Genetic interaction of an origin recognition complex subunit and the Polycomb group gene MEDEA during seed development

The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.     

Disease-associated prion protein elicits immunoglobulin M responses in vivo

Prion diseases such as Creutzfeldt-Jakob disease are believed to result from the misfolding of a widely expressed normal cellular prion protein, PrPc. The resulting disease-associated isoforms, PrP(Sc), have much higher beta-sheet content, are insoluble in detergents, and acquire relative resistance to proteases. Although known to be highly aggregated and to form amyloid fibrils, the molecular architecture of PrP9Sc) is poorly understood. To date, it has been impossible to elicit antibodies to native PrP(Sc) that are capable of recognizing PrP(Sc) without denaturation, even in Pm-P(o/o) mice that are intolerant of it. Here we demonstrate that antibodies for native PrPc and PrP(Sc) can be produced by immunization of Pm-P(o/o) mice with partially purified PrPc and PrP(Sc) adsorbed to immunomagnetic particles using high-affinity anti-PrP monoclonal antibodies (mAbs). Interestingly, the polyclonal response to PrP(Sc) was predominantly of the immunoglobulin M (IgM) isotype, unlike the immunoglobulin G (IgG) responses elicited by PrP(c) or by recombinant PrP adsorbed or not to immunomagnetic particles, presumably reflecting the polymeric structure of disease-associated prion protein. Although heat-denatured PrP(Sc) elicited more diverse antibodies with the revelation of C-terminal epitopes, remarkably, these were also predominantly IgM suggesting that the increasing immunogenicity, acquisition of protease sensitivity, and reduction in infectivity induced by heat are not associated with dissociation of the PrP molecules in the diseased-associated protein. Adsorbing native proteins to immunomagnetic particles may have general applicability for raising polyclonal or monoclonal antibodies to any native protein, without attempting laborious purification steps that might affect protein conformation.     

Rational targeting for prion therapeutics

Prions--pathogens that are lethal to humans and other animals--are thought to be conformational isomers of the cellular prion protein. Their unique biology, and the potential for a wider pathobiological significance of prion-like mechanisms, has motivated much research into understanding prion neurodegeneration. Moreover, concerns that extensive dietary exposure to bovine spongiform encephalopathy (BSE) prions might have infected many individuals--who might eventually develop its human counterpart, variant Creutzfeldt-Jakob disease (vCJD)--has focused much interest on therapeutics. The challenge of interrupting this aggressive, diffuse and uniformly fatal neurodegenerative process is daunting. However, the recent finding that the onset of clinical disease in established neuroinvasive prion infection in a mouse model can be halted and early pathology reversed is a source for considerable optimism. A therapeutic focus on the cellular prion protein, rather than prions themselves, which might not be directly neurotoxic, is suggested.     

Restoration genetics of the vernal pool endemic Lasthenia conjugens (Asteraceae)

Restoration of habitat for endangered species often involves translocation of seeds or individuals from source populations to an area targeted for revegetation. Long-term persistence of a species is dependent on the maintenance of sufficient genetic variation within and among populations. Thus, knowledge and maintenance of genetic variability within rare or endangered species is essential for developing effective conservation and restoration strategies. Genetic monitoring of both natural and restored populations can provide an assessment of restoration protocol success in establishing populations that maintain levels of genetic diversity similar to those in natural populations. California’s vernal pools are home to many endangered plants, thus conservation and restoration are large components of their management. Lasthenia conjugens (Asteraceae) is a federally endangered self-incompatible vernal pool annual with gravity- dispersed seeds. Using the molecular technique of intersimple sequence repeats (ISSRs), this study assessed levels and patterns of genetic variability present within natural and restored populations of L. conjugens. At Travis Air Force Base near Fairfield, California, a vernal pool restoration project is underway. Genetic success of the ecologically based seeding protocol was examined through genetic monitoring of natural and restored populations over a three-year period. Genetic diversity remained constant across the three sampled generations. Diversity was also widely distributed across all populations. We conclude that the protocol used to establish restored populations was successful in capturing similar levels and patterns of genetic diversity to those seen within natural pools. This study also demonstrates how genetic markers can be used to inform conservation and restoration decisions.     

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.     

Dynamic life cycle assessment: framework and application to an institutional building

Purpose

This paper uses a dynamic life cycle assessment (DLCA) approach and illustrates the potential importance of the method using a simplified case study of an institutional building. Previous life cycle assessment (LCA) studies have consistently found that energy consumption in the use phase of a building is dominant in most environmental impact categories. Due to the long life span of buildings and potential for changes in usage patterns over time, a shift toward DLCA has been suggested.

Methods

We define DLCA as an approach to LCA which explicitly incorporates dynamic process modeling in the context of temporal and spatial variations in the surrounding industrial and environmental systems. A simplified mathematical model is used to incorporate dynamic information from the case study building, temporally explicit sources of life cycle inventory data and temporally explicit life cycle impact assessment characterization factors, where available. The DLCA model was evaluated for the historical and projected future environmental impacts of an existing institutional building, with additional scenario development for sensitivity and uncertainty analysis of future impacts.

Results and discussion

Results showed that overall life cycle impacts varied greatly in some categories when compared to static LCA results, generated from the temporal perspective of either the building's initial construction or its recent renovation. From the initial construction perspective, impacts in categories related to criteria air pollutants were reduced by more than 50 % when compared to a static LCA, even though nonrenewable energy use increased by 15 %. Pollution controls were a major reason for these reductions. In the future scenario analysis, the baseline DLCA scenario showed a decrease in all impact categories compared with the static LCA. The outer bounds of the sensitivity analysis varied from slightly higher to strongly lower than the static results, indicating the general robustness of the decline across the scenarios.

Conclusions

These findings support the use of dynamic modeling in life cycle assessment to increase the relevance of results. In some cases, decision making related to building design and operations may be affected by considering the interaction of temporally explicit information in multiple steps of the LCA. The DLCA results suggest that in some cases, changes during a building's lifetime can influence the LCA results to a greater degree than the material and construction phases. Adapting LCA to a more dynamic approach may increase the usefulness of the method in assessing the performance of buildings and other complex systems in the built environment.     

Do pathogens reduce genetic diversity of their hosts? Variable effects of sylvatic plague in black‐tailed prairie dogs

Introduced diseases can cause dramatic declines in—and even the loss of—natural populations. Extirpations may be followed by low recolonization rates, leading to inbreeding and a loss of genetic variation, with consequences on population viability. Conversely, extirpations may create vacant habitat patches that individuals from multiple source populations can colonize, potentially leading to an influx of variation. We tested these alternative hypotheses by sampling 15 colonies in a prairie dog metapopulation during 7 years that encompassed an outbreak of sylvatic plague, providing the opportunity to monitor genetic diversity before, during and after the outbreak. Analysis of nine microsatellite loci revealed that within the metapopulation, there was no change in diversity. However, within extirpated colonies, patterns varied: In half of the colonies, allelic richness after recovery was less than the preplague conditions, and in the other half, richness was greater than the preplague conditions. Finally, analysis of variation within individuals revealed that prairie dogs present in recolonized colonies had higher heterozygosity than those present before plague. We confirmed plague survivorship in six founders; these individuals had significantly higher heterozygosity than expected by chance. Collectively, our results suggest that high immigration rates can maintain genetic variation at a regional scale despite simultaneous extirpations in spatially proximate populations. Thus, virulent diseases may increase genetic diversity of host populations by creating vacant habitats that allow an influx of genetic diversity. Furthermore, even highly virulent diseases may not eliminate individuals randomly; rather, they may selectively remove the most inbred individuals.