Mitochondrial DNA defects in Saccharomyces cerevisiae caused by functional interactions between DNA polymerase gamma mutations associated with disease in human

The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and Ery(R) mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1(G651S) (hG848S) mutant the high mtDNA instability and increased frequency of point Ery(R) mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1(A692T-E900G) (hA889T-hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1(H734Y)/mip1(G807R) (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1(E900G) (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.     

Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis

Directional transport of the phytohormone auxin is established primarily at the point of cellular efflux and is required for the establishment and maintenance of plant polarity. Studies in whole plants and heterologous systems indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins mediate the cellular efflux of natural and synthetic auxins. However, aromatic anion transport resulting from PGP and PIN expression in nonplant systems was also found to lack the high level of substrate specificity seen in planta. Furthermore, previous reports that PGP19 stabilizes PIN1 on the plasma membrane suggested that PIN-PGP interactions might regulate polar auxin efflux. Here, we show that PGP1 and PGP19 colocalized with PIN1 in the shoot apex in Arabidopsis thaliana and with PIN1 and PIN2 in root tissues. Specific PGP-PIN interactions were seen in yeast two-hybrid and coimmunoprecipitation assays. PIN-PGP interactions appeared to enhance transport activity and, to a greater extent, substrate/inhibitor specificities when coexpressed in heterologous systems. By contrast, no interactions between PGPs and the AUXIN1 influx carrier were observed. Phenotypes of pin and pgp mutants suggest discrete functional roles in auxin transport, but pin pgp mutants exhibited phenotypes that are both additive and synergistic. These results suggest that PINs and PGPs characterize coordinated, independent auxin transport mechanisms but also function interactively in a tissue-specific manner.     

Molecular phylogeny and evolution of the order Tribonematales (Heterokonta, Xanthophyceae) based on analysis of plastidial genes rbcL and psaA

Tribonematales is an order of filamentous algae in the class Xanthophyceae (Heterokonta). Few molecular studies, all with a limited taxon sampling, have previously investigated its evolutionary history and phylogenetic relationships. We sequenced the chloroplast-encoded rbcL and psaA genes of several tribonematalean species and of several coccoid and siphonous forms that previous studies revealed to be strictly related to Tribonematales. Multiple alignments included mostly new sequences obtained from 42 taxa. Phylogenetic reconstructions were performed using the maximum likelihood method. The rbcL and psaA data sets were analyzed independently and combined in a single multiple alignment. Neither rbcL nor psaA genes showed intraspecific sequence variation. The former proved to be a better diagnostic marker than the latter for characterization of species. We explored effects produced on phylogenetic outcomes by selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on the combined data set. There is strong statistical support for trees that show several currently recognized taxonomic groups to be polyphyletic. The siphonous orders Botrydiales and Vaucheriales do not form a clade. Botrydiales and Tribonematales are polyphyletic as are the families Botrydiaceae, Centritractaceae and Tribonemataceae and the genera Xanthonema and Bumilleriopsis. We tentatively define new boundaries of the Tribonematales to include both coccoid and filamentous species having a bipartite cell wall and also the siphonous members of the genus Botrydium. Also, our results support morphological convergence at all taxonomic ranks in the evolution of the Xanthophyceae.     

Genotyping of the lactase-phlorizin hydrolase c/t-13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography-based assay in healthy subjects and colorectal cancer patients

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T(-13910) upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)-based assay versus conventional genotype sequencing in detecting the C/T(-13910) polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T(-13910) polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C(-13913)) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C(-13910)) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and labor-saving screening tool for genotyping C/T(-13910) polymorphism, with high success, low cost, and reproducibility.     

Influence of Clonal Variation on the Efficiency of Arundo donax Propagation Methods

The recent growth of interest in Arundo donax L. (Giant reed) as an energy crop is due to its great vegetative vigor and high biomass productivity. This perennial rhizomatous plant is able to grow in a wide range of pedo-climatic conditions and it has been employed by man for a great number of purposes. This has promoted its worldwide diffusion, despite its sexual sterility. Field establishment represents the most expensive phase of the whole cultivation cycle, because of the high cost of the propagules. In this work three agamic propagation methods, rhizome transplantation, hydroponic cultivation, and in vitro propagation, were compared with the aim of improving their efficiency, and reducing costs and time required. We focused our attention on in vitro and hydroponic cultivation, finding clones characterized by a high response to these propagation methods. In vitro propagation is the most widespread technique adopted in the large-scale production of propagules; according to our results hydroponic cultivation also appears to be a valid and cheap propagation method, making it possible to obtain in about 2 months, vigorous plants ready to be transplanted into the field. We found a strong effect of the clone used on the success rate of the specific propagation method, suggesting that clonal selection could be particularly useful in improving the efficiency of propagation techniques, contributing to cost reduction. Evaluation of field performance of plants propagated by tissue culture is an important matter, considering the possible residual effects of hormones on plants’ growth, and the importance of first year growth for field establishment. These experiments highlighted the lower biomass production from plants obtained by tissue culture. A novel cytokinin-like hormone, meta-topolin, was tested in vitro, in a preliminary study to evaluate its possible use for A. donax L. propagation.

     

Anthropogenic disturbance in a changing environment: modelling lifetime reproductive success to predict the consequences of multiple stressors on a migratory population

Animals make behavioural and reproductive decisions that maximise their lifetime reproductive success, and thus their fitness, in light of periodic and stochastic variability of the environment. Modelling the variation of an individual's energy levels formalises this tradeoff and helps to quantify the population‐level consequences of stressors (e.g. disturbance from human activities and environmental change) that can affect behaviour or physiology. In this study, we develop a dynamic state variable model for the spatially explicit behaviour, physiology and reproduction of a female, long‐lived, migratory marine vertebrate. The model can be used to investigate the spatio‐temporal patterns of behaviour and reproduction that allow an individual to maximise its overall reproductive output. We parametrised the model for eastern North Pacific blue whales Balaenoptera musculus, and used it to predict the effects of changing environmental conditions and increasing human disturbance on the population's vital rates. In baseline conditions, the model output had high fidelity to observed energy dynamics, movement patterns and reproductive strategies. Simulated scenarios suggested that environmental changes could have severe consequences on the population's vital rates, but that individuals could tolerate high levels of anthropogenic disturbance. However, this ability depended on where, when and how often disturbance occurred. In scenarios with both environmental change and anthropogenic disturbance, synergistic interactions caused stronger effects than in isolation. In general, larger body size offered a buffer against stochasticity and disturbance, and, consequently, we predicted juveniles to be more susceptible to disturbance. We also predicted that females prioritise their own survival at the expense of the current reproductive attempt, presumably the result of their long lifespan. Our approach provides a general framework to make predictions of the cumulative and synergistic effects of human disturbance and climate change on migratory populations, which can inform effective management and conservation efforts.     

Effects of altered river network connectivity on the distribution of Salmo trutta: Insights from a metapopulation model

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