Short RNAs repress translation after initiation in mammalian cells

MicroRNAs (miRNAs) are predicted to regulate 30% of mammalian protein-encoding genes by interactions with their 3' untranslated regions (UTRs). We use partially complementary siRNAs to investigate the mechanism by which miRNAs mediate translational repression in human cells. Repressed mRNAs are associated with polyribosomes that are engaged in translation elongation, as shown by puromycin sensitivity. The inhibition appears to be postinitiation because translation driven by the cap-independent processes of HCV IRES and CrPV IRES is repressed by short RNAs. Further, metabolic labeling suggests that silencing occurs before completion of the nascent polypeptide chain. In addition, silencing by short RNAs causes a decrease in translational readthrough at a stop codon, and ribosomes on repressed mRNAs dissociate more rapidly after a block of initiation of translation than those on control mRNAs. These results suggest that repression by short RNAs, and thus probably miRNAs, is primarily due to ribosome drop off during elongation of translation.     

Mutations in hepatitis C virus RNAs conferring cell culture adaptation

As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. These replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the replication levels of these RNAs within selected cells were high, the number of G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified several adaptive mutations enhancing the efficiency of colony formation by several orders of magnitude. Adaptive mutations were found in nearly every nonstructural protein but not in the 5' or 3' nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, increasing the number of colonies approximately 500-fold. This mutation was conserved with RNAs isolated from one cell line, in contrast to other amino acid substitutions enhancing the efficiency of colony formation to a much lesser extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation drastically, suggesting that some adaptive mutations are not compatible.     

Remarks on the environmental adaptation of man

Summary In this paper the geographical variability of some morphological, physiological and serological traits of man is discussed with respect to the question, how far this variability can be considered as the result of selective adaptation processes. Though there is already some evidence supporting such an assumption, much more detailed and exactly planned research is necessary to clear the indubitable relations between the distribution patterns of anthropological traits and the various environmental conditions of human biotops. Particularly much more work is required to understand the causative mechanisms on which these relations are based.

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Zusammenfassung In dieser zusammenfassenden Untersuchung wird die geographische Variabilität in der Verteilung morphologischer, physiologischer und serologischer Merkmale des Menschen diskutiert und zwar unter dem Aspekt, inwieweit diese Variabilität als Ergebnis selektiver Adaptationsprozesse interpretiert werden kann. Obwohl es bereits eine Reihe von stichhaltigen Gründen für eine solche Annahme gibt, sind weitere, intensive Untersuchungen zur Klärung der zweifellos vorhandenen Beziehungen zwischen dem geographischen Verteilungsmuster teilungsmuster anthropologischer Merkmale und den unterschiedlichen Umweltbedingungen der verschiedenen menschlichen Biotope erforderlich. Insbesondere müssen sich künftige Untersuchungen noch stärker um eine exakte Erfassung der möglichen Ursachen bemühen, die den Beziehungen zwischen Merkmalsverteilung und Umweltgegebenheiten zugrunde liegen.

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Based on a lecture at the International Symposium on Human Genetics at Waltair (India), March 2–6, 1971.     

Cleavage of Short RNAs Containing Higher Ordered Structures by Hammerhead Ribozymes

Abstract

Cleavage of two types of secondary structure-forming substrates by their cognate hammerhead ribozymes were studied by measuring their kinetic parameters. A substrate with a self-complementary structure (GGUCCUAGGA, CL-3) was slowly cleaved by a two-stranded ribozyme. An isomer having no complementary sequence (GGUCGUAGCA, CL-3N) was cleaved more than 10 times faster than the self-complementary substrate. A newly designed ribozyme which contained a stable loop and stem cleaved the self-complementary decamer 40 times faster than the two-stranded ribozyme. A 15 mer which derived from a ras mRNA was found to have an intermolecular base pairs and was used to design more efficient ribozymes. Gel mobility shift assay was employed to investigate the binding properties of substrates to ribozymes. Investigations of the thermodynamic stability of the ribozyme-substrate complex are essential in the design of ribozymes that efficiently cleave RNA.

     

High-throughput screening of microbial adaptation to environmental stress

We developed a microwell plate, high-throughput, screening method aimed at quantitating the tolerance of a panel of Gram-positive and Gram-negative bacteria to metals (Frankia sp., Escherichia coli, Cupriavidus metallidurans, Rhizobium leguminosarum, and Streptomyces scabies). Microbial viability was quantified using MTS; a tetrazolium salt converted to a water-soluble formazan through microbial reduction. In this paper, we present the stepwise development of the method, highlighting the main elements underlying its reliability, and compare results obtained with literature. We conclude the method is well suited to efficiently screen bacteria, including those that are filamentous and slow-growing, without the need for large amounts of inoculum which may not always be available. The method allows testing of compound gradients with sufficient replicates to generate statistically robust results, and is transposable to other types of cell proliferation assays such as those for antimicrobial susceptibility, and chemoresistance.     

Dormant stages in freshwater bryozoans--an adaptation to transcend environmental constraints

Freshwater invertebrates often disperse between discrete habitat patches via the production of dormant propagules. Being dispersed passively by animal vectors or wind, certain adaptations for exposures to terrestrial and aerial conditions like desiccation and freezing are required. In the present study, we investigate the mechanisms of survival and physiological adaptations due to desiccation and low temperatures in the statoblasts of two populations of the freshwater bryozoan Cristatella mucedo. Our results show that both sessoblasts and floatoblasts tolerate almost complete desiccation and subzero temperatures. Trehalose, a non-reducing disaccharide which has been related to desiccation tolerance, was detected by amperometric chromatography. However, due to the low concentrations found, it is unlikely that trehalose is playing a major part in desiccation tolerance of bryozoan statoblasts. Vitrification is assumed to be important in the survival of desiccation tolerant organisms. Differential scanning calorimetry revealed thermal transitions (T_g onset around 70 °C) in desiccated statoblasts, indicating that a vitreous matrix is present. During the exposure to subzero temperatures, freeze tolerance of statoblasts was confirmed by the detection of internal ice formation, which took place at a crystallisation temperature of between −6 °C and −12 °C.     

Temperature-responsive release of thyroxine and its environmental adaptation in Australians

The hormone thyroxine that regulates mammalian metabolism is carried and stored in the blood by thyroxine-binding globulin (TBG). We demonstrate here that the release of thyroxine from TBG occurs by a temperature-sensitive mechanism and show how this will provide a homoeostatic adjustment of the concentration of thyroxine to match metabolic needs, as with the hypothermia and torpor of small animals. In humans, a rise in temperature, as in infections, will trigger an accelerated release of thyroxine, resulting in a predictable 23% increase in the concentration of free thyroxine at 39°C. The in vivo relevance of this fever-response is affirmed in an environmental adaptation in aboriginal Australians. We show how two mutations incorporated in their TBG interact in a way that will halve the surge in thyroxine release, and hence the boost in metabolic rate that would otherwise occur as body temperatures exceed 37°C. The overall findings open insights into physiological changes that accompany variations in body temperature, as notably in fevers.     

The effects of prenatal and postnatal environmental interaction: Prenatal environmental adaptation hypothesis

Adverse antenatal maternal environments during pregnancy influence fetal development that consequently increases risks of mental health problems including psychiatric disorders in offspring. Therefore, behavioral and brain alterations caused by adverse prenatal environmental conditions are generally considered as deficits. In this article, we propose a novel hypothesis, along with summarizing a body of literatures supporting it, that fetal neurodevelopmental alterations, particularly synaptic network changes occurring in the prefrontal cortex, associated with adverse prenatal environmental conditions may be adaptation to cope with expected severe postnatal environments, and therefore, psychiatric disorders may be able to be understood as adaptive strategies against severe environmental conditions through evolution. It is hoped that the hypothesis presented in this article stimulates and opens a new venue on research toward understanding of biological mechanisms and therapeutic treatments of psychiatric disorders.     

The moose,purine degradation,and environmental adaptation

It is accepted that allantoin is the end-product of purine degradation in mammals, except that uricase activity has been lost during the evolution of humans in which uric acid protects the brain from oxidative damage. However, we have found that the moose Alces americanus excretes extremely low urinary concentrations of allantoin and high concentrations of uric acid very similar to those of humans. Exposure to extreme cold is known to cause oxidative damage, and we suggest that the retention of uric acid by the moose represents an adaptation enabling the species to survive at high latitudes.