In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA ÷ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the sah-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain >70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.

The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR ␣ subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two ␣ types (UNC-38 and UNC-63) and two non-␣ types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.

The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of actIII occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis alone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).

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1 Blocking actions of the novel insecticide, fipronil, were examined on GABA responses recorded from Xenopus oocytes expressing either wild type (dieldrin-sensitive) or mutant (dieldrin-resistant) forms of the Drosphila melanogaster GABA-gated chloride channel homo-oligomer, RDL (the product of the resistance to dieldrin locus: Rdt).2 In the case of the wild type receptor, fipronil blocked GABA-induced currents inducing both a shift to the right in the GABA dose-response curve and depressing the maximum amplitude of responses to GABA. The potency of fipronil was dependent on the GABA concentration but was unaffected by membrane potential. 3 Mutant RDL GABA-receptors, which have a naturally occurring amino acid substitution (A302--S) in the putative ion-channel lining region, conferring resistance to dieldrin and picrotoxinin, were markedly less sensitive to fipronil than the wild-type receptors. 4 Fipronil antagonism is qualitatively similar to that produced by the structurally distinct compound, picrotoxinin. As the mutation A302_.S reduces the potency of both fipronil and picrotoxinin, homooligomeric RDL receptors should facilitate detailed studies of the molecular basis of convulsant/ insecticide antagonist actions on GABA receptors.

Spinal muscular atrophy (SMA) is a common disorder characterized by loss of lower motor neurones of the spinal cord. The disease is caused by mutations in the survival motor neurone ( SMN ) gene. SMN is ubiquitously expressed and evolutionarily conserved, and its role in RNA processing has been well established. However, these properties do not explain the observed specificity of motor neurone death. To gain further insight into the function of SMN, we have isolated and characterized the Caenorhabditis elegans orthologue of the SMN gene ( CeSMN ). Here we show that CeSMN is transmitted maternally as a predominantly nuclear factor, which remains present in all the blastomeres throughout embryonic development and onwards into adulthood. In adult nematodes, a CeSMN-green fluorescent protein fusion protein is expressed in a number of cell types including the germline. Both disruption of the endogenous CeSMN function and overexpression of the gene result in a severe decrease in the number of progeny and in locomotive defects. In addition, its transient knockdown leads to sterility caused by a defect in germ cell maturation. The expression pattern and functional properties so far observed for CeSMN, together with its unusual behaviour in the germline, indicate that SMN may be involved in specific gene expression events at these very early developmental stages. We have also identified a deletion in the CeSMN promoter region in egl-32. This mutant may become a useful genetic tool with which to explore regulation of CeSMN and hence provide possible clues for novel therapeutic strategies for SMA.

1 Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (a4b2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal a subunit (SAD) with the chicken b2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (7)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. 2 Imidacloprid alone of the 4 agonists behaved as a partial agonist on the a4b2 receptor; (+)-epibatidine, (7)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken b2 receptor, as was (7)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. 3 The EC 50 of imidacloprid was decreased by replacing the chicken a4 subunit with the Drosophila SAD a subunit. This a subunit substitution also resulted in an increase in the EC 50 for (+)-epibatidine, (7)-nicotine and acetylcholine. Thus, the Drosophila (SAD) a subunit contributes to the greater apparent anity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. 4 Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADb2 hybrid receptors and as a weak potentiator of ACh responses mediated by a4b2 receptors. This suggests that imidacloprid has complex eects upon these recombinant receptors, determined at least in part by the a subunit.

SUMMARYThe complete small subunit ribosomal RNA (srRNA) gene of Theileria parva was cloned and sequenced. Two primers were designed which permitted the specific amplification of part of the Theileria srRNA gene from Theileria-infected cell line samples which were predominantly (> 95%) bovine DNA. The sequence of the central (variable) region of the srRNA genes of T. annulata, T. taurotragi, T. mutans and two unidentified parasites referred to as Theileria sp. (buffalo) and Theileria sp. (Marula) were obtained. An alignment of the sequences was generated from which 6 oligonucleotide probes, corresponding to species-specific regions, were designed. These probes were demonstrated to provide unequivocal identification of each of the 6 species either by direct detection of parasite srRNA or by hybridization to amplified parasite srRNA genes. The probes were not able to distinguish buffalo-derived T. parva, the causal agent of Corridor disease, from cattle-derived T. parva, the causal agent of East Coast fever.