Interconnecting solvent quality, transcription, and chromosome folding in Escherichia coli.

All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.

EFR3A: a new raft domain organizing protein?

BACKGROUND: Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1. METHODS: We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed. RESULTS: EFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca2+ concentration. CONCLUSIONS: Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.

Evolution and conservation of polycomb repressive complex 1 core components and putative associated factors in the green lineage.

BACKGROUND: Polycomb group (PcG) proteins play important roles in animal and plant development and stress response. Polycomb repressive complex 1 (PRC1) and PRC2 are the key epigenetic regulators of gene expression, and are involved in almost all developmental stages. PRC1 catalyzes H2A monoubiquitination resulting in transcriptional silencing or activation. The PRC1 components in the green lineage were identified and evolution and conservation was analyzed by bioinformatics techniques. RING Finger Protein 1 (RING1), B lymphoma Mo-MLV insertion region 1 homolog (BMI1), Like Heterochromatin Protein 1 (LHP1) and Embryonic Flower 1 (EMF1) are the PRC1 core components and Vernalization 1 (VRN1), VP1/ABI3-Like 1/2/3 (VAL1/2/3), Alfin-like 1-7 (AL1-7), Inhibitor of growth 1/2 (ING1/2), and Early Bolting in Short Days (EBS) / Short Life (SHL) are the associated factors. RESULTS: Each PRC1 subunit possesses special domain organizations, such as RING and the ring finger and WD40-associated ubiquitin-like (RAWUL) domains for RING1 and BMI1, chromatin organization modifier (CHROMO) and chromo shadow (ChSh) domains for LHP1, one or two B3 DNA binding domain(s) for VRN1, B3 and zf-CW domains for VAL1/2/3, Alfin and Plant HomeoDomain (PHD) domains for AL1-7, ING and PHD domains for ING1/2, Bromoadjacent homology (BAT) and PHD domains for EBS/SHL. Six new motifs are uncovered in EMF1. The PRC1 core components RING1 and BMI1, and the associated factors VAL1/2/3, AL1-7, ING1/2, and EBS/SHL exist from alga to higher plants, whereas LHP1 only occurs in higher plants. EMF1 and VRN1 are present only in eudicots. PRC1 components undergo duplication in the plant evolution. Most of plants carry the homologous core component LHP1, the associated factor EMF1, and several homologs in RING1, BMI1, VRN1, AL1-7, ING1/2/3, and EBS/SHL. Cabbage, cotton, poplar, orange and maize often exhibit more gene copies than other species. Domain organization analysis shows that duplicated gene functions may be of diverse. CONCLUSIONS: The PRC1 core components RING1 and BMI1, and the associated factors VAL1/2/3, AL1-7, ING1/2, and EBS/SHL originate from algae. The core component LHP1 is from moss and the associated factors EMF1 and VRN1 are from dicotyledon. PRC1 components are of functional redundancy and diversity in evolution.

Phylogeny-dominant classification of J-proteins in Arabidopsis thaliana and Brassica oleracea.

Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants.

Transgenic tools to characterize neuronal properties of discrete populations of zebrafish neurons.

The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.

Evolution and conservation of JmjC domain proteins in the green lineage.

Histone modification regulates plant development events by epigenetically silencing or activating gene expression, and histone methylation is regulated by histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs). The JmjC domain proteins, an important KDM family, erase methyl marks (CH3-) from histones and play key roles in maintaining homeostasis of histone methylation in vivo. Here, we analyzed 169 JmjC domain proteins from whole genomes of plants ranging from green alga to higher plants together with 36 from two animals (fruit fly and human). The plant JmjC domain proteins were divided into seven groups. Group-I KDM4/JHDM3 and Group-V JMJD6 were found in all the plant species and the other groups were detected mainly in vascular or seed plants. Group-I KDM4/JHDM3 was potentially associated with demethylation of H3K9me2/3, H3K27me2/3, and H3K36me1/2/3, Group-II KDM5A with H3K4me1/2/3, Group-III KDM5B with H3K4me1/2/3 and H3K9me1/2/3, Group-V JMJD6 with H3R2, H4R3, and hydroxylation of H4, and Group-VII KDM3/JHDM2 with H3K9me1/2/3. Group-IV/Group-VI JmjC domain-only A/B proteins were involved in hydroxylation and demethylation of unknown substrate sites. The binding sites for the cofactors Fe(II) and α-ketoglutarate in the JmjC domains also were analyzed. In the α-ketoglutarate binding sites, Thr/Phe/Ser and Lys were conserved and in the Fe(II) binding sites, two His and Glu/Asp were conserved. The results show that JmjC domain proteins are a conserved family in which domain organization and cofactor binding sites have been modified in some species. Our results provide insights into KDM evolution and lay a foundation for functional characterization of KDMs.

Prevalence of the F-type lectin domain.

F-type lectins are fucolectins with characteristic fucose and calcium-binding sequence motifs and a unique lectin fold (the "F-type" fold). F-type lectins are phylogenetically widespread with selective distribution. Several eukaryotic F-type lectins have been biochemically and structurally characterized, and the F-type lectin domain (FLD) has also been studied in the bacterial proteins, Streptococcus mitis lectinolysin and Streptococcus pneumoniae SP2159. However, there is little knowledge about the extent of occurrence of FLDs and their domain organization, especially, in bacteria. We have now mined the extensive genomic sequence information available in the public databases with sensitive sequence search techniques in order to exhaustively survey prokaryotic and eukaryotic FLDs. We report 437 FLD sequence clusters (clustered at 80% sequence identity) from eukaryotic, eubacterial and viral proteins. Domain architectures are diverse but mostly conserved in closely related organisms, and domain organizations of bacterial FLD-containing proteins are very different from their eukaryotic counterparts, suggesting unique specialization of FLDs to suit different requirements. Several atypical phylogenetic associations hint at lateral transfer. Among eukaryotes, we observe an expansion of FLDs in terms of occurrence and domain organization diversity in the taxa Mollusca, Hemichordata and Branchiostomi, perhaps coinciding with greater emphasis on innate immune strategies in these organisms. The naturally occurring FLDs with diverse domain organizations that we have identified here will be useful for future studies aimed at creating designer molecular platforms for directing desired biological activities to fucosylated glycoconjugates in target niches.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins.

The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel ß-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa.

From words to phrases: neural basis of social event semantic composition.

Events are typically composed of at least actions and entities. Both actions and entities have been shown to be represented by neural structures respecting domain organizations in the brain, including those of social/animate (face and body; person-directed action) versus inanimate (man-made object or tool; object-directed action) concepts. It is unclear whether the brain combines actions and entities into events in a (relative) domain-specific fashion or via domain-general mechanisms in regions that have been shown to support semantic and syntactic composition. We tested these hypotheses in a functional magnetic resonance imaging experiment where two domains of verb-noun event phrases (social-person versus manipulation-artifact, e.g., "hug mother" versus "fold napkin") and their component words were contrasted. We found a series of brain region supporting social-composition effects more strongly than the manipulation phrase composition-the bilateral inferior occipital gyrus (IOG), inferior temporal gyrus (ITG) and anterior temporal lobe (ATL)-which either showed stronger activation strength tested by univariate contrast, stronger content representation tested by representation similarity analysis, or stronger relationship between the neural activation patterns of phrases and synthesis (additive and multiplication) of the neural activity patterns of the word constituents. No regions were observed showing evidence of phrase composition for both domains or stronger effects of manipulation phrases. These findings highlight the roles of the visual cortex and ATL in social event compositions, suggesting a domain-preferring, rather than domain-general, mechanisms of verbal event composition.

C2cd6-encoded CatSperτ targets sperm calcium channel to Ca2+ signaling domains in the flagellar membrane.

In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSperτ, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSperτ loss of function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSperτ targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSperτ interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting.

Topography of Visual Features in the Human Ventral Visual Pathway.

Visual object recognition in humans and nonhuman primates is achieved by the ventral visual pathway (ventral occipital-temporal cortex, VOTC), which shows a well-documented object domain structure. An on-going question is what type of information is processed in the higher-order VOTC that underlies such observations, with recent evidence suggesting effects of certain visual features. Combining computational vision models, fMRI experiment using a parametric-modulation approach, and natural image statistics of common objects, we depicted the neural distribution of a comprehensive set of visual features in the VOTC, identifying voxel sensitivities with specific feature sets across geometry/shape, Fourier power, and color. The visual feature combination pattern in the VOTC is significantly explained by their relationships to different types of response-action computation (fight-or-flight, navigation, and manipulation), as derived from behavioral ratings and natural image statistics. These results offer a comprehensive visual feature map in the VOTC and a plausible theoretical explanation as a mapping onto different types of downstream response-action systems.

R2 and Non-Site-Specific R2-Like Retrotransposons of the German Cockroach, Blattella germanica.

The structural and functional organization of the ribosomal RNA gene cluster and the full-length R2 non-LTR retrotransposon (integrated into a specific site of 28S ribosomal RNA genes) of the German cockroach, Blattella germanica, is described. A partial sequence of the R2 retrotransposon of the cockroach Rhyparobia maderae is also analyzed. The analysis of previously published next-generation sequencing data from the B. germanica genome reveals a new type of retrotransposon closely related to R2 retrotransposons but with a random distribution in the genome. Phylogenetic analysis reveals that these newly described retrotransposons form a separate clade. It is shown that proteins corresponding to the open reading frames of newly described retrotransposons exhibit unequal structural domains. Within these retrotransposons, a recombination event is described. New mechanism of transposition activity is discussed. The essential structural features of R2 retrotransposons are conserved in cockroaches and are typical of previously described R2 retrotransposons. However, the investigation of the number and frequency of 5'-truncated R2 retrotransposon insertion variants in eight B. germanica populations suggests recent mobile element activity. It is shown that the pattern of 5'-truncated R2 retrotransposon copies can be an informative molecular genetic marker for revealing genetic distances between insect populations.

In Situ Hi-C for Plants: An Improved Method to Detect Long-Range Chromatin Interactions.

Recently, long noncoding RNAs (lncRNAs) are shown to be implicating nuclear domain organization and gene regulation by mediating long-range chromatin interactions. Chromosome conformation capture (3C) is a method used to study such long-range interaction between two different loci in the 3D nuclear space. Through successive improvement in resolution and throughput, 3C, chromosome conformation capture on chip (4C), and chromosome conformation capture carbon copy (5C) to Hi-C methods were developed to study interactions between loci from one versus one scale to an unprecedented genome-wide resolution. In situ Hi-C is a variant of Hi-C in which proximity ligation is performed at the intact nuclei to improve the signal-to-noise ratio and throughput of the experiment to provide useful genome-wide contact frequency matrix/maps. The contact frequency maps obtained could be used for physical ordering of scaffolds in complex genome assembly projects, in deducing the nuclear domain organization in high resolution and in identifying specific long-range interactions between genomic regions of interest. In this chapter, we describe in detail a protocol for in situ Hi-C used on crops like barley, wheat, rye, oat, and evening primrose.

Conservation and diversification of polycomb repressive complex 2 (PRC2) proteins in the green lineage.

The polycomb group (PcG) proteins are key epigenetic regulators of gene expression in animals and plants. They act in multiprotein complexes, of which the best characterized is the polycomb repressive complex 2 (PRC2), which catalyses the trimethylation of histone H3 at lysine 27 (H3K27me3) at chromatin targets. In Arabidopsis thaliana, PRC2 proteins are involved in the regulation of diverse developmental processes, including cell fate determination, vegetative growth and development, flowering time control and embryogenesis. Here, we systematically analysed the evolutionary conservation and diversification of PRC2 components in lower and higher plants. We searched for and identified PRC2 homologues from the sequenced genomes of several green lineage species, from the unicellular green alga Ostreococcus lucimarinus to more complicated angiosperms. We found that some PRC2 core components, e.g. E(z), ESC/FIE and MSI/p55, are ancient and have multiplied coincidently with multicellular evolution. For one component, some members are newly formed, especially in the Cruciferae. During evolution, higher plants underwent copy number multiplication of various PRC2 components, which occurred independently for each component, without any obvious co-amplification of PRC2 members. Among the amplified members, usually one was well-conserved and the others were more diversified. Gene amplification occurred at different times for different PcG members during green lineage evolution. Certain PRC2 core components or members of them were highly conserved. Our study provides an insight into the evolutionary conservation and diversification of PcG proteins and may guide future functional characterization of these important epigenetic regulators in plants other than Arabidopsis.

Protein NCRII-18: the role of gene fusion in the molecular evolution of restriction endonucleases.

This work first constructed the fusion protein NCRII-18 by fusing the restriction endonuclease Ecl18kI gene and part of the gene coding for the N-terminal domain of the endonuclease EcoRII. The fusion of the EcoRII N-terminal domain leads to a change in the properties of the recombinant protein. Unlike Ecl18kI, which made the basis of NCRII-18, the fusion protein predominantly recognizes the CCWGG sites, having lost the capability of interacting with the CCSGG sites. Experimental data support the hypothesis of a close evolutionary relationship between type IIE and IIP restriction endonucleases via a recombination between domains with active site structure and elements for recognition with domains responsible for recognition of DNA sequences.

Engineered Photoactivatable Genetic Switches Based on the Bacterium Phage T7 RNA Polymerase.

Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects. The split fragments show high modularity when they are combined with different regulatory domains such as those with chemically inducible interaction, enabling chemically controlled switches. To summarize, the T7 RNA polymerase-based switches are powerful tools to implement light-activated gene expression in different contexts. Moreover, results about the studied split positions and domain organizations may facilitate future engineering studies on this and on related proteins.

A survey of HK, HPt, and RR domains and their organization in two-component systems and phosphorelay proteins of organisms with fully sequenced genomes.

Two Component Systems and Phosphorelays (TCS/PR) are environmental signal transduction cascades in prokaryotes and, less frequently, in eukaryotes. The internal domain organization of proteins and the topology of TCS/PR cascades play an important role in shaping the responses of the circuits. It is thus important to maintain updated censuses of TCS/PR proteins in order to identify the various topologies used by nature and enable a systematic study of the dynamics associated with those topologies. To create such a census, we analyzed the proteomes of 7,609 organisms from all domains of life with fully sequenced and annotated genomes. To begin, we survey each proteome searching for proteins containing domains that are associated with internal signal transmission within TCS/PR: Histidine Kinase (HK), Response Regulator (RR) and Histidine Phosphotranfer (HPt) domains, and analyze how these domains are arranged in the individual proteins. Then, we find all types of operon organization and calculate how much more likely are proteins that contain TCS/PR domains to be coded by neighboring genes than one would expect from the genome background of each organism. Finally, we analyze if the fusion of domains into single TCS/PR proteins is more frequently observed than one might expect from the background of each proteome. We find 50 alternative ways in which the HK, HPt, and RR domains are observed to organize into single proteins. In prokaryotes, TCS/PR coding genes tend to be clustered in operons. 90% of all proteins identified in this study contain just one of the three domains, while 8% of the remaining proteins combine one copy of an HK, a RR, and/or an HPt domain. In eukaryotes, 25% of all TCS/PR proteins have more than one domain. These results might have implications for how signals are internally transmitted within TCS/PR cascades. These implications could explain the selection of the various designs in alternative circumstances.