Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

Interaction of Sesbania mosaic virus movement protein with the coat protein - implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

Background: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. Methodology/Principal Findings: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), beta-galactosidase (beta-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational upregulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. Conclusions/Significance: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.

Development and characterization of monoclonal antibodies against WbSXP-1 for the detection of circulating filarial antigens

The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the World Health Organization. Presently, few immunodiagnostics are available for filarial monitoring programmes. The Wuchereria bancrofti (Wb) SXP-1 parasite protein, with 84% homology to Brugia malayi (Bm) SXP-1, was found to be highly immunogenic. WbSXP-1 is one among the diagnostic candidate molecules that were used for developing a rapid-antibody-flow-through diagnostic kit for filariasis. Studies were initiated with the aim of developing monoclonal antibodies against recombinant WbSXP-1 and prospective applications for the detection of both circulating Wb and Bm antigens in serum samples from infected individuals. The monoclones 1A6C2 of subclass IgG1k, and 2A12F8 of class IgM, specifically detected Wb and Bm microfilaria isolated from patients and did not show cross-reactivity with other filarial recombinant antigens. We anticipate that this work will address the problems faced in the rapid diagnosis of human lymphatic filariasis in endemic areas in developing countries.

Cloning and sequence of a cDNA encoding a novel hybrid prolme-rich protein associated with cytokinin-induced haustoria formation in Cuscuta reflexa

A complete cDNA encoding a novel hybrid Pro-rich protein (HyPRP) was identified by differentially screening 3x10(4) recombinant plaques of a Cuscuta reflexa cytokinin-induced haustorial cDNA library constructed in lambda gt10. The nucleotide (nt) sequence consists of: (i) a 424-bp 5'-non coding region having five start codons (ATGs) and three upstream open reading frames (uORFs); (ii) an ORF of 987 bp with coding potential for a 329-amino-acid (aa) protein of M(r), 35203 with a hydrophobic N-terminal region including a stretch of nine consecutive Phe followed by a Pro-rich sequence and a Cys-rich hydrophobic C terminus; and (iii) a 178-bp 3'-UTR (untranslated region). Comparison of the predicted aa sequence with the NBRF and SWISSPROT databases and with a recent report of an embryo-specific protein of maize [Jose-Estanyol et al., Plant Cell 4 (1992) 413-423] showed it to be similar to the class of HyPRPs encoded by genes preferentially expressed in young tomato fruits, maize embryos and in vitro-cultured carrot embryos. Northern analysis revealed an approx. 1.8-kb mRNA of this gene expressed in the subapical region of the C. reflexa vine which exhibited maximum sensitivity to cytokinin in haustorial induction.

Solution structure of BTK-2, a novel hK(v)1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK(v)1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C-alpha, C') and all heavy atoms are 0.81 +/- 0.23 angstrom and 1.51 +/- 0.29 angstrom respectively. The backbone dihedral angles (phi and psi) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the alpha-beta-beta, structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure-function relationships among this important class of voltage-gated potassium channel inhibiting peptides. (C) 2011 Elsevier B.V. All rights reserved.

Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae

Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high efficiency, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter On infection with vBmluc, luciferase was expressed at very high levels, 170 mu g/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating ''glowing silkworms''.

CDNA Cloning, Overexpression in Escherichia coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The K-m values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.

Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three Fl vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay. (C) 1995 Academic Press Limited

Glutathione peroxidase3 of Saccharomyces cerevisiae protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 mu M cadmium (Cd)-treatment, whereas an untreated gpx3 Delta strain showed a drastic reduction in overall phospholipids which was further reduced with 50 mu M Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3 Delta cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3 Delta. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3 Delta cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3 Delta cells during Cd-treatment.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

The genetics of Mycobacterium tuberculosis

Gene manipulation in Mycobacterium tuberculosis has been slow in coming of age owing to the inherent difficulties associated with working on this aerosol-transmitted pathogen, in addition to the paucity of molecular tools such as plasmids and transposons. One of the early approaches to overcome these difficulties was the development of phasmids, which combined the properties of phages and plasmids and allowed introduction of recombinant genes into mycobacteria. The lone plasmid pAL5000 of mycobacteria has been exploited to its fullest potential in the construction of a plethora of vectors. Above all, the single most important achievement has been the development of elegant and innovative approaches to overcome the problem of illegitimate recombination which threatened the success of allelic-exchange mutagenesis in the slow-growing pathogenic mycobacterial species. In this review I discuss the current status of conditionally replicating plasmid and transposon vectors and their application in generating targeted mutations in mycobacteria.

Structures of the catalytic site mutants D99A and H48Q and the calcium-loop mutant D49E of phospholipase A(2)

Crystal structures of the active-site mutants D99A and H48Q and the calcium-loop mutant D49E of bovine phospholipase A(2) have been determined at around 1.9 Angstrom resolution. The D99A mutant is isomorphous to the orthorhombic recombinant enzyme, space group P2(1)2(1)2(1), The H48Q and the calcium-loop mutant D49E are isomorphous to the trigonal recombinant enzyme, space group P3(1)21, The two active-site mutants show no major structural perturbations. The structural water is absent in D99A and, therefore, the hydrogen-bonding scheme is changed. In H48Q, the catalytic water is present and hydrogen bonded to Gln48 N, but the second water found in native His48 is absent. In the calcium-loop mutant D49E, the two water molecules forming the pentagonal bipyramid around calcium are absent and only one O atom of the Glu49 carboxylate group is coordinated to calcium, resulting in only four ligands.

Transplanting the n-terminus from Kv1.4 to Kv1.1 generates an inwardly rectifying K+ channel

A chimeric channel, 4N/1, was generated from two outwardly rectifying K+ channels by linking the N-terminal cytoplasmic domain of hKv1.4 (N terminus ball and chain of hKv1.4) with the transmembrane body of hKvl.l (Delta 78N1 construct of hKvl.l). The recombinant channel has properties similar to the six transmembrane inward rectifiers and opens on hyperpolarization with a threshold of activation at -90 mV. Outward currents are seen on depolarization provided the channel is first exposed to a hyperpolarizing pulse of -100mV or more. Hyperpolarization at and beyond -130mV provides evidence of channel deactivation. Delta 78N1 does not show inward currents on hyperpolarization but does open on depolarizing from -80mV with characteristics similar to native hKvl.l. The outward currents seen in both Delta 78N1 and 4N/1 inactivate slowly at rates consistent with C-type inactivation. The inward rectification of the 4N/1 chimera is consistent with the inactivation gating mechanism. This implies that the addition of the N-terminus from hKv1.4 to hKvl.l shifts channel activation to hyperpolarizing potentials. These results suggest a mechanism involving the N-terminal cytoplasmic domain for conversion of outward rectifiers to inward rectifiers. (C) 1999 Lippincott Williams & Wilkins.

Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus FIN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination. (C) 2002 Elsevier Science (USA).