Mammalian DNMTs in the male germ line DNA of Drosophila

It is controversial whether DNA methylation plays a functional role in Drosophila. We have studied testis DNA of Drosophila melanogaster Meigen, 1830 with antisera against 5-methylcytosine (5mC) and found no evidence for the presence of significant amounts of 5mC. Reactions occur only with 1 of 3 5mC antisera, but they are restricted to nuclear regions without detectable amounts of DNA. The antisera apparently cross-react with other nuclear components. If the murine de novo DNA methyltransferases, DNMT3A and DNMT3B, are expressed under the control of the spermatocyte-specific beta2-tubulin promoter in testes, DNA methylation is not increased and no effects on the fertility of the fly are seen. DNA methylation has, therefore, no functional relevance in the male germ line of Drosophila.     

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.     

Structural and Functional Analyses of PAS Domain Interactions of the Clock Proteins Drosophila PERIOD and Mouse PERIOD2

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal α-helices (αE and αF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix αF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the αF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B β-sheet surface including tryptophane 419 (equivalent to Trp482_dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B β-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B β-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.     

Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis

Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed “triploid block.” Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin–specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues.     

Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling

Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing ～19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH‐sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin‐tagged proteins. While the native proteins had a clustered distribution, the GFP‐tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin‐tagged vesicle proteins are less well integrated in clusters.     

Natural CMT2 Variation Is Associated With Genome-Wide Methylation Changes and Temperature Seasonality

As Arabidopsis thaliana has colonized a wide range of habitats across the world it is an attractive model for studying the genetic mechanisms underlying environmental adaptation. Here, we used public data from two collections of A. thaliana accessions to associate genetic variability at individual loci with differences in climates at the sampling sites. We use a novel method to screen the genome for plastic alleles that tolerate a broader climate range than the major allele. This approach reduces confounding with population structure and increases power compared to standard genome-wide association methods. Sixteen novel loci were found, including an association between Chromomethylase 2 (CMT2) and temperature seasonality where the genome-wide CHH methylation was different for the group of accessions carrying the plastic allele. Cmt2 mutants were shown to be more tolerant to heat-stress, suggesting genetic regulation of epigenetic modifications as a likely mechanism underlying natural adaptation to variable temperatures, potentially through differential allelic plasticity to temperature-stress.     

The Relationship between Brain Morphology and Polysomnography in Healthy Good Sleepers

BackgroundNormal sleep continuity and architecture show remarkable inter-individual variability. Previous studies suggest that brain morphology may explain inter-individual differences in sleep variables.MethodThirty-eight healthy subjects spent two consecutive nights at the sleep laboratory with polysomnographic monitoring. Furthermore, high-resolution T1-weighted MRI datasets were acquired in all participants. EEG sleep recordings were analyzed using standard sleep staging criteria and power spectral analysis. Using the FreeSurfer software for automated segmentation, 174 variables were determined representing the volume and thickness of cortical segments and the volume of subcortical brain areas. Regression analyses were performed to examine the relationship with polysomnographic and spectral EEG power variables.ResultsThe analysis did not provide any support for the a-priori formulated hypotheses of an association between brain morphology and polysomnographic variables. Exploratory analyses revealed that the thickness of the left caudal anterior cingulate cortex was positively associated with EEG beta2 power (24–32 Hz) during REM sleep. The volume of the left postcentral gyrus was positively associated with periodic leg movements during sleep (PLMS).ConclusionsThe function of the anterior cingulate cortex as well as EEG beta power during REM sleep have been related to dreaming and sleep-related memory consolidation, which may explain the observed correlation. Increased volumes of the postcentral gyrus may be the result of increased sensory input associated with PLMS. However, due to the exploratory nature of the corresponding analyses, these results have to be replicated before drawing firm conclusions.     

The Effects of Heart Rate Variability Biofeedback in Patients with Preterm Labour

Preterm birth is a highly prevalent phenomenon that was shown to be associated with mental stress during pregnancy (Rich-Edwards and Grizzard in Am J Obstet Gynecol 192(5 Suppl):S30–S35, 2005). We aimed to assess the effects of heart rate variability (HRV)-biofeedback in patients with preterm labour. Therefore, we conducted a controlled randomized parallel group study in 48 female patients aged 19–38 years (median = 29) with preterm labour at gestational week 24th–32nd (median = 29th). In this study, one group (n = 24) attended six sessions of HRV-biofeedback over 2 weeks whereas patients of the other group (n = 24) were assigned to control sessions. In the HRV-biofeedback treated group, perception of chronic stress was decreased 4 weeks after completion of training compared to baseline (p < 0.05) but there was no change in the control group. In the HRV-biofeedback group, preterm birth was seen in 3 patients (13 %) whereas in the control group, preterm delivery occurred in 8 patients (33 %, p = n.s.). There was no difference in birth weight between groups and HRV remained unchanged. In conclusion, our study demonstrates that HRV-biofeedback can reduce chronic stress in patients with preterm labour when administered as an adjunct to routine care. However, it remains unclear whether stress reduction through HRV-biofeedback has a beneficial effect on preterm birth.