Aminoacylation and conformational properties of yeast mitochondrial tRNA mutants with respiratory deficiency

We report the identification and characterization of eight yeast mitochondrial tRNA mutants, located in mitochondrial tRNA(Gln), tRNA(Arg2), tRNA(Ile), tRNA(His), and tRNA(Cys), the respiratory phenotypes of which exhibit various degrees of deficiency. The mutations consist in single-base substitutions, insertions, or deletions, and are distributed all over the tRNA sequence and structure. To identify the features responsible for the defective phenotypes, we analyzed the effect of the different mutations on the electrophoretic mobility and efficiency of acylation of the mutated tRNAs in comparison with the respective wild-type molecules. Five of the studied mutations determine both conformational changes and defective acylation, while two have neither or limited effect. However, variations in structure and acylation are not necessarily correlated; the remaining mutation affects the tRNA conformation, but not its acylation properties. Analysis of tRNA structures and of mitochondrial and cytoplasmic yeast tRNA sequences allowed us to propose explanations for the observed defects, which can be ascribed to either the loss of identity nucleotides or, more often, of specific secondary and/or tertiary interactions that are largely conserved in native mitochondrial and cytoplasmic tRNAs.     

An artificial riboswitch for controlling pre-mRNA splicing

Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.     

A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses

Prunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.     

Combined bromodeoxyuridine immunocapture and terminal-restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation or plant species

High numbers of bacteria are associated with arbuscular mycorrhizal (AM) fungi, but their functions and in situ activities are largely unknown and most have never been characterized. The aim of the present study was to study the impact of Glomus mosseae inoculation and plant type on the active bacterial communities in soil by using a molecular approach, bromodeoxyuridine (BrdU) immunocapture in combination with terminal-restriction fragment length polymorphism (T-RFLP). This approach combined with sequence information from clone libraries, enabled the identification of actively growing populations, within the total bacterial community. Distinct differences in active bacterial community compositions were found according to G. mosseae inoculation, treatment with an antifungal compound (Benomyl) and plant type. The putative identities of the dominant bacterial species that were activated as a result of G. mosseae inoculation were found to be mostly uncultured bacteria and Paenibacillus species. These populations may represent novel bacterial groups that are able to influence the AM relationship and its subsequent effect on plant growth.     

Calculation of multipoint likelihoods using flanking marker data: a simulation study

The calculation of multipoint likelihoods is computationally challenging, with the exact calculation of multipoint probabilities only possible on small pedigrees with many markers or large pedigrees with few markers. This paper explores the utility of calculating multipoint likelihoods using data on markers flanking a hypothesized position of the trait locus. The calculation of such likelihoods is often feasible, even on large pedigrees with missing data and complex structures. Performance characteristics of the flanking marker procedure are assessed through the calculation of multipoint heterogeneity LOD scores on data simulated for Genetic Analysis Workshop 14 (GAW14). Analysis is restricted to data on the Aipotu population on chromosomes 1, 3, and 4, where chromosomes 1 and 3 are known to contain disease loci. The flanking marker procedure performs well, even when missing data and genotyping errors are introduced.     

Relative stability of quadruplexes containing different number of G-tetrads

The aim of this work is to compare the physicochemical properties of three oligonucleotidic sequences, d(TGGGT), d(TGGGGT) and d(TGGGGGT), which assemble to form quadruplex structures with the same molecularity, but containing three, four, and five G-quartets, respectively. The addition of one or two G-tetrads greatly increases both the enthalpy and Tm values of the quadruplex dissociation.     

The absence of ABCA1 decreases soluble ApoE levels but does not diminish amyloid deposition in two murine models of Alzheimer disease

ABCA1, a cholesterol transporter expressed in the brain, has been shown recently to be required to maintain normal apoE levels and lipidation in the central nervous system. In addition, ABCA1 has been reported to modulate beta-amyloid (Abeta) production in vitro. These observations raise the possibility that ABCA1 may play a role in the pathogenesis of Alzheimer disease. Here we report that the deficiency of ABCA1 does not affect soluble or guanidine-extractable Abeta levels in Tg-SwDI/B or amyloid precursor protein/presenilin 1 (APP/PS1) mice, but rather is associated with a dramatic reduction in soluble apoE levels in brain. Although this reduction in apoE was expected to reduce the amyloid burden in vivo, we observed that the parenchymal and vascular amyloid load was increased in Tg-SwDI/B animals and was not diminished in APP/PS1 mice. Furthermore, we observed an increase in the proportion of apoE retained in the insoluble fraction, particularly in the APP/PS1 model. These data suggested that ABCA1-mediated effects on apoE levels and lipidation influenced amyloidogenesis in vivo.