Analysis of the role of RecQ helicases in RNAi in mammals

The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.     

Spatial and population genetic structure of microsatellites in white pine

We evaluated the population genetic structure of seven microsatellite loci for old growth and second growth populations of eastern white pine (Pinus strobus). From each population, located within Hartwick Pines State Park, Grayling, Michigan, USA, 120-122 contiguous trees were sampled for genetic analysis. Within each population, genetic diversity was high and inbreeding low. When comparing these populations, there is a significant, but small (less than 1%), genetic divergence between populations. Spatial distance between populations or timber harvest at the second growth site were reasonable explanations for the observed minor differences in allele frequencies between populations. Spatial autocorrelation analysis suggested that, for the old growth population, weak positive structuring at 15 m fits the isolation by distance model for a neighbourhood size of about 100 individuals. In comparison, genotypes were randomly distributed in the second growth population. Thus, logging may have decreased spatial structuring at the second growth site, suggesting that management practices may be used to alter natural spatial patterns. In addition, the amount of autocorrelation in the old growth population appears to be lower for some of the microsatellites, suggesting higher numbers of rare alleles and that higher mutation rates may have directly affected spatial statistics by reducing structure.     

Systems biology of yeast cell death

Programmed cell death (PCD) (including apoptosis) is an essential process, and many human diseases of high prevalence such as neurodegenerative diseases and cancer are associated with deregulations in the cell death pathways. Yeast Saccharomyces cerevisiae, a unicellular eukaryotic organism, shares with multicellular organisms (including humans) key components and regulators of the PCD machinery. In this article, we review the current state of knowledge about cell death networks, including the modeling approaches and experimental strategies commonly used to study yeast cell death. We argue that the systems biology approach will bring valuable contributions to our understanding of regulations and mechanisms of the complex cell death pathways.     

Genetic association with high‐resolution climate data reveals selection footprints in the genomes of barley landraces across the Iberian Peninsula

Landraces are local populations of crop plants adapted to a particular environment. Extant landraces are surviving genetic archives, keeping signatures of the selection processes experienced by them until settling in their current niches. This study intends to establish relationships between genetic diversity of barley (Hordeum vulgare L.) landraces collected in Spain and the climate of their collection sites. A high‐resolution climatic data set (5 × 5 km spatial, 1‐day temporal grid) was computed from over 2,000 temperature and 7,000 precipitation stations across peninsular Spain. This data set, spanning the period 1981–2010, was used to derive agroclimatic variables meaningful for cereal production at the collection sites of 135 barley landraces. Variables summarize temperature, precipitation, evapotranspiration, potential vernalization and frost probability at different times of the year and time scales (season and month). SNP genotyping of the landraces was carried out combining Illumina Infinium assays and genotyping‐by‐sequencing, yielding 9,920 biallelic markers (7,479 with position on the barley reference genome). The association of these SNPs with agroclimatic variables was analysed at two levels of genetic diversity, with and without taking into account population structure. The whole data sets and analysis pipelines are documented and available at https://eead-csic-compbio.github.io/barley-agroclimatic-association . We found differential adaptation of the germplasm groups identified to be dominated by reactions to cold temperature and late‐season frost occurrence, as well as to water availability. Several significant associations pointing at specific adaptations to agroclimatic features related to temperature and water availability were observed, and candidate genes underlying some of the main regions are proposed.     

More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes

Pore-forming proteins (PFPs) punch holes in their target cell membrane to alter their permeability. Permeabilization of lipid membranes by PFPs has received special attention to study the basic molecular mechanisms of protein insertion into membranes and the development of biotechnological tools. PFPs act through a general multi-step mechanism that involves (i) membrane partitioning, (ii) insertion into the hydrophobic core of the bilayer, (iii) oligomerization, and (iv) pore formation. Interestingly, PFPs and membranes show a dynamic interplay. As PFPs are usually produced as soluble proteins, they require a large conformational change for membrane insertion. Moreover, membrane structure is modified upon PFPs insertion. In this context, the toroidal pore model has been proposed to describe a pore architecture in which not only protein molecules but also lipids are directly involved in the structure. Here, we discuss how PFPs and lipids cooperate and remodel each other to achieve pore formation, and explore new evidences of protein-lipid pore structures.     

A new nuclear protease with cathepsin L properties is present in HeLa and Caco‐2 cells

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell‐cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH‐protease) participates in male chromatin remodeling and in cell‐cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38–42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco‐2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non‐proliferative cells. J. Cell. Biochem. 111: 1099–1106, 2010. © 2010 Wiley‐Liss, Inc.     

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

The cytokinin content in fruit tissue of the kiwifruit ( Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high-performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O - and 9-glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography-mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O - and 9-glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g⁻¹ fresh weight at anthesis to 41 pmol g⁻¹ fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g⁻¹ fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g⁻¹ fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.     

Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter

The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly.Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation.