Restriction Endonucleases BamHI Research Articles

The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes.

The substitution-inert polynuclear platinum(II) complex (PPC) series, [{trans-Pt(NH3)2(NH2(CH2)nNH3)}2-μ-(trans-Pt(NH3)2(NH2(CH2)nNH2)2}](NO3)8, where n = 5 (AH78P), 6 (AH78 TriplatinNC) and 7 (AH78H), are potent non-covalent DNA binding agents where nucleic acid recognition is achieved through use of the ‘phosphate clamp' where the square-planar tetra-am(m)ine Pt(II) coordination units all form bidentate N–O–N complexes through hydrogen bonding with phosphate oxygens. The modular nature of PPC–DNA interactions results in high affinity for calf thymus DNA (Kapp ∼5 × 107 M−1). The phosphate clamp–DNA interactions result in condensation of superhelical and B-DNA, displacement of intercalated ethidium bromide and facilitate cooperative binding of Hoechst 33258 at the minor groove. The effect of linker chain length on DNA conformational changes was examined and the pentane-bridged complex, AH78P, was optimal for condensing DNA with results in the nanomolar region. Analysis of binding affinity and conformational changes for sequence-specific oligonucleotides by ITC, dialysis, ICP-MS, CD and 2D-1H NMR experiments indicate that two limiting modes of phosphate clamp binding can be distinguished through their conformational changes and strongly suggest that DNA condensation is driven by minor-groove spanning. Triplatin-DNA binding prevents endonuclease activity by type II restriction enzymes BamHI, EcoRI and SalI, and inhibition was confirmed through the development of an on-chip microfluidic protocol.

Synthesis, in-vitro cytotoxic activity and DNA interactions of new dicarboxylatoplatinum(II) complexes with 2-hydroxymethylbenzimidazole as carrier ligands.

The aim of this study was to investigate the in-vitro cytotoxic activity of new platinum(II) complexes on the human HeLa (ER-), MCF-7 (ER+) and MDA-MB 231 (ER-) cell lines. Furthermore, we investigated plasmid DNA interactions and inhibition of BamHI and HindIII restriction enzyme activity of the complex 1-4,7. Platinum(II) complexes were synthesised from precursor complexes of [PtL2 Cl2 ] and [PtL2 I2 ]. Their cytotoxic activity was tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Their plasmid DNA interactions and restriction enzyme activities were also investigated using the agarose gel electrophoresis method. The growth inhibitory effect results showed that the cytotoxicity of complex 2 was found to be the most active complex among the synthesised complexes. The MTT results showed that complex 2 was found to be cytotoxic equal to cisplatin and higher than carboplatin against the MCF-7 and MDA-MB-231 cell lines. Furthermore, the estrogen or progesterone co-treatment slightly increased the cytotoxicity of complex 2, the cisplatin and carboplatin compared with the complex 2 tested alone in 50 μm concentration. According to plasmid DNA interaction and the restriction studies, complexes 1-4,7 modified the tertiary structure of pBR322 plasmid DNA, and complexes 2-4 prevented enzyme digestion at high concentrations.

Flocculation of Bacillus amyloliquefaciens H Disintegrates with Cationized Starch and Aminated Hydroxyethylcellulose

The ability of cationized hydroxyethylated starch (CHES) and aminated hydroxyethylcellulose (DEAE-HEC) to flocculate cell disintegrate of Bacillus amyloliquefaciens H (BamHI) was investigated. The efficiency of flocculation was compared to that of synthetic polymer polyethyleneimine (PEI) and natural polysaccharide chitosan. The influence of salt concentration and biomass concentration on flocculation efficiency was investigated. It was found that the efficiency of flocculation with CHES and DEAE-HEC was similar to that of PEI but better compared to chitosan. Recovery of total soluble proteins at higher than 0.3% concentration of flocculant decreased by more than 18.8% and 42.3% compared to PEI and chitosan, respectively. The yield of BamHI restriction endonuclease activity with all flocculants was similar except for chitosan where 13.1% lower yield was obtained. Meanwhile, efficiency of flocculation with CHES and DEAE-HEC depends drastically on the salt concentration, that is, flocculation diminishes if NaCl concentrations higher than 0.2 M (for CHES) or 0.1 M (for DEAE-HEC) are used. The results have shown that CHES and DEAE-HEC are promising flocculants of cell disintegrates if higher yield of protein is of great concern.

Cloning and expression of the enzymatic region of Streptococcal hyaluronidase.

Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity. The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein) plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied. Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair) was inserted correctly into the recombinant vector. The expressed protein (65 KDa) was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient's sera. In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

Frequency distribution of high risk alleles of CYP2C19, CYP2E1, CYP3A4 genes in Haryana population

The genotype of an individual can significantly influence the disposition of a chemical, and determine their susceptibility to its toxicity. Many enzymes involved in either activation or detoxification of chemical carcinogen metabolism are polymorphically expressed, with the alleles presenting different enzymatic activities and some of them having been associated with susceptibility to cancer. Cytochrome P450 (P450 or CYP) constitutes the most important phase I enzyme group responsible for the metabolism of endogenous and exogenous (xenobiotics) substances. The present study was aimed to analyze the frequencies of commonly known polymorphisms of human xenobiotic metabolizing genes (XMG) in the Haryana State population of North India. The study was conducted in 308 healthy Haryana volunteers. DNA was extracted from leucocytes and the genetic polymorphisms in CYP2C19*2, CYP2C19*3, CYP2E1*5B and CYP3A4*1B were determined by digesting the PCR Product with restriction enzymes BamHI, SmaI, PstI and PstI respectively. The genotype frequencies of CYP2C19*2, CYP2C19*3, CYP2E1*5B, and CYP3A4*1B were found to be 22.0%, 0.0%, 2.11% and 2.0% respectively. The North Indian population which is known to be Caucasoid Aryans is ethnically different from South Indians known as Caucasoid Dravidians but no significant difference in genetic polymorphism was found.

Cloning, expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25mM IPTG after 8h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His6 was active in a wide temperature (19.7–79.4°C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2°C, which is 10–18°C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His6 preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9min at 85°C), suggesting that E34Tt-His6 has potential for industrial applications.

Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

First report of the association of a ‘Candidatus Phytoplasma ulmi’ isolate with a witches' broom disease of reed in China

Phragmites australis (Poaceae) is widely distributed around the world. It is not only a perennial weed, but also a raw material for the paper industry, a medicinal plant, animal fodder, an ornamental in landscaping and a building material. It now covers approximately two million ha in China, including ten thousand ha in Xinjiang Province. During September 2012, symptoms of witches' broom, stunting, and smaller leaves (Fig. 1A) were observed in over 57% of P. australis plants at the Shihezi University Experimental Station (Shihezi, Xinjiang Province). Leaf samples were collected from both symptomless plants and plants showing symptoms. Total DNA was extracted from 0.5 g fresh leaf midribs from P. australis plants with and without symptoms, using the CTAB method (Angelini et al., 1). The DNA extracts were tested for phytoplasma infection by a nested polymerase chain reaction (PCR) assay with phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 2) and R16F2n/R16R2 (Gundersen & Lee, 3). The two primer pairs produced PCR products of 1.8 and 1.2 kb, respectively, from most of the plants with symptoms (4/5), but not from the symptomless plants (2/2). R16F2n/R16R2 amplicons were cloned (pMD18-T simple vector, TaKaRa, China) and sequenced. All of the phytoplasma R16F2n/R16R2 sequences were 100% identical to each other. BLAST comparisons showed that the P. australis witches' broom phytoplasma 16S rDNA consensus sequence (GenBank Accession No. KC331052) had 99% sequence identity with those of members of group 16SrV (‘Candidatus Phytoplasma ulmi’). Similarity coefficients and in silico restriction fragment length polymorphism (RFLP) analyses of the R16F2n/R16R2 sequence with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, I, and TaqI restriction endonucleases (iPhyClassifier, http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) (Fig. 2), indicated that P. australis contained a phytoplasma strain identical to those of the reference strain AB052876, ‘Ca. Phytoplasma ulmi’, subgroup 16SrV-B. A neighbour-joining dendrogram (MEGA 5.0, USA) based on the 16S rDNA sequences of the P. australis witches' broom phytoplasma and twenty-five reference phytoplasmas in NCBI database supported the PCR and RFLP results since the P. australis witches' broom phytoplasma grouped within the 16SrV-B phytoplasma clade (Fig. 3). The group 16SrV was previously associated with witches' broom diseases of several plant species in China including jujube (Zhu et al., 6), paper mulberry (Liu et al., 4), and Chinese honeylocust (Min et al., 5). This is the first report of a ‘Ca. Phytoplasma ulmi‘-related strain affecting P. australis in China. This research was supported by the National Key Technology R&D Program of China (2011BAD48B00).

Construction of eukaryotic expression vector of heme oxygenase-1 and its expression in rat peritoneal mesothelial cells

Objective To construct the eukaryotic expression vector of heme oxygenase-1(HO-l) gene,and transfect the recombinant expression vector to rat peritoneal mesothelial cells(RPMCs) in vitro.Methods Total RNA was extracted from SD rat spleen,and the full length fragment of HO-1 CDS part was amplified by RT-PCR.The product of PCR was cloned to pMD-18T vector.The pMD-18T-HO-1 and pcDNA3.1(-) vector were digested with restriction endonuclease BamHⅠ and KpnⅠ,then T4 DNA ligase was used to ligate HO-1 gene fragment and digested pcDNA3.1(-) vector to construct the eukaryotic expression vector pcDNA3.1(-)-HO-1.The recombinant plasmid pcDNA3.1(-)-HO-1 was transfected into RPMCs.The expression of HO-1 in RPMCs was detected by RT-PCR and Western blotting respectively.Results The full length of rat HO-1 gene was cloned,and the eukaryotic expression vector pcDNA3.1(-)-HO-1 was successfully constructed.Restriction endonucleases digestion confirmed the target DNA fragment was inserted into the expression vector,and was verified by DNA sequencing.RT-PCR and Western blotting revealed that HO-1 was highly expressed in RPMCs after transfection with pcDNA3-HO-1.Conclusion HO-1 eukaryotic expression vector has been successfully constructed,and HO-1 expresses effectively in RPMCs,which lays a foundation for the further study of the effect of HO-1 in RPMCs injury during peritoneal dialysis.

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Several gram-positive pathogenic bacteria employ near-iron transporter (NEAT) domains to acquire heme from hemoglobin during infection. However, the structural requirements and mechanism of action for NEAT-mediated heme extraction remains unknown. Bacillus anthracis exhibits a rapid growth rate during systemic infection, suggesting that the bacterium expresses efficient iron acquisition systems. To understand how B. anthracis acquires iron from heme sources, which account for 80% of mammalian iron stores, we investigated the properties of the five-NEAT domain hemophore IsdX2. Using a combination of bioinformatics and site-directed mutagenesis, we determined that the heme extraction properties of IsdX2 are dependent on an amino acid with an amide side chain within the 310-helix of the NEAT domain. Additionally, we used a spectroscopic analysis to show that IsdX2 NEAT domains only scavenge heme from methemoglobin (metHb) and that autoxidation of oxyhemoglobin to metHb must occur prior to extraction. We also report the crystal structures of NEAT5 wild type and a Q29T mutant and present surface plasmon resonance data that indicate that the loss of this amide side chain reduces the affinity of the NEAT domain for metHb. We propose a model whereby the amide side chain is first required to drive an interaction with metHb that destabilizes heme, which is subsequently extracted and coordinated in the aliphatic heme-binding environment of the NEAT domain. Because an amino acid with an amide side chain in this position is observed in NEAT domains of several genera of gram-positive pathogenic bacteria, these results suggest that specific targeting of this or nearby residues may be an entry point for inhibitor development aimed at blocking bacterial iron acquisition during infection.

Development of a Novel PCR Restriction Analysis of thehsp65Gene as a Rapid Method To Screen for the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in High-Burden Countries

The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.

Hierarchical Accessibility of Double-Helical RNA and DNA Processing Signals in Highly Dense Self-Assemblies

The ability to accurately detect biomolecules in single-cell amounts is an important goal of basic and translational research. The inherent capability of nucleic acids to self-assemble allows the spontaneous formation of highly dense, self-assembled monolayers (SAMs). These assemblages can provide the basis for development of nano-based devices with programmable, single-molecule detection capability. Double-stranded(ds) RNA is an important biomarker of viral infection and certain cancers. While dsRNA behavior in solution has been extensively characterized by diverse physicochemical approaches, the properties of highly dense assemblies of dsRNA are largely unknown, and may be qualitatively different from those in solution. High-density, 39 bp, chimeric dsRNA:dsDNA (ds-chimera) SAMs have been prepared on modified gold surfaces. using atomic force microscopy (AFM)-based nanomanipulation and analysis, and fluorescence microscopy, we demonstrate the preferential binding of ethidium to the ds-chimera SAM compared to the single-stranded form. As revealed by AFM detection of height change, the ds-chimera SAM is reactive towards the dsRNA-specific RNase III of Aquifex aeolicus (Aa-RNase III) and restriction endonuclease BamHI, each having a recognition site in the dsRNA and dsDNA segments, respectively. We also show that the reactivity of the BamHI cleavage site, which is proximal to the gold surface, can be controlled by SAM density, and that access to the BamHI site is dependent upon the prior action of RNase III at its site in the center of the ds-chimera. These results reveal novel properties of protein-nucleic acid interactions within a high-density array environment that are relevant to nanoscale detection methodologies.

Toward detection of DNA-bound proteins using solid-state nanopores: insights from computer simulations.

Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.

Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 μm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways.

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.

Biological Characterization and Complete Genome Sequence of a Possible Strain of Indian cassava mosaic virus from Jatropha curcas in India

AbstractThe outbreak of a severe mosaic disease with a significant incidence was noticed on Jatropha curcas plants growing in Lucknow, Northern India. The causal virus was successfully transmitted by whiteflies (Bemisia tabaci) and grafting from naturally infected to healthy J. curcas plants. The association of Begomovirus with the mosaic disease of J. curcas was detected by PCR using primers specific to DNA‐A of Begomoviruses. Further, full‐length DNA‐A genome of ∼2.7 kb was amplified by RCA followed by digestion with Bam HI restriction enzyme. Cloning and sequencing of obtained amplicons resulted in 2740 nucleotides of complete DNA‐A consisting of six ORFs and IR region (GenBank Accession HM230683). The sequence analysis revealed highest 85% similarities with Jatropha curcas mosaic virus, 77–84% with Indian cassava mosaic virus and 73–76% with Sri Lankan cassava mosaic virus isolates. Phylogenetic analysis of the Begomovirus isolate also showed a clear‐cut distinct relationship with earlier reported Begomoviruses from Jatropha curcas and other Begomoviruses. On the basis of the guidelines of the International Committee on Taxonomy of Viruses (ICTV‐2008), our virus isolate was identified as a possible strain of Indian cassava mosaic virus, and its name Jatropha mosaic India virus (JMIV) is proposed.

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαβ) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαβ was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.

Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region

TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a "landmark" of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321-366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331-369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.