Highly divergent genes for methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection

The origins and the evolutionary history of tetrahydromethanopterin-linked C1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.     

Analysis of male meiotic "sex body" proteins during XY female meiosis provides new insights into their functions

During male meiosis in mammals the X and Y chromosomes become condensed to form the sex body (XY body), which is the morphological manifestation of the process of meiotic sex chromosome inactivation (MSCI). An increasing number of sex body located proteins are being identified, but their functions in relation to MSCI are unclear. Here we demonstrate that assaying male sex body located proteins during XY female mouse meiosis, where MSCI does not take place, is one way in which to begin to discriminate between potential functions. We show that a newly identified protein, "Asynaptin" (ASY), detected in male meiosis exclusively in association with the X and Y chromatin of the sex body, is also expressed in pachytene oocytes of XY females where it coats the chromatin of the asynapsed X in the absence of MSCI. Furthermore, in pachytene oocytes of females carrying a reciprocal autosomal translocation, ASY associates with asynapsed autosomal chromatin. Thus the location of ASY to the sex body during male meiosis is likely to be a response to the asynapsis of the non-homologous regions [outside the pseudoautosomal region (PAR)] of the heteromorphic X-Y bivalent, rather than being related to MSCI. In contrast to ASY, the previously described sex body protein XY77 proved to be male sex body specific. Potential functions for MSCI and the sex body are discussed together with the possible roles of these two proteins.     

A degron-based strategy reveals new insights into Aurora B function in C. elegans

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2’s major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.     

Phylogenomic analysis of the hemp family (Cannabaceae) reveals deep cyto-nuclear discordance and provides new insights into generic relationships

Cannabaceae are a relatively small family of angiosperms, but they include several species of huge economic and cultural significance: marijuana or hemp (Cannabis sativa) and hops (Humulus lupulus). Previous phylogenetic studies have clarified the most deep relationships in Cannabaceae, but relationships remain ambiguous among several major lineages. Here, we sampled 82 species representing all genera of Cannabaceae and utilized a new dataset of 90 nuclear genes and 82 chloroplast loci from Hyb-Seq to investigate the phylogenomics of Cannabaceae. Nuclear phylogenetic analyses revealed a robust and consistent backbone for Cannabaceae. We observed nuclear gene-tree conflict at several deep nodes in inferred species trees, also cyto-nuclear discordance concerning the relationship between Gironniera and Lozanella and the relationships among Trema s.l. (including Parasponia), Cannabis + Humulus, and Chaetachme + Pteroceltis. Coalescent simulations and network analyses suggest that observed deep cyto-nuclear discordances were most likely to stem from incomplete lineage sorting (ILS); nuclear gene-tree conflict might be caused by both ILS and gene flow between species. All genera of Cannabaceae were recovered as monophyletic, except for Celtis, which consisted of two distinct clades: Celtis I (including most Celtis species) and Celtis II (including Celtis gomphophylla and Celtis schippii). We suggest that Celtis II should be recognized as the independent genus Sparrea based on both molecular and morphological evidence. Our work provides the most comprehensive and reliable phylogeny to date for Cannabaceae, enabling further exploration of evolutionary patterns across this family and highlighting the necessity of comparing nuclear with chloroplast data to examine the evolutionary history of plant groups.     

Overexpressing the ANR1 MADS-box gene in transgenic plants provides new insights into its role in the nitrate regulation of root development

The expression of the ANR1 MADS-box gene was manipulated in transgenic plants to investigate its role in the NO(3)(-)-dependent regulation of root development in Arabidopsis thaliana. Constitutive overexpression of ANR1 in roots, achieved using GAL4 enhancer trap lines, resulted in more rapid early seedling development, increased lengths and numbers of lateral roots and increased shoot fresh weight. Based on results obtained with five different enhancer trap lines, the overexpression of ANR1 in the lateral root tips appears to be more important for this phenotype than its level of expression in the developing lateral root primordia. Dexamethasone-mediated induction of ANR1 in lines expressing an ANR1-GR (glucocorticoid receptor) fusion protein stimulated lateral root growth but not primary root growth. Short-term (24 h) dexamethasone treatments led to prolonged stimulation of lateral root growth, whether the lateral roots were already mature or still unemerged at the time of treatment. In split-root experiments, localized application of dexamethasone to half of the root system of an ANR1-GR line elicited a localized increase in both the length and numbers of lateral roots, mimicking the effect of a localized NO(3)(-) treatment. In both types of transgenic line, the root phenotype was strongly dependent on the presence of NO(3)(-), indicating that there are additional components involved in ANR1 function that are NO(3)(-) regulated. The implications of these results for our understanding of ANR1's mode of action in the root response to localized NO(3)(-) are discussed.     

Finding pathogenicity islands and gene transfer events in genome data

MOTIVATION: There is a growing literature on wavelet theory and wavelet methods showing improvements on more classical techniques, especially in the contexts of smoothing and extraction of fundamental components of signals. G+C patterns occur at different lengths (scales) and, for this reason, G+C plots are usually difficult to interpret. Current methods for genome analysis choose a window size and compute a chi(2) statistics of the average value for each window with respect to the whole genome. RESULTS: Firstly, wavelets are used to smooth G+C profiles to locate characteristic patterns in genome sequences. The method we use is based on performing a chi(2) statistics on the wavelet coefficients of a profile; thus we do not need to choose a fixed window size, in that the smoothing occurs at a set of different scales. Secondly, a wavelet scalogram is used as a measure for sequence profile comparison; this tool is very general and can be applied to other sequence profiles commonly used in genome analysis. We show applications to the analysis of Deinococcus radiodurans chromosome I, of two strains of Helicobacter pylori (26695, J99) and two of Neisseria meningitidis (serogroup B strain MC58 and serogroup A strain Z2491). We report a list of loci that have different G+C content with respect to the nearby regions; the analysis of N. meningitidis serogroup B shows two new large regions with low G+C content that are putative pathogenicity islands. AVAILABILITY: Software and numerical results (profiles, scalograms, high and low frequency components) for all the genome sequences analyzed are available upon request from the authors.     

Cell cholesterol efflux: integration of old and new observations provides new insights

Numerous studies using a variety of cell/acceptor combinations have demonstrated differences in cholesterol efflux among cells. These studies also show that different acceptors, ranging from simple molecules like cyclodextrins to serum, stimulate efflux through a variety of mechanisms. By combining early observations with data derived from recent studies, it is now possible to formulate a model for cell cholesterol efflux which proposes that an array of different mechanisms, including aqueous diffusion, lipid-free apolipoprotein membrane microsolubilization, and SR-BI-mediated cholesterol exchange contribute to cholesterol flux. In this model the relative importance of each mechanism would be determined both by the cell type and the nature of the extracellular cholesterol acceptor.     

Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.     

The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (R_g) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.     

Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.     

Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin

Human chorionic gonadotropin (hCG), is a placental hormone which exerts its major effect by stimulating progesterone production, crucially sustaining the early weeks of pregnancy. Detection of hCG with specific monoclonal antibodies (mAbs) has become the chosen means for pregnancy diagnosis. We have used antibody Fv fragments derived from two high-affinity mAbs, one against the alpha and the other against the beta-hCG subunit to enable the crystallisation of intact or desialylated hCG. Crystals of a ternary complex composed of Fv anti-alpha/hCG/Fv anti-beta were found to diffract to 3.5 A resolution, and the structure was solved by molecular replacement. In the crystal, the two Fvs keep hCG as in a molecular cage, providing good protein-protein contacts and leaving enough space for the saccharides to be accommodated in the cell solvent. The two Fvs were found not to interact directly through their complementary-determining regions with the hCG saccharides, but only with the protein. The hCG structure in the ternary complex was very close to that of the HF partially deglycosylated hormone, thus indicating that neither the saccharides nor the Fvs had any substantial influence on hormone structure.     

The physical map of wheat chromosome 1BS provides insights into its gene space organization and evolution

Background

The wheat genome sequence is an essential tool for advanced genomic research and improvements. The generation of a high-quality wheat genome sequence is challenging due to its complex 17 Gb polyploid genome. To overcome these difficulties, sequencing through the construction of BAC-based physical maps of individual chromosomes is employed by the wheat genomics community. Here, we present the construction of the first comprehensive physical map of chromosome 1BS, and illustrate its unique gene space organization and evolution.

Results

Fingerprinted BAC clones were assembled into 57 long scaffolds, anchored and ordered with 2,438 markers, covering 83% of chromosome 1BS. The BAC-based chromosome 1BS physical map and gene order of the orthologous regions of model grass species were consistent, providing strong support for the reliability of the chromosome 1BS assembly. The gene space for chromosome 1BS spans the entire length of the chromosome arm, with 76% of the genes organized in small gene islands, accompanied by a two-fold increase in gene density from the centromere to the telomere.

Conclusions

This study provides new evidence on common and chromosome-specific features in the organization and evolution of the wheat genome, including a non-uniform distribution of gene density along the centromere-telomere axis, abundance of non-syntenic genes, the degree of colinearity with other grass genomes and a non-uniform size expansion along the centromere-telomere axis compared with other model cereal genomes. The high-quality physical map constructed in this study provides a solid basis for the assembly of a reference sequence of chromosome 1BS and for breeding applications.     

Disruption of FEM1C-W gene in zebra finch: evolutionary insights on avian ZW genes

Sex chromosome genes control sex determination and differentiation, but the mechanisms of sex determination in birds are unknown. In this study, we analyzed the gene FEM1C which is highly conserved from Caenorhabditis elegans to higher vertebrates and interacts with the sex determining pathway in C. elegans. We found that FEM1C is located on the Z and W chromosome of zebra finches and probably other Passerine birds, but shows only Z linkage in other avian orders. In the zebra finch, FEM1C-W is degraded because of a point mutation and possibly because of loss of the first exon containing the start methionine. Thus, FEM1C-W appears to have degenerated or been lost from most bird species. FEM1C-Z is expressed in a cytoplasmic location in zebra finch fibroblast cells, as in C. elegans. FEM1C represents an interesting example of evolutionary degradation of a W chromosome gene.