High level stable expression of rat brain type IIA sodium channel ?-subunit in CHO cells

The neuronal sodium channels are responsible for the rising phase of action potential and are composed of three subunits, of which the alpha-subunit has been shown to be adequate for most of its functional properties. We have stably expressed the rat brain type IIA sodium channel alpha-subunit in CHO cell tine using a CMV promoter-based vector. The expression was confirmed by detecting a 6.5 kb RNA corresponding to sodium channel alpha-subunit using Northern hybridization. The cells stably expressing the alpha-subunit, yield isolated sodium currents of amplitudes greater than 4nA when studied in whole-cell configuration of the patch-clamp technique. The sodium currents are characterized by activation and inactivation properties similar to neuronal sodium channels, and are blocked by the voltage gated sodium channel blocker tetrodotoxin (TTX).

Mycobacterium tuberculosis groE promoter controls the expression of the bicistronic groESL1 operon and shows differential regulation under stress conditions

Heat shock promoters of mycobacteria are strong promoters that become rapidly upregulated during macrophage infection and thus serve as valuable candidates for expressing foreign antigens in recombinant BCG vaccine. In the present study, a new heat shock promoter controlling the expression of the groESL1 operon was identified and characterized. Mycobacterium tuberculosis groESL1 operon codes for the immunodominant 10 kDa (Rv3418c, GroES/Cpn10/Hsp10) and 60 kDa (Rv3417c, GroEL1/Cpn60.1/Hsp60) heat shock proteins. The basal promoter region was 115 bp, while enhanced activity was seen only with a 277-bp fragment. No promoter element was seen in the groES-groEL1 intergenic region. This operon codes for a bicistronic mRNA transcript as determined by reverse transcriptase-PCR and Northern blot analysis. Primer extension analysis identified two transcriptional start sites (TSSs) TSS1 (-236) and TSS2 (-171), out of which one (TSS2) was heat inducible. The groE promoter was more active than the groEL2 promoter in Mycobacterium smegmatis. Further, it was found to be differentially regulated under stress conditions, while the groEL2 promoter was constitutive.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

A DC-link Capacitor Voltage Balancing with CMV Elimination using only the Switching State Redundancies for a Reduced Switch Count Multi-Level Inverter fed IM Drive

Common mode voltage (CMV) variations in PWM inverter-fed drives generate unwanted shaft and bearing current resulting in early motor failure. Multilevel inverters reduce this problem to some extent, with higher number of levels. But the complexity of the power circuit increases with an increase in the number of inverter voltage levels. In this paper a five-level inverter structure is proposed for open-end winding induction motor (IM) drives, by cascading only two conventional two-level and three-level inverters, with the elimination of the common mode voltage over the entire modulation range. The DC link power supply requirement is also optimized by means of DC link capacitor voltage balancing, with PWM control., using only inverter switching state redundancies. The proposed power circuit gives a simple power bits structure.

Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOXI promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool `PromPredict'. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

Transcriptional activation of the Escherichia coli bgl operon: negative regulation by DNA structural elements near the promoter

The bgl operon of Escherichia coil is transcriptionally inactive in wild-type cells. DNA insertion sequences (IS) constitute a major class of spontaneous mutations that activate the cryptic bgl promoter. In an attempt to study the molecular mechanism of activation mediated by insertion sequences, transcription of the bgl promoter was carried out in vitro. Stimulation of transcription is observed when a plasmid containing an insertionally activated bgl promoter is used as a template in the absence of proteins other than RNA polymerase. Deletions that remove sequences upstream of the bgl promoter, and insertion of a 1.2 kb DNA fragment encoding resistance to kanamycin, activate the promoter. Point mutations within a region of dyad symmetry upstream of the promoter, which has the potential to extrude into a cruciform structure under torsional stress, also lead to activation, Introduction of a sequence with dyad symmetry, upstream of an activated bgl promoter carrying a deletion of upstream sequences, results in a fourfold reduction in transcription, These results suggest that the cryptic nature of the bgl promoter is because of the presence of DNA structural elements near the promoter that negatively affect transcription.

Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P-rrnAPCL1, P-rrnB and P-gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Functional Identification of Regulatory Elements within the Promoter Region of Platelet-Derived Growth Factor 2

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2,the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure.In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

High-quality annotation of promoter regions for 913 bacterial genomes

Motivation: The number of bacterial genomes being sequenced is increasing very rapidly and hence, it is crucial to have procedures for rapid and reliable annotation of their functional elements such as promoter regions, which control the expression of each gene or each transcription unit of the genome. The present work addresses this requirement and presents a generic method applicable across organisms. Results: Relative stability of the DNA double helical sequences has been used to discriminate promoter regions from non-promoter regions. Based on the difference in stability between neighboring regions, an algorithm has been implemented to predict promoter regions on a large scale over 913 microbial genome sequences. The average free energy values for the promoter regions as well as their downstream regions are found to differ, depending on their GC content. Threshold values to identify promoter regions have been derived using sequences flanking a subset of translation start sites from all microbial genomes and then used to predict promoters over the complete genome sequences. An average recall value of 72% (which indicates the percentage of protein and RNA coding genes with predicted promoter regions assigned to them) and precision of 56% is achieved over the 913 microbial genome dataset.

Mutations in beta ` subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

P>Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the beta' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the beta' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the beta' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.