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In JoVE (2)
- Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
- Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
Other Publications (37)
- Molecular Vision
- Functional & Integrative Genomics
- Molecular Vision
- FEMS Microbiology Reviews
- Current Genetics
- Molecular Genetics and Genomics : MGG
- Human Gene Therapy
- Toxicological Sciences : an Official Journal of the Society of Toxicology
- Journal of Theoretical Biology
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- Scandinavian Journal of Gastroenterology
- Circulation Research
- The Journal of General Virology
- Plant Physiology
- BMC Genomics
- Nature Methods
- Virus Research
- Virology Journal
- FEMS Microbiology Reviews
- PLoS Biology
- Methods in Molecular Biology (Clifton, N.J.)
- Methods in Molecular Biology (Clifton, N.J.)
- Human Gene Therapy
- Molecular BioSystems
- Journal of Molecular Evolution
- European Journal of Clinical Investigation
- Science (New York, N.Y.)
- Structure (London, England : 1993)
- Molecular Microbiology
- PloS One
- Methods in Molecular Biology (Clifton, N.J.)
- Methods in Molecular Biology (Clifton, N.J.)
- British Journal of Haematology
- PLoS Genetics
Articles by Mohan Babu in JoVE
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Alla Gagarinova*1, Mohan Babu*2,3, Jack Greenblatt1,2, Andrew Emili1,2
1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina
Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.
Published November 12, 2012. Keywords: Genetics, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
Mohan Babu1,2, Olga Kagan1, Hongbo Guo1, Jack Greenblatt1,3, Andrew Emili1,3
1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto
Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.
Published November 12, 2012. Keywords: Genetics, Molecular Biology, Medicine, Biochemistry, Microbiology, affinity purification, Escherichia coli, gram-negative bacteria, cytosolic proteins, SPA-tagging, homologous recombination, mass spectrometry, protein interaction, protein complex
Other articles by Mohan Babu on PubMed
Molecular Vision. Jul, 2004 | Pubmed ID: 15257268
Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare eye genetic disorder caused by mutations in the FOXL2 gene located at chromosome 3q23. The purpose of the present study was to carry out genetic analysis of BPES in a five-generation Indian family.
Functional & Integrative Genomics. Oct, 2004 | Pubmed ID: 15349794
A large-scale comparative genomic analysis of unisequence sets obtained from an Ustilago maydis EST collection was performed against publicly available EST and genomic sequence datasets from 21 species. We annotated 70% of the collection based on similarity to known sequences and recognized protein signatures. Distinct grouping of the ESTs, defined by the presence or absence of similar sequences in the species examined, allowed the identification of U. maydis sequences present only (1) in fungal species, (2) in plants but not animals, (3) in animals but not plants, or (4) in all three eukaryotic lineages assessed. We also identified 215 U. maydis genes that are found in the ascomycete but not in the basidiomycete genome sequences searched. Candidate genes were identified for further functional characterization. These include 167 basidiomycete-specific sequences, 58 fungal pathogen-specific sequences (including 37 basidiomycete pathogen-specific sequences), and 18 plant pathogen-specific sequences, as well as two sequences present only in other plant pathogen and plant species.
Genetic Analysis of a Four Generation Indian Family with Usher Syndrome: a Novel Insertion Mutation in MYO7A
Molecular Vision. Nov, 2004 | Pubmed ID: 15592175
Usher syndrome (USH) is a rare autosomal recessive disorder characterized by deafness and retinitis pigmentosa. The purpose of this study was to determine the genetic cause of USH in a four generation Indian family.
FEMS Microbiology Reviews. Apr, 2005 | Pubmed ID: 15808743
The helix-turn-helix (HTH) domain is a common denominator in basal and specific transcription factors from the three super-kingdoms of life. At its core, the domain comprises of an open tri-helical bundle, which typically binds DNA with the 3rd helix. Drawing on the wealth of data that has accumulated over two decades since the discovery of the domain, we present an overview of the natural history of the HTH domain from the viewpoint of structural analysis and comparative genomics. In structural terms, the HTH domains have developed several elaborations on the basic 3-helical core, such as the tetra-helical bundle, the winged-helix and the ribbon-helix-helix type configurations. In functional terms, the HTH domains are present in the most prevalent transcription factors of all prokaryotic genomes and some eukaryotic genomes. They have been recruited to a wide range of functions beyond transcription regulation, which include DNA repair and replication, RNA metabolism and protein-protein interactions in diverse signaling contexts. Beyond their basic role in mediating macromolecular interactions, the HTH domains have also been incorporated into the catalytic domains of diverse enzymes. We discuss the general domain architectural themes that have arisen amongst the HTH domains as a result of their recruitment to these diverse functions. We present a natural classification, higher-order relationships and phyletic pattern analysis of all the major families of HTH domains. This reconstruction suggests that there were at least 6-11 different HTH domains in the last universal common ancestor of all life forms, which covered much of the structural diversity and part of the functional versatility of the extant representatives of this domain. In prokaryotes the total number of HTH domains per genome shows a strong power-equation type scaling with the gene number per genome. However, the HTH domains in two-component signaling pathways show a linear scaling with gene number, in contrast to the non-linear scaling of HTH domains in single-component systems and sigma factors. These observations point to distinct evolutionary forces in the emergence of different signaling systems with HTH transcription factors. The archaea and bacteria share a number of ancient families of specific HTH transcription factors. However, they do not share any orthologous HTH proteins in the basal transcription apparatus. This differential relationship of their basal and specific transcriptional machinery poses an apparent conundrum regarding the origins of their transcription apparatus.
Current Genetics. May, 2005 | Pubmed ID: 15809875
When fungi interact with plants as pathogens or as symbionts, there are often changes in fungal cell morphology and nuclear state. This study establishes the use of cDNA microarrays to detect gene expression changes in Ustilago maydis cells that differ in structure and nuclear content. Categorizing differentially expressed genes on the basis of function indicated that U. maydis cell types vary most in the expression of genes related to metabolism. We also observed that more genes are up-regulated in the filamentous dikaryon than in the filamentous diploid, relative to non-pathogenic budding cells. Our comparison of pathogenic development indicated that the dikaryon is more virulent than the diploid. Other identified expression patterns suggest a cell-specific difference in nutrient acquisition, cell metabolism and signal transduction. The relevance of gene expression change to cell type biology is discussed.
Molecular Genetics and Genomics : MGG. Jun, 2005 | Pubmed ID: 15887033
Ustilago maydis is a model fungal pathogen that induces the formation of tumors in maize. The tumor provides an environment for hyphal differentiation, leading to the formation of thick-walled, diploid teliospores. Such spores serve as a dispersal agent for smut and rust fungi, and their germination leads to new rounds of infection. The morphological changes that occur during teliospore germination in U. maydis have been described in detail. However, the specific molecular events that facilitate this process have not been identified. Through the construction and hybridization of microarrays containing a set of 3918 non-redundant cDNAs, we have identified genes that are differentially regulated during teliospore germination. Teliospores induced to germinate for 4 and 11 h were selected for comparison with dormant teliospores. Genes identified as differentially expressed included many that are presumably involved in as yet undescribed molecular events during teliospore germination, as well as characterized genes previously shown to be required for the process. This study represents the first large-scale investigation of changes in gene expression during teliospore germination.
Adenovirus-mediated Gene Transfer of Placental Growth Factor to Perivascular Tissue Induces Angiogenesis Via Upregulation of the Expression of Endogenous Vascular Endothelial Growth Factor-A
Human Gene Therapy. Dec, 2005 | Pubmed ID: 16390273
Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family that binds specifically to VEGF receptor (VEGFR)-1. However, the mechanism of PlGF- and VEGFR-1-mediated angiogenesis has remained unclear and some in vitro studies suggest that VEGF-A/VEGFR-2 signaling may also play a role in PlGF-mediated angiogenesis. To clarify these issues we evaluated angiogenic responses in a well-characterized periadventitial angiogenesis model using adenovirus-mediated PlGF-2 (AdvPlGF-2) gene transfer. We also investigated the roles of VEGFR-1 and VEGFR-2 in PlGF-2-mediated angiogenesis. Using a periadventitial collar technique, AdvPlGF-2 (1 x 10(9) plaque-forming units/ml) was transferred to the adventitia of New Zealand White rabbits alone or together with adenoviruses encoding soluble VEGFR-1 (sVEGFR-1) or soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were killed 7 days after gene transfer. Increased neo-vessel formation, upregulation of endogenous VEGF-A expression, and a significant inflammatory response were seen in AdvPlGF-2-transduced arteries. The neo-vessels were large and well perfused. sVEGFR-1 and sVEGFR-2 suppressed the angiogenic response of PlGF-2 by 80 and 71.7%, respectively. We conclude that adenovirus-mediated PlGF-2 gene transfer to vascular tissue increases endogenous VEGF-A expression and produces significant angiogenesis. Both sVEGFR-1 and sVEGFR-2 can inhibit PlGF-2-mediated angiogenesis. PlGF-2 is a potentially useful candidate for the induction of therapeutic angiogenesis in vivo.
Radiation Exposure Impairs Luteinizing Hormone Signal Transduction and Steroidogenesis in Cultured Human Leydig Cells
Toxicological Sciences : an Official Journal of the Society of Toxicology. Jun, 2006 | Pubmed ID: 16569731
Therapeutic, accidental, and experimental radiation exposures decreased serum testosterone in males, leading to various sexual problems. Since testicular Leydig cells are the predominant source of circulating testosterone, findings on the direct effects of radiation on Leydig cell steroidogenesis and the mechanism behind such effects would be of greater importance to the use of safer radiation doses in cancer therapy and to adopt preventive or therapeutic measures to alleviate postirradiation lesions, respectively. Therefore, this study was undertaken to explore the same using cultured human Leydig cells. Testicles removed from advanced prostatic carcinoma patients were used for isolation and purification of Leydig cells. Purified Leydig cells were cultured and then exposed to different doses (2, 4, 6, 8, and 10 Gy) of fractioned gamma radiation. Normal and irradiated cells were used for luteinizing hormone (LH) receptor quantification or total RNA isolation to study LH receptor mRNA expression or LH/cyclic AMP (cAMP) stimulation test. While LH-stimulated cells were used for cAMP assay, LH- and cAMP-stimulated cells were used for the estimation of steroidogenic enzymes, testosterone and estradiol production. Radiation exposure caused adverse effects on Leydig cell steroidogenesis in a dose-dependent manner. While lower doses (2 and 4 Gy) were ineffective, higher doses (6 Gy and above) drastically decreased LH receptor, basal and LH-stimulated cAMP generation, and basal, LH-, and cAMP-stimulated steroidogenesis. While 2 Gy of radiation exposure increased the LH receptor mRNA level, other doses did not induce any significant change. Therefore, it is concluded that higher doses of radiation impair Leydig cell steroidogenesis by affecting LH signal transduction at the level of both pre- and post-cAMP generation. Decreased level of LH receptors following higher doses of radiation exposure is not coupled with impaired expression of its mRNA.
Pseudo Amino Acid Composition and Multi-class Support Vector Machines Approach for Conotoxin Superfamily Classification
Journal of Theoretical Biology. Nov, 2006 | Pubmed ID: 16890961
Conotoxins are disulfide rich small peptides that target a broad spectrum of ion-channels and neuronal receptors. They offer promising avenues in the treatment of chronic pain, epilepsy and cardiovascular diseases. Assignment of newly sequenced mature conotoxins into appropriate superfamilies using a computational approach could provide valuable preliminary information on the biological and pharmacological functions of the toxins. However, creation of protein sequence patterns for the reliable identification and classification of new conotoxin sequences may not be effective due to the hypervariability of mature toxins. With the aim of formulating an in silico approach for the classification of conotoxins into superfamilies, we have incorporated the concept of pseudo-amino acid composition to represent a peptide in a mathematical framework that includes the sequence-order effect along with conventional amino acid composition. The polarity index attribute, which encodes information such as residue surface buriability, polarity, and hydropathy, was used to store the sequence-order effect. Several methods like BLAST, ISort (Intimate Sorting) predictor, least Hamming distance algorithm, least Euclidean distance algorithm and multi-class support vector machines (SVMs), were explored for superfamily identification. The SVMs outperform other methods providing an overall accuracy of 88.1% for all correct predictions with generalized squared correlation of 0.75 using jackknife cross-validation test for A, M, O and T superfamilies and a negative set consisting of short cysteine rich sequences from different eukaryotes having diverse functions. The computed sensitivity and specificity for the superfamilies were found to be in the range of 84.0-94.1% and 80.0-95.5%, respectively, attesting to the efficacy of multi-class SVMs for the successful in silico classification of the conotoxins into their superfamilies.
VEGF-A, VEGF-D, VEGF Receptor-1, VEGF Receptor-2, NF-kappaB, and RAGE in Atherosclerotic Lesions of Diabetic Watanabe Heritable Hyperlipidemic Rabbits
FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Oct, 2006 | Pubmed ID: 16935942
Plaque angiogenesis may be associated with the development of unstable and vulnerable plaques. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. Our objective was to determine the effect of diabetes on atherosclerosis and on the expression of angiogenesis-related genes in atherosclerotic lesions. Alloxan was used to induce diabetes in male Watanabe heritable hyperlipidemic (WHHL) rabbits that were sacrificed 2 and 6 months after the induction of diabetes. Nondiabetic WHHL rabbits served as controls. Blood glucose (Glc), serum-free fatty acids (FFA), and serum triglyceride levels were significantly higher in diabetic rabbits. Accelerated atherogenesis was observed in the diabetic WHHL rabbits together with increased intramyocellular lipids (IMCL), as determined by 1H-NMR spectroscopy. Atherosclerotic lesions in the diabetic rabbits had an increased content of macrophages and showed significant increases in immunostainings for vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, RAGE, and NF-kappaB. VEGF-A165 and VEGFR-2 mRNA levels were significantly increased in aortas of the diabetic rabbits, where a trend toward increased plaque vascularization was also observed. These results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, and VEGF receptor-2 expression, and increases NF-kappaB, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.
Screening for Human Cationic Trypsinogen (PRSS1) and Trypsinogen Inhibitor Gene (SPINK1) Mutations in a Finnish Family with Hereditary Pancreatitis
Scandinavian Journal of Gastroenterology. Aug, 2007 | Pubmed ID: 17613931
Mutations in the cationic trypsinogen gene (PRSS1) have been linked with hereditary pancreatitis (HP). A change in R122H in the third exon is one of the mutations most frequently associated with HP. A mutation N34S in the serine protease inhibitor Kazal type 1 gene has also been shown to be linked with HP. The purpose of this study was to report on the incidence of PRSS1 and SPINK1 mutations in a Finnish family with HP and to correlate the findings to the clinical symptoms.
15-lipoxygenase-1 Prevents Vascular Endothelial Growth Factor A- and Placental Growth Factor-induced Angiogenic Effects in Rabbit Skeletal Muscles Via Reduction in Growth Factor MRNA Levels, NO Bioactivity, and Downregulation of VEGF Receptor 2 Expression
Circulation Research. Feb, 2008 | Pubmed ID: 17991885
Human 15-lipoxygenase-1 (15-LO-1) is an oxidizing enzyme capable of producing reactive lipid hydroperoxides. 15-LO-1 and its products have been suggested to be involved in many pathological conditions, such as inflammation, atherogenesis, and carcinogenesis. We used adenovirus-mediated gene transfers to study the effects of 15-LO-1 on vascular endothelial growth factor (VEGF)-A165- and placental growth factor (PlGF)-induced angiogenesis in rabbit skeletal muscles. 15-LO-1 significantly decreased all angiogenic effects induced by these growth factors, including capillary perfusion, vascular permeability, vasodilatation, and an increase in capillary number. The effects are attributable to the reduction in the amount of VEGF-A165 and PlGF transcripts by 15-LO-1, resulting in reduced protein expression. The most likely mediator of the VEGF family-induced capillary vasodilatation is nitric oxide (NO), which is produced by NO synthases. Endothelial NO synthase protein expression and NO synthase activity were significantly induced by VEGF-A165, and these inductions were reduced by 15-LO-1. VEGF-A165 induces its angiogenic effects primarily via vascular endothelial growth factor receptor (VEGFR)2, and also PlGF mediates angiogenic signaling via VEGFR2, even though it binds to VEGFR1. VEGFR2 expression is induced by peroxisome proliferator-activating receptor . We showed by quantitative RT-PCR and immunohistochemistry that expression of endogenous rabbit peroxisome proliferator-activating receptor and VEGFR2 were significantly increased in the growth factor-transduced muscles, but these inductions were efficiently prevented by 15-LO-1. In conclusion, the results suggest that expression of 15-LO-1 has an efficient antiangiogenic effect in vivo via reduction in growth factor mRNA levels, NO bioactivity, and VEGFR2 expression.
Association of the Transcriptional Response of Soybean Plants with Soybean Mosaic Virus Systemic Infection
The Journal of General Virology. Apr, 2008 | Pubmed ID: 18343851
Compatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. To assess transcriptional changes in soybean plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation (p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean leaves with SMV infection at different time points were associated with the observed symptom development. By using stringent selection criteria (>or=2- or
The Arabidopsis BRAHMA Chromatin-remodeling ATPase is Involved in Repression of Seed Maturation Genes in Leaves
Plant Physiology. Jul, 2008 | Pubmed ID: 18508955
Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue.
Altered Gene Expression Changes in Arabidopsis Leaf Tissues and Protoplasts in Response to Plum Pox Virus Infection
BMC Genomics. 2008 | Pubmed ID: 18613973
Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled.
Nature Methods. Sep, 2008 | Pubmed ID: 18677321
Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.
Identification and Molecular Characterization of Two Naturally Occurring Soybean Mosaic Virus Isolates That Are Closely Related but Differ in Their Ability to Overcome Rsv4 Resistance
Virus Research. Dec, 2008 | Pubmed ID: 18793685
A naturally occurring Rsv4 resistance-breaking isolate (L-RB) and a closely related non-resistance-breaking isolate (L) of Soybean mosaic virus (SMV) were identified in soybean fields in London, Ontario, Canada. The viral genomes of L and L-RB were completely sequenced. Each isolate has a 9585-nucleotide genome with a single open reading frame encoding a polyprotein of approximately 350 kDa. L-RB and L have a very high sequence similarity (99.6%) at both the nucleotide and amino acid levels. Phylogenetic analysis showed that the two isolates belong to the G2 pathotype. Pathogenicity predictions of all virus/soybean combinations, based on the phylogenetic profile, were confirmed by pathogenicity tests using L and L-RB isolates and soybeans carrying different resistance genes, with an exception that L-RB infected a soybean cultivar carrying Rsv4 resistance. The temporal and spatial proximity of L and L-RB and their high sequence similarity suggest L-RB was likely derived from the SMV-L quasispecies. Recombination analysis did not reveal the evidence of genetic recombination for the emergence of L-RB. Mutations introduced by virus-encoded RNA-dependent RNA polymerase during viral genome replication and selection pressure probably contributed to the occurrence of L-RB.
Recombination Analysis of Soybean Mosaic Virus Sequences Reveals Evidence of RNA Recombination Between Distinct Pathotypes
Virology Journal. 2008 | Pubmed ID: 19036160
RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV), the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.
Computational and Experimental Approaches to Chart the Escherichia Coli Cell-envelope-associated Proteome and Interactome
FEMS Microbiology Reviews. Jan, 2009 | Pubmed ID: 19054114
The bacterial cell-envelope consists of a complex arrangement of lipids, proteins and carbohydrates that serves as the interface between a microorganism and its environment or, with pathogens, a human host. Escherichia coli has long been investigated as a leading model system to elucidate the fundamental mechanisms underlying microbial cell-envelope biology. This includes extensive descriptions of the molecular identities, biochemical activities and evolutionary trajectories of integral transmembrane proteins, many of which play critical roles in infectious disease and antibiotic resistance. Strikingly, however, only half of the c. 1200 putative cell-envelope-related proteins of E. coli currently have experimentally attributed functions, indicating an opportunity for discovery. In this review, we summarize the state of the art of computational and proteomic approaches for determining the components of the E. coli cell-envelope proteome, as well as exploring the physical and functional interactions that underlie its biogenesis and functionality. We also provide a comprehensive comparative benchmarking analysis on the performance of different bioinformatic and proteomic methods commonly used to determine the subcellular localization of bacterial proteins.
PLoS Biology. Apr, 2009 | Pubmed ID: 19402753
One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.
Systematic Characterization of the Protein Interaction Network and Protein Complexes in Saccharomyces Cerevisiae Using Tandem Affinity Purification and Mass Spectrometry
Methods in Molecular Biology (Clifton, N.J.). 2009 | Pubmed ID: 19521826
Defining protein complexes is a vital aspect of cell biology because cellular processes are often carried out by stable protein complexes and their characterization often provides insights into their function. Accurate identification of the interacting proteins in macromolecular complexes is easiest after purification to near homogeneity. To this end, the tandem affinity purification (TAP) system with subsequent protein identification by high-throughput mass spectrometry was developed (1, 2) to systematically characterize native protein complexes and transient protein interactions under near-physiological conditions. The TAP tag containing two adjacent affinity purification tags (calmodulin-binding peptide and Staphylococcus aureus protein A) separated by a tobacco etch virus (TEV) protease cleavage site is fused with the open reading frame of interest. Using homologous recombination, a fusion library was constructed for the yeast Saccharomyces cerevisiae (3) in which the carboxy-terminal end of each predicted open reading frame is individually tagged in the chromosome so that the resulting fusion proteins are expressed under the control of their natural promoters (3). In this chapter, an optimized protocol for systematic protein purification and subsequent mass spectrometry-based protein identification is described in detail for the protein complexes of S. cerevisiae (4-6).
Sequential Peptide Affinity Purification System for the Systematic Isolation and Identification of Protein Complexes from Escherichia Coli
Methods in Molecular Biology (Clifton, N.J.). 2009 | Pubmed ID: 19544035
Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).
Intravitreal Adenoviral 15-lipoxygenase-1 Gene Transfer Prevents Vascular Endothelial Growth Factor A-induced Neovascularization in Rabbit Eyes
Human Gene Therapy. Dec, 2009 | Pubmed ID: 19694557
Excessive angiogenesis mediated by vascular endothelial growth factor (VEGF) plays an important role in angioproliferative ocular diseases. We have previously developed a large animal model for these diseases by intravitreal adenoviral gene transfer of VEGF-A(165). 15-Lipoxygenase-1 (15-LO-1), an oxidizing enzyme producing reactive lipid hydroperoxides, has been shown to induce aberrant angiogenesis in cancer models of transgenic mice overexpressing human 15-LO-1. Our purpose was to study the effects of 15-LO-1 on VEGF-A(165)-induced angiogenesis in New Zealand White rabbit eyes, using intravitreal adenovirus-mediated gene transfers. AdCMV and Adh15-LO-1 alone served as controls. As determined by immunohistochemistry, VEGF-A(165) significantly increased the number and size of the capillaries in various compartments of the eyes. 15-LO-1 efficiently inhibited VEGF-A(165)-induced neovascularization and pathological changes by reducing VEGF-A(165) mRNA and protein expression, determined by RT-PCR, ELISA, and immunohistochemistry. 15-LO-1, which produces endogenous ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), also prevented VEGF-A(165)-induced expression of PPARgamma and VEGF receptor-2, as measured by quantitative RT-PCR. In conclusion, our findings show that 15-LO-1 prevents VEGF-A(165)-induced angiogenesis and consequent pathology in the eyes, suggesting that intravitreal 15-LO-1 gene transfer could be a potential new strategy for the treatment of neovascular complications in the eyes.
Systems-level Approaches for Identifying and Analyzing Genetic Interaction Networks in Escherichia Coli and Extensions to Other Prokaryotes
Molecular BioSystems. Dec, 2009 | Pubmed ID: 19763343
Molecular interactions define the functional organization of the cell. Epistatic (genetic, or gene-gene) interactions, one of the most informative and commonly encountered forms of functional relationships, are increasingly being used to map process architecture in model eukaryotic organisms. In particular, 'systems-level' screens in yeast and worm aimed at elucidating genetic interaction networks have led to the generation of models describing the global modular organization of gene products and protein complexes within a cell. However, comparable data for prokaryotic organisms have not been available. Given its ease of growth and genetic manipulation, the Gram-negative bacterium Escherichia coli appears to be an ideal model system for performing comprehensive genome-scale examinations of genetic redundancy in bacteria. In this review, we highlight emerging experimental and computational techniques that have been developed recently to examine functional relationships and redundancy in E. coli at a systems-level, and their potential application to prokaryotes in general. Additionally, we have scanned PubMed abstracts and full-text published articles to manually curate a list of approximately 200 previously reported synthetic sick or lethal genetic interactions in E. coli derived from small-scale experimental studies.
Journal of Molecular Evolution. Apr, 2010 | Pubmed ID: 20333370
Bacterial lipoproteins, characterized by the N-terminal N-acyl S-diacylglyceryl Cysteine, are key membrane proteins in bacterial homeostasis. It is generally thought that during the modification lipoprotein precursors are translocated via the Sec-machinery in an unfolded state. The recent discovery of twin-arginine translocation (TAT) machinery, meant for exporting folded-proteins, and the presence of TAT-type signal sequences in co-factor-containing (hence already folded) lipoproteins, prompted us to investigate its role and significance in lipoprotein biosynthesis. We systematically analyzed 696 prokaryotic genomes using an algorithm based on DOLOP and TatP rules to predict TAT-pathway-dependent lipoprotein substrates. Occurrence of the deduced TAT-pathway-dependent lipoprotein substrates in relation to genome size, presence or absence of TAT machinery, and extent of its usage for lipoprotein export and habitat types revealed that unlike the host-obligates, the free-living prokaryotes in complex hostile environments (e.g., soil) depend more on TAT-exported lipoproteins. Functional classification of the predicted TAT-dependent lipoproteins revealed enrichment in hydrolases and oxido-reductases, which are fast-folding and co-factor-containing proteins. The role of the TAT pathway in the export of folded-lipoproteins and in niche-specific adaptation for survival has important implications not only in lipoprotein biosynthesis, but also for protein and metabolic engineering applications.
European Journal of Clinical Investigation. May, 2010 | Pubmed ID: 20534064
Arterial occlusive disease is often associated with diabetes mellitus and hypercholesterolaemia which may reduce angiogenic potential of several growth factors. Accordingly, the usefulness of therapeutic angiogenesis in the presence of diabetes and hypercholesterolaemia has remained unclear. We evaluated angiogenic effects of the mature form of vascular endothelial growth factor-D (VEGF-D(deltaNdeltaC)) in skeletal muscles in the presence of severe diabetes and hypercholesterolaemia.
Science (New York, N.Y.). Jul, 2010 | Pubmed ID: 20671182
Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers and are difficult to detect in single cells. We carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.
Structure of a SLC26 Anion Transporter STAS Domain in Complex with Acyl Carrier Protein: Implications for E. Coli YchM in Fatty Acid Metabolism
Structure (London, England : 1993). Nov, 2010 | Pubmed ID: 21070944
Escherichia coli YchM is a member of the SLC26 (SulP) family of anion transporters with an N-terminal membrane domain and a C-terminal cytoplasmic STAS domain. Mutations in human members of the SLC26 family, including their STAS domain, are linked to a number of inherited diseases. Herein, we describe the high-resolution crystal structure of the STAS domain from E. coli YchM isolated in complex with acyl-carrier protein (ACP), an essential component of the fatty acid biosynthesis (FAB) pathway. A genome-wide genetic interaction screen showed that a ychM null mutation is synthetically lethal with mutant alleles of genes (fabBDHGAI) involved in FAB. Endogenous YchM also copurified with proteins involved in fatty acid metabolism. Furthermore, a deletion strain lacking ychM showed altered cellular bicarbonate incorporation in the presence of NaCl and impaired growth at alkaline pH. Thus, identification of the STAS-ACP complex suggests that YchM sequesters ACP to the bacterial membrane linking bicarbonate transport with fatty acid metabolism.
Molecular Microbiology. Jan, 2011 | Pubmed ID: 21219465
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.
Ribosome-dependent ATPase Interacts with Conserved Membrane Protein in Escherichia Coli to Modulate Protein Synthesis and Oxidative Phosphorylation
PloS One. 2011 | Pubmed ID: 21556145
Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.
Array-based Synthetic Genetic Screens to Map Bacterial Pathways and Functional Networks in Escherichia Coli
Methods in Molecular Biology (Clifton, N.J.). 2011 | Pubmed ID: 21815091
Cellular processes are carried out through a series of molecular interactions. Various experimental approaches can be used to investigate these functional relationships on a large-scale. Recently, the power of investigating biological systems from the perspective of genetic (gene-gene or epistatic) interactions has been evidenced by the ability to elucidate novel functional relationships. Examples of functionally related genes include genes that buffer each other's function or impinge on the same biological process. Genetic interactions have traditionally been investigated in bacteria by combining pairs of mutations (e.g., gene deletions) and assessing deviation of the phenotype of each double mutant from an expected neutral (or no interaction) phenotype. Fitness is a particularly convenient phenotype to measure: when the double mutant grows faster or slower than expected, the two mutated genes are said to show alleviating or aggravating interactions, respectively. The most commonly used neutral model assumes that the fitness of the double mutant is equal to the product of individual single mutant fitness. A striking genetic interaction is exemplified by the loss of two nonessential genes that buffer each other in performing an essential biological function: deleting only one of these genes produces no detectable fitness defect; however, loss of both genes simultaneously results in systems failure, leading to synthetic sickness or lethality. Systematic large-scale genetic interaction screens have been used to generate functional maps for model eukaryotic organisms, such as yeast, to describe the functional organization of gene products into pathways and protein complexes within a cell. They also reveal the modular arrangement and cross talk of pathways and complexes within broader functional neighborhoods (Dixon et al., Annu Rev Genet 43:601-625, 2009). Here, we present a high-throughput quantitative Escherichia coli Synthetic Genetic Array (eSGA) screening procedure, which we developed to systematically infer genetic interactions by scoring growth defects among large numbers of double mutants in a classic Gram-negative bacterium. The eSGA method exploits the rapid colony growth, ease of genetic manipulation, and natural efficient genetic exchange via conjugation of laboratory E. coli strains. Replica pinning is used to grow and mate arrayed sets of single gene mutant strains and to select double mutants en masse. Strain fitness, which is used as the eSGA readout, is quantified by the digital imaging of the plates and subsequent measuring and comparing single and double mutant colony sizes. While eSGA can be used to screen select mutants to probe the functions of individual genes, using eSGA more broadly to collect genetic interaction data for many combinations of genes can help reconstruct a functional interaction network to reveal novel links and components of biological pathways as well as unexpected connections between pathways. A variety of bacterial systems can be investigated, wherein the genes impinge on a essential biological process (e.g., cell wall assembly, ribosome biogenesis, chromosome replication) that are of interest from the perspective of drug development (Babu et al., Mol Biosyst 12:1439-1455, 2009). We also show how genetic interactions generated by high-throughput eSGA screens can be validated by manual small-scale genetic crosses and by genetic complementation and gene rescue experiments.
Adventitial Gene Transfer of VEGFR-2 Specific VEGF-E Chimera Induces MCP-1 Expression in Vascular Smooth Muscle Cells and Enhances Neointimal Formation
Atherosclerosis. Nov, 2011 | Pubmed ID: 21862016
The role of vascular endothelial growth factors (VEGFs) in neointimal formation has been controversial. VEGF receptor (R)-2 signaling pathway is crucial in bringing about the effects of VEGFs including vasodilatation, endothelial cell migration and proliferation. In this study we have used an established adventitial gene transfer technique, in vitro studies and a novel VEGF-E/PlGF chimera that binds specifically to VEGFR-2, to investigate the role of VEGFR-2 in neointimal formation.
Array-based Synthetic Genetic Screens to Map Bacterial Pathways and Functional Networks in Escherichia Coli
Methods in Molecular Biology (Clifton, N.J.). 2011 | Pubmed ID: 21877280
Cellular processes are carried out through a series of molecular interactions. Various experimental approaches can be used to investigate these functional relationships on a large-scale. Recently, the power of investigating biological systems from the perspective of genetic (gene-gene, or epistatic) interactions has been evidenced by the ability to elucidate novel functional relationships. Examples of functionally related genes include genes that buffer each other's function or impinge on the same biological process. Genetic interactions have traditionally been investigated in bacteria by combining pairs of mutations (for example, gene deletions) and assessing deviation of the phenotype of each double mutant from an expected neutral (or no interaction) phenotype. Fitness is a particularly convenient phenotype to measure: when the double mutant grows faster or slower than expected, the two mutated genes are said to show alleviating or aggravating interactions, respectively. The most commonly used neutral model assumes that the fitness of the double mutant is equal to the product of individual single mutant fitness. A striking genetic interaction is exemplified by the loss of two nonessential genes that buffer each other in performing an essential biological function: deleting only one of these genes produces no detectable fitness defect; however, loss of both genes simultaneously results in systems failure, leading to synthetic sickness or lethality. Systematic large-scale genetic interaction screens have been used to generate functional maps for model eukaryotic organisms, such as yeast, to describe the functional organization of gene products into pathways and protein complexes within a cell. They also reveal the modular arrangement and cross-talk of pathways and complexes within broader functional neighborhoods (Dixon et al. Annu Rev Genet 43:601-625, 2009). Here, we present a high-throughput quantitative Escherichia coli synthetic genetic array (eSGA) screening procedure, which we developed to systematically infer genetic interactions by scoring growth defects among large numbers of double mutants in a classic gram-negative bacterium. The eSGA method exploits the rapid colony growth, ease of genetic manipulation, and natural efficient genetic exchange via conjugation of laboratory E. coli strains. Replica pinning is used to grow and mate arrayed sets of single-gene mutant strains as well as to select double mutants en mass. Strain fitness, which is used as the eSGA readout, is quantified by the digital imaging of the plates and subsequent measuring and comparing single and double mutant colony sizes. While eSGA can be used to screen select mutants to probe the functions of individual genes; using eSGA more broadly to collect genetic interaction data for many combinations of genes can help reconstruct a functional interaction network to reveal novel links and components of biological pathways as well as unexpected connections between pathways. A variety of bacterial systems can be investigated, wherein the genes impinge on a essential biological process (e.g., cell wall assembly, ribosome biogenesis, chromosome replication) that are of interest from the perspective of drug development (Babu et al. Mol Biosyst 12:1439-1455, 2009). We also show how genetic interactions generated by high-throughput eSGA screens can be validated by manual small-scale genetic crosses and by genetic complementation and gene rescue experiments.
Sorafenib Monotherapy Gives Sustainable Suppression of FLT3 Clone in Untreated Patients with FLT3-internal Tandem Duplication Positive Acute Myeloid Leukaemia
British Journal of Haematology. Nov, 2011 | Pubmed ID: 22050655
Genetic Interaction Maps in Escherichia Coli Reveal Functional Crosstalk Among Cell Envelope Biogenesis Pathways
PLoS Genetics. Nov, 2011 | Pubmed ID: 22125496
As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.
Cell. Aug, 2012 | Pubmed ID: 22939629
Cellular processes often depend on stable physical associations between proteins. Despite recent progress, knowledge of the composition of human protein complexes remains limited. To close this gap, we applied an integrative global proteomic profiling approach, based on chromatographic separation of cultured human cell extracts into more than one thousand biochemical fractions that were subsequently analyzed by quantitative tandem mass spectrometry, to systematically identify a network of 13,993 high-confidence physical interactions among 3,006 stably associated soluble human proteins. Most of the 622 putative protein complexes we report are linked to core biological processes and encompass both candidate disease genes and unannotated proteins to inform on mechanism. Strikingly, whereas larger multiprotein assemblies tend to be more extensively annotated and evolutionarily conserved, human protein complexes with five or fewer subunits are far more likely to be functionally unannotated or restricted to vertebrates, suggesting more recent functional innovations.
Nature. Sep, 2012 | Pubmed ID: 22940862
Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.