Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation.

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.     

Sequence studies of nonradioactive Mycoplasma tRNAPhe with the aid of polynucleotide phosphorylase and polynucleotide kinase

The known methods of enzymatic phosphorylation with [(32)P]phosphate of the 3'- or 5'-hydroxyl group of an oligonucleotide have been applied to oligonucleotides derived from Mycoplasma tRNA(Phe). The fingerprints obtained by both methods are very similar to each other and to that of uniformly labelled tRNA. The sequence of some oligonucleotides was determined by partial digestion of the 3'-phosphorylated fragment with spleen phosphodiesterase and of the corresponding 5'-phosphorylated fragment with venom phosphodiesterase.     

Genome-wide networks of amino acid covariances are common among viruses

Coordinated variation among positions in amino acid sequence alignments can reveal genetic dependencies at noncontiguous positions, but methods to assess these interactions are incompletely developed. Previously, we found genome-wide networks of covarying residue positions in the hepatitis C virus genome (R. Aurora, M. J. Donlin, N. A. Cannon, and J. E. Tavis, J. Clin. Invest. 119:225-236, 2009). Here, we asked whether such networks are present in a diverse set of viruses and, if so, what they may imply about viral biology. Viral sequences were obtained for 16 viruses in 13 species from 9 families. The entire viral coding potential for each virus was aligned, all possible amino acid covariances were identified using the observed-minus-expected-squared algorithm at a false-discovery rate of ≤1%, and networks of covariances were assessed using standard methods. Covariances that spanned the viral coding potential were common in all viruses. In all cases, the covariances formed a single network that contained essentially all of the covariances. The hepatitis C virus networks had hub-and-spoke topologies, but all other networks had random topologies with an unusually large number of highly connected nodes. These results indicate that genome-wide networks of genetic associations and the coordinated evolution they imply are very common in viral genomes, that the networks rarely have the hub-and-spoke topology that dominates other biological networks, and that network topologies can vary substantially even within a given viral group. Five examples with hepatitis B virus and poliovirus are presented to illustrate how covariance network analysis can lead to inferences about viral biology.     

Ox-PAPC activation of PMET system increases expression of heme oxygenase-1 in human aortic endothelial cell

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) has been demonstrated to accumulate in atherosclerotic lesions and regulates expression of more than 1,000 genes in human aortic endothelial cell (HAEC). Among the most highly induced is heme oxygenase-1 (HO-1), a cell-protective antioxidant enzyme, which is sensitively induced by oxidative stress. To identify the pathway by which Ox-PAPC induces HO-1, we focused on the plasma membrane electron transport (PMET) complex, which contains ecto-NADH oxidase 1 (eNOX1) and NADPH:quinone oxidoreductase 1 (NQO1) and affects cellular redox status by regulating levels of NAD(P)H. We demonstrated that Ox-PAPC and its active components stimulated electron transfer through the PMET complex in HAECs from inside to outside [as determined by extracellular 2-(4-iodophenyl)-3-(44-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) reduction] and from outside to inside of the cell (as determined by intracellular NBT reduction). Chemical inhibitors of PMET system and siRNAs to PMET components (NQO1 and eNOX1) significantly decreased HO-1 induction by Ox-PAPC. We present evidence that Ox-PAPC activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in HAEC plays an important role in the induction of HO-1 and PMET inhibitors blocked Nrf2 activation by Ox-PAPC. We hypothesized that PMET activation by Ox-PAPC causes intracellular NAD(P)H depletion, which leads to the increased oxidative stress and HO-1 induction. Supporting this hypothesis, cotreatment of cells with exogenous NAD(P)H and Ox-PAPC significantly decreased oxidative stress and HO-1 induction by Ox-PAPC. Taken together, we demonstrated that the PMET system in HAEC plays an important role in the regulation of cellular redox status and HO-1 expression by Ox-PAPC.     

Edgetic perturbation models of human inherited disorders

Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as ‘nodes’ and ‘edges’, respectively. Better understanding of genotype‐to‐phenotype relationships in human disease will require modeling of how disease‐causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products (‘node removal’) and interaction‐specific or edge‐specific (‘edgetic’) alterations. Global computational analyses of ～50 000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies.     

HLA-Associated Immune Escape Pathways in HIV-1 Subtype B Gag,Pol and Nef Proteins

Background

Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA)-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution.

Methodology/Principal Findings

HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q≤0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association), followed by Gag then Pol (where ∼15–20% of codons exhibited HLA associations), confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q≤0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations.

Conclusions/Significance

The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate research in HIV epitope discovery and host-pathogen co-evolution, and are relevant to the continued search for an effective CTL-based AIDS vaccine.     

Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development

Background  

The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.     

An experimental metagenome data management and analysis system

The application of shotgun sequencing to environmental samples has revealed a new universe of microbial community genomes (metagenomes) involving previously uncultured organisms. Metagenome analysis, which is expected to provide a comprehensive picture of the gene functions and metabolic capacity for microbial communities, needs to be conducted in the context of a comprehensive data management and analysis system. We present in this paper IMG/M, an experimental metagenome data management and analysis system that is based on the Integrated Microbial Genomes (IMG) system. IMG/M provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative context. IMG/M is available at http://img.jgi.doe.gov/m.