Howard A. Baylis

The stringent response in Streptomyces coelicolor A3(2).

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 act III occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis atone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).

Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences

The 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. mutants 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.

Actions of the insecticide fipronil, on dieldrin-sensitive and- resistant GABA receptors of Drosophila melanogaster.

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: Rdl). 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→SS) 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, homo‐oligomeric RDL receptors should facilitate detailed studies of the molecular basis of convulsant/ insecticide antagonist actions on GABA receptors.

Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co‐expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)‐epibatidine, (−)‐nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two‐electrode, voltage‐clamp electrophysiology. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)‐epibatidine, (−)‐nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)‐nicotine, whereas (+)‐epibatidine and acetylcholine were full agonists. The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)‐epibatidine, (−)‐nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh‐mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.

Reduction of Caenorhabditis elegans frataxin increases sensitivity to oxidative stress, reduces lifespan, and causes lethality in a mitochondrial complex II mutant

Friedreich ataxia is an autosomal recessive neurological disorder caused by deficiency of the mitochondrial protein frataxin. Studies in patient cells, mouse knockout animals, and Saccharomyces cerevisiae models have suggested several hypotheses on the frataxin function, but the full physiology of frataxin in mitochondria has not been well established yet. We have characterized the genomic structure of frh‐1, the Caenorhabditis elegans frataxin gene, and we have developed a transient knockdown model of C. elegans frataxin deficiency by RNA interference. frh‐1(RNAi) worms show a consistent pleiotropic phenotype that includes slow growth, lethargic behavior, egg laying defects, reduced brood size, abnormal pharyngeal pumping, and altered defecation. Lifespan is significantly reduced, and worms have increased sensitivity to oxidative stress that, in turn, might explain the reduction of longevity of the worms. We also demonstrate synthetic genetic interaction between frh‐1 and mev‐1, the gene encoding the succinate dehydrogenase cytochrome b subunit of complex II in mitochondria, suggesting a possible role of the C. elegans frataxin in the electron transport chain; thus, the respiratory chain might be involved in the pathogenesis of the disease. We propose that this C. elegans model may be a useful biological tool for drug screening in Friedreich ataxia.

Detection of a carrier state in Theileria parva-infected cattle by the polymerase chain reaction

Two sets of oligonucleotide primers, one derived from a repetitive sequence and the other from the gene encoding a 67 kDa sporozoite antigen of Theileria parva, were used to amplify parasite DNA from the blood of T. parva-infected carrier cattle using the polymerase chain reaction (PCR). PCR amplification products were obtained from 15 carrier cattle infected with one of 4 different T. parva stocks. Successful amplifications were performed using DNA from 2 cattle infected with T. p. parva Pemba Mnarani, 10 cattle infected with T. p. parva Marikebuni, 2 cattle infected with T. p. bovis Boleni and 1 animal infected with T. p. lawrencei 7014. No amplification products were obtained from any of 7 cattle which had been infected with the T. p. parva Muguga stock. A synthetic oligonucleotide, which hybridized specifically to T. p. parva Marikebuni DNA among 6 T. parva stocks tested, was designed using sequence data from within the region of the T. parva genome amplified by the repetitive sequence primers. The oligonucleotide was used to probe PCR products and to increase the sensitivity and specificity of carrier animal detection. Southern blot analysis using a T. parva repetitive sequence probe demonstrated the existence of restriction fragment length polymorphisms between parasites isolated from T. p. parva Marikebuni-infected carrier cattle. The use of the PCR and other methods of carrier animal detection are discussed.

Infection with Theileria annulata induces expression of matrix metalloproteinase 9 and transcription factor AP-1 in bovine leucocytes.

Theileria annulata infects bovine leucocytes and results in their reversible transformation such that they become immortalised and metastatic. The present study describes parasite-induced changes in host cell gene expression which have a direct bearing on this transformation process. T. annulata-infected leucocytes produce a number of novel metalloproteinase activities. One of these, previously called B1, is a 97-kDa protein which is secreted in large amounts and has been purified from protein-free, conditioned medium. An antiserum to this enzyme was used to isolate a cDNA clone. The predicted protein sequence of B1 is 81% identical to human matrix metalloproteinase 9 (MMP9), demonstrating that it is the bovine homologue of this enzyme. RNAase protection assays demonstrated that the MMP9 activity, unique to infected cells, is due to increased MMP9 mRNA levels. We also assayed the levels of transcription factor AP-1 and demonstrated that it was constitutively present in increased amounts in Theileria-infected cells. In addition we assayed the level of mRNA encoding c-Fos, a common component of AP-1 and observed that it was indeed up-regulated in infected cells. Since AP-1 is implicated in the control of the cell cycle, and MMP9 can confer metastatic properties, these results are of considerable significance with respect to the transformed phenotype induced by Theileria infection.

Transcriptional analysis of the 16S rRNA gene of the rrnD gene set of Streptomyces coelicolor A3(2)

The nucleotide sequence of 2.5 kb of the Streptomyces coelicolor A3(2) rRNA gene set rrnD, extending from upstream of the 16S rRNA gene to the putative 5′ end of the 23S rRNA gene, has been determined (Baylis and Bibb, 1987; this paper). In addition to locating the 5′ end of the 16S rRNA gene, nuclease S1 mapping identified seven RNA 5′ end‐points upstream of the 16S rRNA gene; four of these were coincident with transcriptional initiation points for S. coelicolor A3(2) RNA poiymerase in vitro and were consequently regarded as in vivo transcription start points for promoters p1 to p4. One end‐point identified by nuclease S1 mapping localized a putative processing site analogous to those found upstream of 16S rRNA genes in other eubacteria. Sequence motifs similar to those discovered in low G+C Gram‐positive bacteria were found associated with two of the promoters and the processing site. A probable protein coding region was observed upstream of the promoter region.

Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line

RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wild-type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor by GABAergic insecticides.

Improved characterization of Theileria parva isolates using the polymerase chain reaction and oligonucleotide probes

Theileria parva DNA was purified from piroplasms isolated from cattle infected with 5 different East African isolates of the parasite, including the East Coast fever reference stock T. p. parva Muguga. Total cellular DNA was prepared from T. parva schizont-infected bovine lymphoblastoid cell cultures (11 isolates). Two probes, previously isolated from T. p. parva Muguga repetitive genomic DNA, were hybridized to restriction digests; closely similar restriction fragment length polymorphism (RFLP) patterns were produced, and no two isolates had the same RFLP pattern. The DNA sequences of probe PMB3, two further copies of the repeated sequence from T. p. parva Muguga, and homologous regions from T. p. parva Kiambu 4 and T. p. lawrencei 3081, were determined. Oligonucleotides were synthesized corresponding to two conserved sections flanking a region which varied between isolates. These oligonucleotides were used as primers in the polymerase chain reaction to amplify the variable region. Further oligonucleotides corresponding to sequences in this variable region were able to distinguish between isolates and no sample hybridized to both oligonucleotides. This is the first unequivocal plus/minus discrimination between groups of isolates to be achieved for T. parva.