the Krebs cycle and itaconic acid which inhibits the glyoxylate cycle were tested individually and in combination against C. elegans. If the potent reproduction effect of fluoroacetic acid relative to lethality is caused by inhibiting both the Krebs and glyoxylate cycles, the combination of malonic acid and itaconic acid would be expected to produce results qualitatively similar to those obtained with fluoroacetic acid. For the mixture, a large ratio of the LC(50) to EC(50) for reproduction would indicate synergy. Concentration-response curves for the survival assay and the reproduction assay were developed for each test compound and combination of compounds. The probit method was used to calculate the LC(50) and EC(50) for reproduction. The ratio of the LC(50) to EC(50) was calculated for each and is presented in the Table. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid, possibly one that is specific to reproduction and possibly one which affects aconitase.
[
Worm Breeder's Gazette,
1996]
We have isolated a C. elegans cDNA (accession no. U24189) that encodes a putative 388 amino-acid RNA-binding protein with two RNA recognition motifs (RRMs). The RNA-binding protein shows extensive similarity to the spliceosome-associated protein 49 (SAP 49) which has been shown to be tightly involved in U2 snRNP function in mammalian cells (Champion-Arnaud and Reed, Genes Dev. 8, 1974-83, 1994). Surprisingly, the cDNA also contains two additional ORFs upstream of the coding region for the C. elegans SAP 49 homologue (cSAP49). The product deduced from the first ORF shows no homology with any known protein, whereas the product of the second ORF encodes an actin-related protein. Since very short spacer regions containing the possible poly(A) and 3' splice sites are present between these ORFs, and since these ORFs are continuous in the genome sequence (CEC08B11; accession no. Z46676), it is suggested that these ORFs are first transcribed in a single primary transcript and then processed into three independent mRNAs by trans-splicing mechanism. Indeed, we confirmed three different mRNAs corresponding to the three putative proteins by Northernblot analysis. By microinjection of the GFP- and LacZ-fusion constructs of cSAP49 under the control of the native promoter, we have found that it is expressed at the restricted regions of the developing worm, mainly in the nuclei of intestine tissues. This expression pattern is puzzling because the human SAP 49 is thought to bear the essential function in all cells. In addition, using an in vitro selection method, we have demonstrated that cSAP49 possesses specific RNA binding ability which is attributed to the second RRM. Its RNA binding is in essentially a sequence-specific manner but is affected by the surrounding sequence context, suggesting that cSAP49 may recognize the secondary or tertiary structure as an additive information. We speculate that a possible target for the cSAP49 may be the U2 snRNA since it is highly structured and contains a sequence similar to a part of the consensus sequence of cSAP49-selected RNAs. We are currently examining whether cSAP49 really binds to the U2 snRNA in the presence or absence of another U2-associated protein SAP 145, and whether there would be other tissue-specific SAP 49 homologue in C. elegans.