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V Nagaraja

Publications and source records attributed to V Nagaraja.

At least 19 recordsLinked to original sources
PubMed1996

Sequence-specific DNA binding of the phage Mu C protein: footprinting analysis reveals altered DNA conformation upon protein binding.

The mom gene of bacteriophage Mu, which codes for a DNA modification function, is regulated in a complex manner at both transcriptional and translational levels. The phage-encoded C protein functions as an activator of mom transcription. The mom promoter has features of an activator-dependent weak promoter, and the C binding site is located upstream and overlapping the -35 region and includes the palindromic sequence TTAT(N)6ATAA. The interactions of this activator protein at its binding site in Pmom has been investigated using four different chemical footprinting reagents. The protein footprint spans a region of 18 to 25 bp, depending on the nature of the chemical reagent used. Dimethylsulfate protection experiments revealed the base-specific interactions. The protected guanines are separated by 15 bp and are located beyond the interrupted palindromic sequence. A tripartite footprint was observed with hydroxyl radical, generated by Fe(II)-EDTA, which shows the binding of the protein to one face of the helix. The extent of protection conferred by the bound protein, however, is not uniform, suggesting that the interaction is asymmetric. The chemical nuclease 1,10-phenanthroline-copper, a minor groove specific ligand, shows hyper-reactivity upon protein binding in the top strand nucleotide triplet CAC, again confirming the protein-induced alterations in DNA conformation. Gel exclusion chromatography and chemical crosslinking experiment with the purified protein suggest that this mode of interaction is accomplished by a dimeric protein. This observation is supported by electrophoretic mobility shift assay using heterodimer of pure C protein and staphylococcal protein A-C fusion. The deletion analysis implicates a role for the carboxyl-terminal region of the protein in DNA binding.

Base Sequence↗
PubMed1996

Theoretical permutation gel electrophoretic analysis of a curved DNA fragment located in circular permutation.

Using the theoretical model for DNA curvature, we analyzed a set of fragments with a curved insert located in circular permutation. The theoretical permutation analysis of each of the cyclically located fragments reveals the presence of a shifting molecular bend locus. The delineation of the molecular bend locus associated with the fragments obtained by a second permutation helps in providing an explanation for the differential mobility behavior of the fragments.

Animals↗
PubMed1995

Differential binding of RNA polymerase to the wild type Mu mom promoter and its C independent mutant: a theoretical analysis.

Using the theoretical model for DNA bending we have analyzed the Mu mom promoter wild type and its mutant tin7 which showed differential binding to the RNA polymerase. We have demonstrated here the structural change as a result of the point mutation which may be responsible for the altered binding of RNA polymerase. Analysis using both sets of parameters essentially gives the same result.

Bacteriophage mu↗
PubMed1995

Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli.

The cloning and characterization of DNA gyrase genes from Mycobacterium smegmatis is described. The DNA sequence of 5119 bp encoding both gyrB and gyrA genes was determined. The gene gyrB precedes gyrA with a short intergenic region of 29 nucleotides. The proteins encoded, GyrB and GyrA, exhibit 45-80% identity to gyrase polypeptides from other bacteria. The genes were further engineered for overexpression in Escherichia coli. Both genes were individually cloned into a phage T7 expression system and overexpressed. The expressed GyrB and GyrA proteins had molecular masses of 75 and 95 kDa, respectively, in agreement with that calculated from the ORFs. The extracts from the overexpressing clones were fractionated to enrich the subunits and assayed for enzyme activity. While the individual extracts showed no detectable activity, the combined extract exhibited a strong DNA supercoiling activity. This activity was ATP-dependent and novobiocin-sensitive. The identity of the genes was also confirmed by complementation analysis.

Amino Acid Sequence↗
PubMed1995

DNA methylation in mycobacteria: absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences.

The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.

5-Methylcytosine↗
PubMed1994

On the mobility behavior of a curved DNA fragment located in circular permutation.

Experimental and theoretical investigations on the mobility behavior of a set of permuted fragments with a K-DNA insert is reported. The fragments with the permuted flanking sequences have the K-DNA insert located differentially with respect to the fragment ends. The fragment wherein the insert is located in the center showed maximum retardation as compared to fragments where the insert was at the end. The experimental analysis is also in accord with the theoretical investigation.

Base Sequence↗
PubMed1994

Overproduction and purification of C protein, the late gene transcription activator from phage Mu.

We report here the high-level overproduction and single-step purification for the C protein of bacteriophage Mu. Attempts to secrete the protein using the pelB signal sequence, in a T7 expression system, failed to yield the processed product. Moreover, the overexpressed fusion protein was inactive in DNA binding assays. In order to obtain the native protein, the sequences coding for the signal peptide were removed. The clones thus obtained upon induction overproduced the C protein, a significant amount of which was present in the S20 pellet fraction. The protein was recovered from this pellet by high salt extraction and purified by specific immunoaffinity chromatography. The purified protein was active in DNA binding assay. The final yield of the protein was 9 mg of approximately 95% purity from 1 g wet wt cells.

Bacteriophage mu↗
PubMed1994

Mode of binding of quercetin to DNA.

The difference spectrum of the quercetin--DNA complex versus quercetin alone was characterized by a peak at 395 nm. An increase in the magnitude of difference spectrum was seen with increased ionic strength. Spectrophotometric changes in absorbance and fluorescence of quercetin showed that ethidium bromide is able to displace quercetin from the quercetin--DNA complex. These results indicate that the binding of quercetin to DNA does not involve electrostatic interactions but may be intercalative in nature. Experiments using DNase I footprinting technique showed that the flavonoid does not possess any preferred sites of binding in DNA. Strand scission in DNA by the quercetin--Cu(II) system gave a generally uniform cutting pattern of internucleotide bonds. This led to the observation that the quercetin--Cu(II) cleavage reaction has the potential of being used as preferred DNA footprinting reagent.

Animals↗
PubMed1994

Engineering hyperexpression of bacteriophage Mu C protein by removal of secondary structure at the translation initiation region.

The structure at the translation initiation region (TIR) of mRNA has pronounced regulatory effects on gene expression. Our attempts to overexpress the C gene of bacteriophage Mu in a variety of expression vectors resulted in low yields of protein. Analysis of Mu C mRNA shows the potential to form a secondary structure involving a ribosome binding site and AUG codon. We have engineered the overproduction of the protein using a PCR-aided cloning approach to remove the sequences involved in the formation of this secondary structure. The overexpressing clone, under the control of T7 gene 10 promoter in a T7 expression system yielded > 30% of total cell protein. The difference in mRNA structure between expressing and non-expressing clones was confirmed by electrophoretic analysis of run-off transcripts. The overexpressed protein was purified in a single step by site-specific DNA affinity chromatography. The purified recombinant protein was active in band shift assays. DNA binding activity required Mg2+ and was weak in the presence of Mn2+. Cd2+ or Zn2+ could not support DNA binding. Under optimal conditions, the equilibrium binding constant (Kapp) was determined to be 2 x 10(12) M-1.

Bacteriophage mu↗
PubMed1994

Molecular cloning of gyrA and gyrB genes of Mycobacterium tuberculosis: analysis of nucleotide sequence.

We have recently reported the cloning of gyrA and gyrB genes from Mycobacterium tuberculosis H37Ra [Curr. Science, (1994) 66, 664-667]. Here, we present the complete nucleotide sequence of gyrB gene from M. tuberculosis H37Ra along with the flanking regions. The gyrA gene has been located 34 nucleotides downstream of gyrB and has been partially sequenced; both the genes seem to be transcribed from the promoter elements located upstream of gyrB coding sequence. The gyrB gene encodes a polypeptide of 714 amino acids. The deduced amino acid sequences of gyrB and a part of gyrA show extensive homology to the corresponding genes from other bacterial species. The DNA gyrase of M. tuberculosis could be utilised to develop new line of antitubercular drugs.

Amino Acid Sequence↗
PubMed1994

DNA topoisomerase I from Mycobacterium smegmatis.

DNA topoisomerase I has been purified from Mycobacterium smegmatis to near homogeneity using different column chromatographic techniques. The enzyme activity relaxes form I DNA into form IV DNA, requiring Mg2+, but not ATP or any other cofactors for its activity. Several properties of the enzyme were found to be similar to that of the prototype enzyme, Escherichia coli topoisomerase I.

DNA Topoisomerases, Type I↗
PubMed1994

Employing L-lactic acid powder in the preparation of a dry "acid concentrate" for use in a bicarbonate-based dialysis solution-generating system: experience in hemodialysis patients.

By replacing the liquid acetic acid present in the "acid concentrate" of a bicarbonate-based dialysis solution-generating system with an equimolar amount of solid L-lactic acid and by using the dry forms of the remaining constituents, we were able to create a dry "acid concentrate" just prior to use, and successfully employed this "acid concentrate" to produce a bicarbonate-based solution to hemodialyze patients.

Bicarbonates↗
PubMed1992

Functionally distinct RNA polymerase binding sites in the phage Mu mom promoter region.

Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i) in vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from -74 to -24 with respect to transcription initiation. This overlaps a known strong C-binding site (at -35 to -54). RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to +21. With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from -35 to -54), RNAP bound to P1 independent of C. We conclude that P1 is the 'functional' RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.

Bacteriophage mu↗
PubMed1991

Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA.

Bacteriophage Mu controls an unusual DNA-modification function encoded by the mom gene, which is located in an operon that consists of two overlapping genes. The com gene, located proximal to the 5' end of the common mRNA transcript, encodes a polypeptide of 62 amino acids that is required for translation of mom. Analysis of the derived amino acid sequence reveals that Com contains zinc-binding finger motifs, suggesting that Com may be a zinc-activated regulatory protein. Atomic absorption analysis showed that there is about one zinc bound per molecule of Com. We have subcloned the com gene into an expression vector and thus have overproduced and purified the Com protein. By gel retardation analysis with various 32P-labeled RNAs (made by in vitro transcription with T7 RNA polymerase), we show that Com binds specifically to com-mom mRNA. A single C----U substitution mutation, located 26 nucleotides upstream from the mom translation start codon, abolishes Com binding. The nature of the Com target sequence was deduced from in vitro footprinting analyses. The results are consistent with the existence of a complex stem-loop structure within the overlap of the com-mom open-reading-frames. Com binding to its target site results in the destabilization of a proposed translation-inhibitor stem-loop (TIS) to expose the Shine-Dalgarno sequence and mom translation initiation codon. This suggests that Com interaction with a specific site on its cognate mRNA alters the mRNA secondary structure to activate translation of mom.

Amino Acid Sequence↗
PubMed1985

Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences.

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.

Adenosine Triphosphatases↗
PubMed1985

The nucleotide sequence recognised by the Escherichia coli D type I restriction and modification enzyme.

A type I restriction endonuclease from a new isolate of Escherichia coli (E. coli E166) has been purified and characterised. The enzyme, EcoD, has a recognition sequence similar in overall structure to the previously determined type I enzyme sequences, an exception being that it is degenerate. The sequence is 5'-T-T-A-N-N-N-N-N-N-N-G-T-C-Y-3' 3'-A-A-T-N-N-N-N-N-N-N-C-A-G-R-5' where Y is a pyrimidine, R is a purine and N can be any nucleotide. The enzyme methylates adenosyl residues in both strands of the DNA that are separated by ten base pairs, suggesting that the enzyme interacts with DNA along one face of the helix making contacts in two successive major grooves.

Base Sequence↗