Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur

The self-organizing fractal theory as a universal discovery method: the phenomenon of life

A universal discovery method potentially applicable to all disciplines studying organizational phenomena has been developed. This method takes advantage of a new form of global symmetry, namely, scale-invariance of self-organizational dynamics of energy/matter at all levels of organizational hierarchy, from elementary particles through cells and organisms to the Universe as a whole. The method is based on an alternative conceptualization of physical reality postulating that the energy/matter comprising the Universe is far from equilibrium, that it exists as a flow, and that it develops via self-organization in accordance with the empirical laws of nonequilibrium thermodynamics. It is postulated that the energy/matter flowing through and comprising the Universe evolves as a multiscale, self-similar structure-process, i.e., as a self-organizing fractal. This means that certain organizational structures and processes are scale-invariant and are reproduced at all levels of the organizational hierarchy. Being a form of symmetry, scale-invariance naturally lends itself to a new discovery method that allows for the deduction of missing information by comparing scale-invariant organizational patterns across different levels of the organizational hierarchy

Genome-scale spatiotemporal analysis of Caenorhabditis elegans microRNA promoter activity

The Caenorhabditis elegans genome encodes more than 100 microRNAs (miRNAs). Genetic analyses of miRNA deletion mutants have only provided limited insights into miRNA function. To gain insight into the function of miRNAs, it is important to determine their spatiotemporal expression pattern. Here, we use miRNA promoters driving the expression of GFP as a proxy for miRNA expression. We describe a set of 73 transgenic C. elegans strains, each expressing GFP under the control of a miRNA promoter. Together, these promoters control the expression of 89 miRNAs (66% of all predicted miRNAs). We find that miRNA promoters drive GFP expression in a variety of tissues and that, overall, their activity is similar to that of protein-coding gene promoters. However, miRNAs are expressed later in development, which is consistent with functions after initial body-plan specification. We find that miRNA members belonging to families are more likely to be expressed in overlapping tissues than miRNAs that do not belong to the same family, and provide evidence that intronic miRNAs may be controlled by their own, rather than a host gene promoter. Finally, our data suggest that post-transcriptional mechanisms contribute to differential miRNA expression. The data and strains described here will provide a valuable guide and resource for the functional analysis of C. elegans miRNAs

Gene-centered regulatory networks

Differential gene expression plays a critical role in the development and physiology of multicellular organisms. At a ‘systems level’ (e.g. at the level of a tissue, organ or whole organism), this process can be studied using gene regulatory network (GRN) models that capture physical and regulatory interactions between genes and their regulators. In the past years, significant progress has been made toward the mapping of GRNs using a variety of experimental and computational approaches. Here, we will discuss gene-centered approaches that we employed to characterize GRNs and describe insights that we have obtained into the global design principles of gene regulation in complex metazoan systems

Gene-centered regulatory network mapping

The Caenorhabditis elegans hermaphrodite is a complex multicellular animal that is composed of 959 somatic cells. The C. elegans genome contains approximately 20,000 protein-coding genes, 940 of which encode regulatory transcription factors (TFs). In addition, the worm genome encodes more than 100 microRNAs and many other regulatory RNA and protein molecules. Most C. elegans genes are subject to regulatory control, most likely by multiple regulators, and combined, this dictates the activation or repression of the gene and corresponding protein in the relevant cells and under the appropriate conditions. A major goal in C. elegans research is to determine the spatiotemporal expression pattern of each gene throughout development and in response to different signals, and to determine how this expression pattern is accomplished. Gene regulatory networks describe physical and/or functional interactions between genes and their regulators that result in specific spatiotemporal gene expression. Such regulators can act at transcriptional or post-transcriptional levels. Here, I will discuss the methods that can be used to delineate gene regulatory networks in C. elegans. I will mostly focus on gene-centered yeast one-hybrid (Y1H) assays that are used to map interactions between non-coding genic regions, such as promoters, and regulatory TFs. The approaches discussed here are not only relevant to C. elegans biology, but can also be applied to other model organisms and humans

Characterisation of isolates of Haemophilus paragallinarum from Indonesia

Objective: To characterise 18 isolates of Haemophilus paragallinarum isolated from chickens in Indonesia. Procedure: The isolates were identified to species level by traditional phenotypic methods. Six of the isolates were also identified by a species-specific polymerase chain reaction. Fourteen of the isolates were examined for resistance to a panel of seven antimicrobial agents using a disc diffusion method. All 18 isolates were serotyped according to the Page scheme using reference antisera in a haemagglutination inhibition test. Results: Four of the 18 isolates were obtained from indigenous (kampung) chickens, with the remainder being from typical intensive poultry production systems. The 18 isolates were obtained from 11 outbreaks that showed the typical clinical signs of infectious coryza and 11 of the isolates were obtained from chickens that had been vaccinated with infectious coryza vaccines. All 18 isolates were confirmed as H paragallinarumby biochemical testing and six isolates were also identified as H paragallinarum by the polymerase chain reaction test. Eleven isolates were resistant to erythromycin and streptomycin, 10 to neomycin, eight to oxytetracycline, five isolates to doxycycline, three to sulphamethoxazoltrimethoprim but only one to ampicillin. Seven isolates were Page serovar A, four were Page serovar B and seven were Page serovar C. Conclusion: The presence of all three Page serovars (A, B and C) has been confirmed for the first time in Indonesian chickens. As the majority of the infectious coryza vaccines in use in Indonesia contain only serovar A and C, the presence of serovar B in chickens indicates that the protection by these bivalent vaccines would be reduced. The use of trivalent infectious coryza vaccines that contain serovars A, B and C is recommended for use in Indonesia