Analysis of upstream activation of thevnfHpromoter ofAzotobacter vinelandii

1998 ◽  
Vol 44 (5) ◽  
pp. 405-415 ◽  
Author(s):  
Umesh K Bageshwar ◽  
Ramesh Raina ◽  
Nirupam Roy Choudhury ◽  
H K Das
Keyword(s):  
Azotobacter Vinelandii ◽  
Dna Bending ◽  
Polyacrylamide Gel Electrophoresis ◽  
Upstream Region ◽  
Curved Dna ◽  
Low Concentrations ◽  
The Face ◽  
Dna Fragment ◽  
Ofazotobacter Vinelandii ◽  
Subsequent Substitution

BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, where as addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.Key words: Azotobacter vinelandii, vnfA, vnfH, promoter-lacZ fusion, DNA bending.

scholarly journals Identification of a Positive Transcription Regulatory Element within the Coding Region of the nifLA Operon in Azotobacter vinelandii

2005 ◽  
Vol 71 (7) ◽  
pp. 3716-3724 ◽  
Author(s):  
Ranjana Mitra ◽  
Hirendra K. Das ◽  
Aparna Dixit
Keyword(s):  
Binding Sites ◽  
Promoter Activity ◽  
Azotobacter Vinelandii ◽  
Specific Binding ◽  
Regulatory Element ◽  
Upstream Region ◽  
Significant Finding ◽  
Coding Region ◽  
S1 Nuclease ◽  
The Face

ABSTRACT Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the σ54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.

Wake Flow of Single and Multiple Yawed Cylinders

2004 ◽  
Vol 126 (5) ◽  
pp. 861-870 ◽  
Author(s):  
A. Thakur ◽  
X. Liu ◽  
J. S. Marshall
Keyword(s):  
Computational Study ◽  
Three Dimensional ◽  
Side Wall ◽  
Wake Flow ◽  
Upstream Region ◽  
Two Dimensional ◽  
Cylinder Wake ◽  
Dimensional Approximation ◽  
The Face ◽  
Drag And Lift

An experimental and computational study is performed of the wake flow behind a single yawed cylinder and a pair of parallel yawed cylinders placed in tandem. The experiments are performed for a yawed cylinder and a pair of yawed cylinders towed in a tank. Laser-induced fluorescence is used for flow visualization and particle-image velocimetry is used for quantitative velocity and vorticity measurement. Computations are performed using a second-order accurate block-structured finite-volume method with periodic boundary conditions along the cylinder axis. Results are applied to assess the applicability of a quasi-two-dimensional approximation, which assumes that the flow field is the same for any slice of the flow over the cylinder cross section. For a single cylinder, it is found that the cylinder wake vortices approach a quasi-two-dimensional state away from the cylinder upstream end for all cases examined (in which the cylinder yaw angle covers the range 0⩽ϕ⩽60°). Within the upstream region, the vortex orientation is found to be influenced by the tank side-wall boundary condition relative to the cylinder. For the case of two parallel yawed cylinders, vortices shed from the upstream cylinder are found to remain nearly quasi-two-dimensional as they are advected back and reach within about a cylinder diameter from the face of the downstream cylinder. As the vortices advect closer to the cylinder, the vortex cores become highly deformed and wrap around the downstream cylinder face. Three-dimensional perturbations of the upstream vortices are amplified as the vortices impact upon the downstream cylinder, such that during the final stages of vortex impact the quasi-two-dimensional nature of the flow breaks down and the vorticity field for the impacting vortices acquire significant three-dimensional perturbations. Quasi-two-dimensional and fully three-dimensional computational results are compared to assess the accuracy of the quasi-two-dimensional approximation in prediction of drag and lift coefficients of the cylinders.

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide ‘maps’ after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

The isolation of surface array proteins from bacteria

1984 ◽  
Vol 62 (11) ◽  
pp. 1181-1189 ◽  
Author(s):  
S. F. Koval ◽  
R. G. E. Murray
Keyword(s):  
Molecular Weight ◽  
Protein Conformation ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Features ◽  
Proteolytic Degradation ◽  
Low Concentrations ◽  
Single Polypeptide

The methods used for the isolation of regularly structured (RS) surface array proteins of a range of prokaryotes are described. Most RS proteins can be selectively solubilized from envelope preparations with low concentrations of urea or guanidine hydrochloride. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis of the protein extracts shows that most RS arrays are composed of a single polypeptide that may contain carbohydrate. The molecular weight of the proteins varies from 41 000 to 200 000. Possible reasons for the presence of more than one polypeptide in RS protein preparations are discussed, as well as the evidence for proteolytic degradation of some RS proteins during isolation. Structural features of the RS proteins are described and the importance of protein conformation to assembly of the arrays is indicated.

scholarly journals Transcriptional regulation of alpha(1b) adrenergic receptors (alpha(1b)AR) by nuclear factor 1 (NF1): a decline in the concentration of NF1 correlates with the downregulation of alpha(1b)AR gene expression in regenerating liver.

1996 ◽  
Vol 16 (11) ◽  
pp. 5997-6008 ◽  
Author(s):  
B Gao ◽  
L Jiang ◽  
G Kunos
Keyword(s):  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Upstream Region ◽  
Remnant Liver ◽  
Nuclear Factor ◽  
Nucleotide Substitutions ◽  
P2 Promoter ◽  
Hep3b Cells ◽  
Nuclear Factor 1

The 5' upstream region from --490 to --540 (footprint II) within the dominant P2 promoter of the rat alpha(1b) adrenergic receptor (alpha(1b)AR) gene is recognized by a sequence-specific DNA-binding protein (B. Gao, M. S. Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNA-protein) blots of crude nuclear extracts as 32- and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts. Methylation interference analysis revealed strong contact with a TTGGCT hexamer and weak contact with a TGGCGT hexamer in the 3' and 5' portions of footprint II, respectively. Nucleotide substitutions within these hexamers significantly reduced protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity. Gel mobility supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32- and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into Hep3B cells or primary rat hepatocytes of cDNAs of the NF1-like proteins NF1/L, NF1/X, and NF1/Redl resulted in a three- to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2. Partial hepatectomy markedly decreased the levels of NF1 in the remnant liver and its binding to P2, which paralleled declines in the rate of transcription of the alpha(1b)AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcription of the rat alpha(1b)AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms responsible for the reduced expression of the alpha(1b)AR gene during liver regeneration.

The Electrophoretic Separation of Curved Cisplatin-Modifled DNA Fragments on Polyacrylamide Gels Is Dependent on the Voltage Gradient

1998 ◽  
Vol 53 (9-10) ◽  
pp. 921-923
Author(s):  
Julia Yaneva ◽  
Jordanka Zlatanova
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Threshold Level ◽  
Voltage Gradient ◽  
Dna Fragments ◽  
Electrophoretic Separation ◽  
Polyacrylamide Gels ◽  
Curved Dna ◽  
Electrophoretic Behavior ◽  
Param Eters

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The param eters of the electric field do not affect separation of ‘normal’ DNA fragments of comparable length.

scholarly journals Interaction of bleomycin with a bent DNA fragment

1992 ◽  
Vol 284 (3) ◽  
pp. 929-934 ◽  
Author(s):  
K P Nightingale ◽  
K R Fox
Keyword(s):  
Binding Sites ◽  
Dna Bending ◽  
Cobalt Complex ◽  
Dnase I ◽  
Cleavage Sites ◽  
Kinetoplast Dna ◽  
Bent Dna ◽  
Diethyl Pyrocarbonate ◽  
Dnase I Footprinting ◽  
Dna Fragment

The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3′ side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.

scholarly journals Hydrolysis of histones by proteinases

1988 ◽  
Vol 250 (3) ◽  
pp. 859-864 ◽  
Author(s):  
R J Harvima ◽  
K Yabe ◽  
J E Fräki ◽  
K Fukuyama ◽  
W L Epstein
Keyword(s):  
Mast Cell ◽  
Cell Differentiation ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Histone H2a ◽  
Cathepsin H ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Epidermal Cell Differentiation

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.

Sign in / Sign up

Export Citation Format

Share Document