Expression of pheromone binding proteins during antennal development in the gypsy moth Lymantria dispar

We have identified 2 olfactory specific proteins in the gypsy moth Lymantria dispar that are uniquely associated with the male antennae, the principal olfactory organs of this animal. These proteins were the major soluble protein components of the olfactory sensilla, present in equivalent amounts. Both proteins comigrated on SDS-PAGE, showing an apparent molecular mass of 15,000 Da but migrated separately on non-SDS-PAGE, indicating differences in net charge. N-terminal amino acid sequence analysis showed that the 2 proteins share 50% identity, indicating that they are genetically distinct homologs. Both proteins bound the L. dispar sexpheromone, associated with antisera prepared against the previously identified phermone-binding protein (PBP) of the moth Antheraea polyphemus, and shared sequence identity with the A. polyphemus PBP. These 2 proteins are therefore identified as L. dispar PBPs and are termed PBP1 and PBP2 based on their migration differences on non-SDS-PAGE. It is estimated that PBP1 and PBP2 are present in the sensilla lumen at a combined concentration of 13.4 mM. The expression of the L. dispar PBPs was examined during the 11 d development of the adult antenna. PBP1 and PBP2 were first detected by non-SDS-PAGE analysis and Coomassie blue staining 3 d before adult eclosion, on day A-3. Levels increased, reaching a plateau on day A-1 that continued into adult life. In vivo labeling studies indicated that the rate of PBP synthesis increased from A-3 to a plateau on A-2, where it remained into adult life. In vitro translations of antennal mRNAs indicated that translatable PBP mRNA was available at a very low level on day A-4, increased slightly on A-3 and dramatically on A-2, and remained at a high level into adult life. PBP mRNA represented the major translatable mRNA in the antenna during this period. It was estimated that the PBPs undergo a combined steady-state turnover of 8 x 10(7) molecules/hr/sensillum. Cursory in vivo and in vitro translation studies of antennal mRNA from A. polyphemus and Manduca sexta showed similar temporal patterns of PBP expression, suggesting that the L. dispar observations are general

MicroRNA-276a Functions in Ellipsoid Body and Mushroom Body Neurons for Naive and Conditioned Olfactory Avoidance in Drosophila

MicroRNA (miRNA)-mediated gene regulation plays a key role in brain development and function. But there are few cases in which the roles of individual miRNAs have been elucidated in behaving animals. We report a miR-276a::DopR regulatory module in Drosophila that functions in distinct circuits for naive odor responses and conditioned odor memory. Drosophila olfactory aversive memory involves convergence of the odors (conditioned stimulus) and the electric shock (unconditioned stimulus) in mushroom body (MB) neurons. Dopamine receptor DopR mediates the unconditioned stimulus inputs onto MB. Distinct dopaminergic neurons also innervate ellipsoid body (EB), where DopR function modulates arousal to external stimuli. We demonstrate that miR-276a is required in MB neurons for memory formation and in EB for naive responses to odors. Both roles of miR-276a are mediated by tuning DopR expression. The dual role of this miR-276a::DopR genetic module in these two neural circuits highlights the importance of miRNA-mediated gene regulation within distinct circuits underlying both naive behavioral responses and memory

Sequencing the Connectome

Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity. We propose BOINC ("barcoding of individual neuronal connections"), a method for converting the problem of connectivity into a form that can be read out by high-throughput DNA sequencing. The appeal of using sequencing is that its scale--sequencing billions of nucleotides per day is now routine--is a natural match to the complexity of neural circuits. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution could transform neuroscience research

Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of Bacillus subtilis

Development of genetic competence in Bacillus subtilis is controlled by the competence-specific transcription factor ComK. ComK activates transcription of itself and several other genes required for competence. The activity of ComK is controlled by other genes including mecA, clpC, and comS. We have used purified ComK, MecA, ClpC, and synthetic ComS to study their interactions and have demonstrated the following mechanism for ComK regulation. ClpC, in the presence of ATP, forms a ternary complex with MecA and ComK, which prevents ComK from binding to its specific DNA target. This complex dissociates when ComS is added, liberating active ComK. ClpC and MecA function as a molecular switch, in which MecA confers molecular recognition, connecting ClpC to ComK and to ComS

Retrotransposon activation contributes to neurodegeneration in a Drosophila TDP-43 model of ALS

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD

The bdbDC operon of Bacillus subtilis encodes thiol-disulfide oxidoreductases required for competence development

The development of genetic competence in the Gram-positive eubacterium Bacillus subtilis is a complex postexponential process. Here we describe a new bicistronic operon, bdbDC, required for competence development, which was identified by the B. subtilis Systematic Gene Function Analysis program. Inactivation of either the bdbC or bdbD genes of this operon results in the loss of transformability without affecting recombination or the synthesis of ComK, the competence transcription factor. BdbC and BdbD are orthologs of enzymes known to be involved in extracytoplasmic disulfide bond formation. Consistent with this, BdbC and BdbD are needed for the secretion of theEscherichia coli disulfide bond-containing alkaline phosphatase, PhoA, by B. subtilis. Similarly, the amount of the disulfide bond-containing competence protein ComGC is severely reduced in bdbC or bdbD mutants. In contrast, the amounts of the competence proteins ComGA and ComEA remain unaffected by bdbDC mutations. Taken together, these observations imply that in the absence of either BdbC or BdbD, ComGC is unstable and that BdbC and BdbD catalyze the formation of disulfide bonds that are essential for the DNA binding and uptake machinery

Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the bhydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

Expression and regulation of caudal in the lower cyclorrhaphan fly Megaselia

The homeobox gene caudal (cad) regulates posterior development in Drosophila. In early embryos, the cad protein (CAD) is expressed in a posterior-to-anterior concentration gradient, which contributes polarity to the developing embryo. The CAD gradient is complementary to and dependent on the anterior pattern organizer Bicoid (BCD), which represses the translation of ubiquitous maternal cad transcripts in the anterior embryo through a direct interaction with the cad 3′ untranslated region (UTR). Here, we show that early embryos of the lower cyclorrhaphan fly Megaselia express the putative cad orthologue Mab-cad throughout the posterior three quarters of the blastoderm but lack maternal transcripts. In transgenic blastoderm embryos of Drosophila, Mab-cad cis-regulatory DNA drives the expression of a reporter gene in a similar pattern, while Mab-cad 3′ UTR fails to mediate translational repression of a ubiquitously transcribed reporter. For another lower cyclorrhaphan fly (Lonchoptera) and two related outgroup taxa of Cyclorrhapha (Empis, Haematopota), we report maternal cad expression in ovarian follicles. Together, our results suggest that BCD is not required for the translational repression of Mab-cad, and that maternal cad expression was lost in the Megaselia lineage

Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum

As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis