‘RNA walk’ a novel approach to study RNA–RNA interactions between a small RNA and its target

In this study we describe a novel method to investigate the RNA–RNA interactions between a small RNA and its target that we termed ‘RNA walk’. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT–PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by ‘RNA walk’ and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that ‘RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.     

The promoter of Milk vetch dwarf virus component 8 confers effective gene expression in both dicot and monocot plants

The activity of a predicted promoter, PMC8, from Milk vetch dwarf virus was evaluated by comparing it with the cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues.     

Multifactor dimensionality reduction reveals a strong gene–gene interaction between STC1 and COL11A1 genes as a possible risk factor of knee osteoarthritis

Articular cartilage is an avascular tissue with a structure that allows it to support and cushion the overload of the surfaces in contact. It maintains its metabolic functions due to the contribution of different signaling pathways. However, several factors play a role in its deterioration, allowing to the development of osteoarthritis (OA), and one of the major factors is genetic. Our goal was to identify gene–gene interactions (epistasis) between five signaling pathways involved in the articular cartilage metabolism as possible indicators of OA risk. We applied the Multifactor-Dimensionality Reduction (MDR) method to identify and characterize the epistasis between 115 SNPs located in 73 genes related to HIF-1α, Wnt/β-catenin, cartilage extracellular matrix metabolism, oxidative stress, and uric acid transporters. Ninety three patients diagnosed with primary knee OA and 150 healthy controls were included in the study. Genotyping was performed with the OpenArray system, the statistical analysis was carried out with the STATA software v14, and epistasis was analyzed with the MDR software v3.0.2. The MDR analysis revealed that the best interaction model was between polymorphisms rs17786744 of the STC1 gene and rs2615977 of the COL11A1 gene, with an entropy value of 4.44%, CVC 8/10, OR 5.60, 95% CI 3.27–9.59, p?<?0.0001. Under this interaction model, we identified high and low risk genotypes involved in OA development. Our results suggest complex interactions between STC1 and COL11A1 genes that might have an impact on genetic susceptibility to develop OA. Further studies are required to confirm it.

     

The dynamics of trade in live wildlife across the Guangxi border between China and Vietnam during 1993–1996 and its control strategies

A survey on trade in live wild animals was conducted along the Guangxi border between China and Vietnam during 1993–1996. The results showed that there were 55 species, which were underestimated, involved in the trade, including 15 species of mammals, 10 species of birds, 29 species of reptiles and 1 species of amphibian. Many of them were species listed on Appendices I and II of CITES and on China's protection list (known as ASSP). During the investigation, about 2.29–29.325 tons of wildlife per day were imported to China form Vietnam. Some species with a large volume of trade may be over-exploited. Sixty-three percent of middle–high quality restaurants in three border cities and Nanning in Guangxi sold wildlife foods. Twenty kinds of wild animals were eaten at the restaurants. Although numbers of species involved in the trade remained fairly stable over the survey period, numbers of species listed in Appendices I and II of CITES and the state protection list had declined. The scale of wildlife markets in border cities of Guangxi and consumption levels of wildlife in the restaurants had been reduced because border control measures were enforced on the trade by local governments of China. However, the prices of the 5 main species in wildlife markets were still going up. This suggested that demand for wildlife in Guangxi was strong and will facilitate the illegal trade. The key steps to control the illegal wildlife trade between the two countries should focus on (i)suppression of illegal wildlife markets and prohibition of the sale of wildlife food in restaurants consisting of species listed in the ASSP inventory; (ii) international cooperation in the control of the trade; (iii) tighter enforcement of CITES for both countries; (iv) control of invasion of exotic species and epidemics of disease in the trade in China; and (v) education for wildlife conservation in China.     

RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides(Lepidoptera:Crambidae)

Trehalose 6-phosphate synthase(TPS),an enzyme that hydrolyzes two glucose molecules to yield trchalose,plays a pivotal role in various physiological processes.In this study,we cloned the trehalose-6-phosphate synthase gene(HvTPS)and investigated its expression patterns in various tssues and d:velopmental stages in Heortia vitessoides Moore(Lepidoptera:Crambidac).HvTPS was highly expressed in the fat body and after pupation or before molting.We knocked down TPS in H.vitessoides by RNA interference and found that 3.0μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes.Additionally.compared to the controls,TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0μg of dsHIvTPS.Furthermore,the silencing of HvTPS suppressed the cxpression of six key genecs in the chitin biosynthesis pathway and one key gene related to lipid catabolism.The expression levels of two genes associated with lipid biosynthesis were upregulated.These results strongly suggest that HvTPS is essential for the normal growth and development of H.vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.     

The RNA interference—virus interplay: tools of nature for gene modulation,morphogenesis, evolution and a possible mean for aflatoxin control

This article points out, that viruses, in an interplay with RNA interference and as vehicles for intergenic and interspecies gene transfer, may work as agents for intracellular gene modulation, for steering of individual morphogenesis and as a driving force of evolution in the toolbox of nature. This is illustrated in particular in the light of a fungal double-stranded RNA virus that may be employed as a suitable agent for a biological control of aflatoxins, the most carcinogenic natural substances occurring in food and feedstuff.     

Functional expression of a rat homologue of the voltage gated either á go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart.

We have cloned a mammalian (rat) homologue of Drosophila ether á go-go (eag) cDNA, which encodes a distinct type of voltage activated potassium (K) channel. The derived Drosophila and rat eag polypeptides share > 670 amino acids, with a sequence identity of 61%, exhibiting a high degree of similarity at the N-terminus, the hydrophobic core including the pore forming P region and a potential cyclic nucleotide binding site. Rat eag mRNA is specifically expressed in the central nervous system. In the Xenopus oocyte expression system rat eag mRNA gives rise to voltage activated K channels which have distinct properties in comparison with Drosophila eag channels and other voltage activated K channels. Thus, the rat eag channel further extends the known diversity of K channels. Most notably, the kinetics of rat eag channel activation depend strongly on holding membrane potential. Hyperpolarization slows down the kinetics of activation; conversely depolarization accelerates the kinetics of activation. This novel K channel property may have important implications in neural signal transduction allowing neurons to tune their repolarizing properties in response to membrane hyperpolarization.     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

The S. pombe cdc16 gene is required both for maintenance of p34cdc2 kinase activity and regulation of septum formation: a link between mitosis and cytokinesis?

In the fission yeast Schizosaccharomyces pombe, septum formation and cytokinesis are dependent upon the initiation, though not the completion of mitosis. A number of cell cycle mutants which show phenotypes consistent with a defect in the regulation of septum formation have been isolated. A mutation in the S. pombe cdc16 gene leads to the formation of multiple septa without cytokinesis, suggesting that the normal mechanisms that limit the cell to the formation of a single septum in each cycle do not operate. Mutations in the S. pombe early septation mutants cdc7, cdc11, cdc14 and cdc15 lead to the formation of elongated, multinucleate cells, as a result of S phase and mitosis continuing in the absence of cytokinesis. This suggests that in these cells, the normal mechanisms which initiate cytokinesis are defective and that they are unable to respond to this by preventing further nuclear cycles. Genetic analysis has implied that the products of some of these genes may interact with that of the cdc16 gene. To understand how the processes of septation and cytokinesis are regulated and coordinated with mitosis we are studying the early septation mutants and cdc16. In this paper, we present the cloning and analysis of the cdc16 gene. Deletion of the gene shows that it is essential for cell proliferation: spores lacking a functional cdc16 gene germinate, complete mitosis and form multiple septa without undergoing cell cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon–phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida

The phnA gene encoding a novel carbon–phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.