Copy Number Variation in Transcriptionally Active Regions of Sexual and Apomictic Boechera Demonstrates Independently Derived Apomictic Lineages

In asexual (apomictic) plants, the absence of meiosis and sex is expected to lead to mutation accumulation. To compare mutation accumulation in the transcribed genomic regions of sexual and apomictic plants, we performed a double-validated analysis of copy number variation (CNV) on 10 biological replicates each of diploid sexual and diploid apomictic Boechera, using a high-density (>700 K) custom microarray. The Boechera genome demonstrated higher levels of depleted CNV, compared with enriched CNV, irrespective of reproductive mode. Genome-wide patterns of CNV revealed four divergent lineages, three of which contain both sexual and apomictic genotypes. Hence genome-wide CNV reflects at least three independent origins (i.e., expression) of apomixis from different sexual genetic backgrounds. CNV distributions for different families of transposable elements were lineage specific, and the enrichment of LINE/L1 and long term repeat/Copia elements in lineage 3 apomicts is consistent with sex and meiosis being mechanisms for purging genomic parasites. We hypothesize that significant overrepresentation of specific gene ontology classes (e.g., pollen–pistil interaction) in apomicts implies that gene enrichment could be an adaptive mechanism for genome stability in diploid apomicts by providing a polyploid-like system for buffering the effects of deleterious mutations.     

Diversity and evolutionary patterns of immune genes in free-ranging Namibian leopards (Panthera pardus pardus)

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for fitness-related genetic variation in wildlife populations. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, we isolated and characterized genetic variation at the adaptively most important region of MHC class I and MHC class II-DRB genes in 25 free-ranging African leopards from Namibia and investigated the mechanisms that generate and maintain MHC polymorphism in the species. Using single-stranded conformation polymorphism analysis and direct sequencing, we detected 6 MHC class I and 6 MHC class II-DRB sequences, which likely correspond to at least 3 MHC class I and 3 MHC class II-DRB loci. Amino acid sequence variation in both MHC classes was higher or similar in comparison to other reported felids. We found signatures of positive selection shaping the diversity of MHC class I and MHC class II-DRB loci during the evolutionary history of the species. A comparison of MHC class I and MHC class II-DRB sequences of the leopard to those of other felids revealed a trans-species mode of evolution. In addition, the evolutionary relationships of MHC class II-DRB sequences between African and Asian leopard subspecies are discussed.     

Double Knockout Mutants of Arabidopsis Grown under Normal Conditions Reveal that the Plastidial Phosphorylase Isozyme Participates in Transitory Starch Metabolism

In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1 × phs1a and mex1 × phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1 × phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.During the last two decades, biochemical analyses of starch metabolism in higher plants have been favored by the availability of large sets of insertion mutants deficient in a single starch-related gene product. Based on phenotypical characterization of these mutants followed by the identification of the respective locus in the genome, novel starch-related proteins were discovered that reside inside the plastid, in the cytosol, in the nucleus, and in the plastidial envelope membranes. Taken together, these results have largely altered the current view on starch metabolism (Zeeman et al., 2010; Fettke et al., 2012a; Smith, 2012).Despite this progress, phenotypical analyses of starch-related mutants are complex and, under certain circumstances, yield misleading conclusions. Loss of function of metabolic steps may cause the entire starch synthesizing or degrading process to become nonfunctional. In this case, mutants are expected to have starch levels that are significantly altered. If, however, single knockout mutants are capable of partially or fully compensating the loss of function by other routes, the resulting phenotypes are less obvious and more difficult to predict. Carbon fluxes through existing paths may be enhanced, or novel metabolic routes may be established that compensate the lost function. As an example, leaves of Arabidopsis (Arabidopsis thaliana) mutants constitutively lacking the plastidial hexose-phosphate isomerase strongly express a distinct plastidial Glc-6-P/orthophosphate antiporter isoform that in wild-type plants is found only in heterotrophic tissues (Kunz et al., 2010). In mesophyll cells of the mutant, the reductive pentose phosphate cycle cannot drive assimilatory starch biosynthesis, as chloroplasts are unable to convert Fru-6-P to Glc-6-P. However, their capacity of transporting Glc-6-P between the cytosolic and the chloroplastic compartment is strongly increased. Furthermore, nonfunctionality of some starch-related proteins can lead to enlarged or diminished metabolite pools that via sensing processes, lead to cellular alterations distant from central carbon metabolism. This complexity is evidenced by several starch-related Arabidopsis mutants that possess a largely altered plastidial ultrastructure and exhibit premature degradation of the entire chloroplast (Stettler et al., 2009; Cho et al., 2011).Furthermore, several starch-related enzymes are capable of forming homomeric or heteromeric complexes that are functionally relevant but, to some extent, variable (Delatte et al., 2005; Utsumi and Nakamura, 2006; Kubo et al., 2010; Emes and Tetlow, 2012; Nakamura et al., 2012; Streb et al., 2012).In starch or glycogen storing prokaryotic and eukaryotic cells, α-glucan phosphorylase (EC 2.4.1.1) is common. Initially, this enzyme was considered to be the main starch synthesizing activity (Hanes, 1940). Later, both starch and glycogen synthases have been discovered that utilize either ADPglucose or UDPglucose (or both; Deschamps et al., 2006) as hexosyl donor. Ample evidence has been presented that these enzymes are essential biosynthetic enzymes (Ballicora et al., 2003; Zeeman et al., 2010; Roach et al., 2012; Palm et al., 2013). Furthermore, it is widely accepted that in glycogen-storing cells, phosphorylase is indispensible for the degradation of the storage polysaccharide (Hwang et al., 1989; Alonso-Casajús et al., 2006; Wilson et al., 2010; Roach et al., 2012; Gazzerro et al., 2013).In plant cells, the metabolic function of phosphorylase is more complex and far from being clear. In lower and higher plants, two distinct phosphorylase types exist as plastid- and cytosol-specific isozymes and are designated as Pho1 (or, in Arabidopsis, PHS1) and Pho2 (PHS2), respectively. Based on the large differences in the affinities for glycogen, the plastidial and the cytosolic phosphorylases are also named as low-affinity (L-type) and high-affinity (H-type) isozymes, respectively. As starch is restricted to the plastids, only the Pho1 (PHS1) type appears to possess direct access to native starch and/or plastidial starch-derived α-glucans.Conflicting phenotypical features have been reported for several mutants possessing altered levels of the plastidial phosphorylase isozyme(s). In the starch-related mutant4 of the unicellular green alga Chlamydomonas reinhardtii, the lack of one plastidial Pho1 isozyme (designated as PhoB) was associated with a lower cellular starch content, abnormally shaped granules, a modified amylopectin structure, and an elevated amylose-to-amylopectin ratio when the cells were kept under nitrogen limitation (Dauvillée et al., 2006). These phenotypical features suggest an involvement of the plastidial phosphorylase PhoB in the biosynthesis of a storage polysaccharide resembling the reserve starch of higher plants. Similarly, a rapid incorporation of ¹⁴C into starch was observed when tuber discs from various transgenic potato lines were incubated with [U-¹⁴C]Glc-1-P. The rate of starch labeling was found to reflect the activity of the plastidial phosphorylase isozyme Pho1 (Fettke et al., 2010, 2012b). By contrast, transgenic potato (Solanum tuberosum) lines have been generated that due to expression of an antisense construct, possess a largely diminished total Pho1 activity in leaves. Leaf starch content is essentially unchanged compared with that of the wild-type plants, suggesting that under normal growth conditions, the plastidial phosphorylase is not necessarily involved in starch metabolism or, alternatively, can easily be replaced by other enzymes (Sonnewald et al., 1995). Likewise, the phenotype (including leaf starch content) of an Arabidopsis mutant lacking functional PHS1 has been reported not to differ from the wild type when the plants were grown under normal conditions. However, under water stress conditions, significantly more local leaf lesions have been reported to occur (Zeeman et al., 2004).When leaf discs from bean (Phaseolus vulgaris) or Arabidopsis plants were exposed to conditions favoring photorespiration (i.e. an atmosphere consisting of 30% [v/v] O₂ and 70% [v/v] N₂ but lacking CO₂), transitory starch was degraded in the light at a high rate and the plastidial Glc-6-P pool increased. In Arabidopsis mutants deficient in PHS1, the Glc monophosphate pool did not respond to photorespiratory conditions (Weise et al., 2006). These data lead to the conclusion that in illuminated leaves with very high rates of photorespiration, PHS1 is involved in the conversion of starch to Glc monophosphates but does not to participate in the nocturnal starch degradation.When studying several starch-related Arabidopsis mutants, we noticed that two single knockout mutations that both affect the maltose metabolism but differ in the subcellular location of the target protein possess a significantly increased PHS1 activity (Malinova et al., 2011a, 2011b). One mutant constitutively lacks the functional cytosolic transglucosidase (also designated as disproportionating enzyme2; DPE2) and, therefore, the cytosolic route of starch-derived maltose metabolism is impaired (Chia et al., 2004; Lu and Sharkey, 2004). The other mutant does not express the plastidial maltose transporter MEX1, resulting in a massively enlarged maltose pool (Niittylä et al., 2004). Thus, in the two mutants, the metabolism of starch-derived maltose is blocked at different subcellular sites, i.e. the cytosol and the chloroplast. The enhanced PHS1 activity as observed for the two mutants is difficult to explain unless a more general function of the phosphorylase isozyme in starch metabolism is assumed.For a detailed functional analysis of PHS1-related processes, we generated two types of constitutive PHS1-deficient double knockout mutants (DPE2 plus PHS1 or MEX1 plus PHS1) and studied their phenotypes in more detail under various experimental conditions. Shoot growth and leaf chlorophyll content are reduced when the plants are grown under a light-dark regime, but under continuous illumination, both effects are far less pronounced. Based on these data, we propose that the plastidial phosphorylase participates in both the turnover of transitory starch and in the maintenance of intact chloroplasts.     

UniqTag: Content-Derived Unique and Stable Identifiers for Gene Annotation

When working on an ongoing genome sequencing and assembly project, it is rather inconvenient when gene identifiers change from one build of the assembly to the next. The gene labelling system described here, UniqTag, addresses this common challenge. UniqTag assigns a unique identifier to each gene that is a representative k-mer, a string of length k, selected from the sequence of that gene. Unlike serial numbers, these identifiers are stable between different assemblies and annotations of the same data without requiring that previous annotations be lifted over by sequence alignment. We assign UniqTag identifiers to ten builds of the Ensembl human genome spanning eight years to demonstrate this stability. The implementation of UniqTag in Ruby and an R package are available at https://github.com/sjackman/uniqtag sjackman/uniqtag. The R package is also available from CRAN: install.packages ("uniqtag"). Supplementary material and code to reproduce it is available at https://github.com/sjackman/uniqtag-paper.     

Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

An expression‐uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP‐bait protein from the target cells. Here, the TAP‐tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue‐specific binding partners of the protein kinase A catalytic subunit Cβ1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor‐dependent interaction of Cβ1 with the cell cycle and apoptosis regulatory protein‐1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell‐ and tissue‐specific protein complexes.     

Interaction of hypochlorous acid and myeloperoxidase with phosphatidylserine in the presence of ammonium ions

The close association of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes (PMNs) and other apoptotic cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, ammonium ions inhibit in a concentration-dependent manner the hypochlorous acid-mediated formation of aldehyde and nitrile products from 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS). Concomitantly, the formation of monochloramine (NH₂Cl) raises with increasing NH₄⁺ concentrations. A transchlorination from monochlorinated O-phospho-l-serine to NH₄⁺ with the formation of NH₂Cl occurs only when extraordinary high NH₄⁺ concentrations are applied. Due to the low rate of 0.044 M^− 1 s^− 1 for this process, a transhalogenation reaction from transient chlorinated intermediates of the serine moiety to NH₄⁺ can be ruled out as an important process contributing to the HOCl-mediated formation of NH₂Cl. A significant formation of NH₂Cl by myeloperoxidase interacting with DPPS in the presence of ammonium ions takes only place at acidic pH values around 5, a scenario that may occur in phagosomes of macrophages after the uptake of apoptotic PMNs.     

4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells,an experimental model of type-2 diabetes

Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ～ 7-fold improved axial resolution (～ 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.