RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of ~250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.     

Conserved residues R420 and Q428 in a cytoplasmic loop of the citrate/malate transporter CimH of Bacillus subtilis are accessible from the external face of the membrane

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET,(1) which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.     

Impact of the Mg<Superscript>2+</Superscript>-citrate transporter CitM on heavy metal toxicity in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis possesses a secondary transporter, CitM, that is specific for the complex of citrate and Mg(2+) but is also capable of transporting citrate in complex with the heavy metal ions Zn(2+), Ni(2+) and Co(2+). We report on the impact of CitM activity on the toxicity of Zn(2+), Ni(2+) and Co(2+) in B. subtilis. In a citM deletion mutant or under conditions in which CitM is not expressed, the toxic effects of the metals were reduced by the presence of citrate in the medium. In contrast, the presence of citrate dramatically enhanced toxicity when the Mg(2+)-citrate transporter was present in the membrane. It is demonstrated that the complex of Ni(2+) and citrate is transported into the cell and that the uptake is responsible for the enhanced toxicity. At toxic concentrations of the metal ions, the cultures adapted by developing tolerance against these ions. Tolerant cells isolated by exposure to one of the metal ions remained tolerant after growth in the absence of toxic metal ions and were cross-tolerant against the other two toxic ions. Tolerant strains were shown to contain point mutations in the citM gene, which resulted in premature termination of translation.     

Universal,robust, highly quantitative SNP allele frequency measurement in DNA pools

Detecting alleles that confer small increments in susceptibility to disease will require large-scale allelic association studies of single-nucleotide polymorphisms (SNPs) in candidate, or positional candidate, genes. However, current genotyping technologies are one to two orders of magnitude too expensive to permit the analysis of thousands of SNPs in large samples. We have developed and thoroughly validated a highly accurate protocol for SNP allele frequency estimation in DNA pools based upon the SNaPshot (Applied Biosystems) chemistry adaptation of primer extension. Using this assay, we were able to estimate the difference in allele frequencies between pooled cases and controls (Delta) with a mean error of 0.01. Moreover, when we genotyped seven different SNPs in a single multiplex reaction, the results were similar, with a mean error for Delta of 0.008. The assay performed well for alleles of low frequency alleles (f approximately 0.05) and was accurate even with relatively poor quality DNA template extracted from mouthwashes. Our assay conditions are generalisable, universal, robust and, therefore, for the first time, permit high-throughput association analysis at a realistic cost.     

Generation of EST and microarray resources for functional genomic studies on chicken intestinal health

Expressed sequenced tags (ESTs) and microarray resources have a great impact on the ability to study host response in mice and humans. Unfortunately, these resources are not yet available for domestic farm animals. The aim of this study was to provide genomic resources to study chicken intestinal health, in particular malabsorption syndrome (MAS), which affects mainly the intestine. Therefore a normalized and subtracted cDNA library containing more than 7000 clones was prepared. Randomly chosen clones were sequenced for control purposes. New ESTs were found and multiple ESTs not identified in the chicken intestine before were observed. The number of non-specific ESTs in this cDNA library was low. Based on this normalized and subtracted library a cDNA microarray was made. In a preliminary hybridization experiment with the microarray, genes were identified to be up- or downregulated in MAS infected chickens. This indicates that the generated resources are valuable tools to investigate chicken intestinal health by whole genome expression analysis approaches.     

The two-component signal transduction protein Chk1p regulates quorum sensing in Candida albicans

Regulation of hyphal morphogenesis in Candida albicans can occur through quorum sensing (QS). A QS signal, farnesol, is produced during high-density growth and inhibits morphogenesis. However, the signal transduction pathway that regulates QS is unknown. Here, we show that a C. albicans mutant lacking Chk1p but not either the Sln1p or the Nik1p histidine kinase is refractory to the inhibitory effect of farnesol both in cell suspension and during the formation of a biofilm. This study is the first to demonstrate a role for a two-component signal transduction protein in QS by a eukaryotic organism.     

Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction

The Cladosporium fulvum (Cf)-4 gene of tomato confers resistance to the fungus C. fulvum, expressing the corresponding avirulence (Avr)4 gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca(2+) signalling and protein kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf-4 interaction. Using transgenic tobacco cells expressing the tomato Cf-4-resistance gene as a model system, phospholipid signalling pathways were studied by pre-labelling the cells with (32)P(i) and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in (32)P-PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in a Cf-4(+)-, time- and dose-dependent manner, while the non-matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4-induced PA formation, a PLD-specific transphosphatidylation assay and a differential (32)P-labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4-induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4-induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.     

Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools

At present, the cost of genotyping single nucleotide polymorphisms (SNPs) in large numbers of subjects poses a formidable problem for molecular genetic approaches to complex diseases. We have tested the possibility of using primer extension and denaturing high performance liquid chromatography to estimate allele frequencies of SNPs in pooled DNA samples. Our data show that this method should allow the accurate estimation of absolute allele frequencies in pooled samples of DNA and also of the difference in allele frequency between different pooled DNA samples. This technique therefore offers an efficient and cheap method for genotyping SNPs in large case-control and family-based association samples.     

An additional step in the transmission of Yersinia pestis?

Plague, caused by the bacterium Yersinia pestis, is a mammalian vector-borne disease, transmitted by fleas that serve as the vector between rodent hosts. For many pathogens, including Y. pestis, there are strong evolutionary pressures that lead to a reduction in ‘useless genes'', with only those retained that reflect function in the specific environment inhabited by the pathogen. Genetic traits critical for survival and transmission between two environments, the rodent and the flea, are conserved in epizootic/epidemic plague strains. However, there are genes that remain conserved for which no function in the flea–rodent cycle has yet been observed, indicating an additional environment may exist in the transmission cycle of plague. Here, we present evidence for highly conserved genes that suggests a role in the persistence of Y. pestis after death of its host. Furthermore, maintenance of these genes points to Y. pestis traversing a post-mortem path between, and possibly within, epizootic periods and offering insight into mechanisms that may allow Y. pestis an alternative route of transmission in the natural environment.