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S Basak

Publications and source records attributed to S Basak.

At least 19 recordsLinked to original sources
PubMed2005

Studies on codon usage in Thermoplasma acidophilum and its possible implications on the occurrences of lateral gene transfer.

Codon usage studies have been carried out on the coding sequences of Thermoplasma acidophilum, which is an archaeon and grows at very low pH and high temperature. Overall codon usage data analysis indicates that all the four bases are almost equifrequent at the third position of codons, which is expected (since genomic GC % of this genome is about 46%). However, multivariate statistical analysis indicates that there are two major trends in the codon usage variation among the genes in this organism. In the first major trend it is observed that genes having G and C ending codons are clustered at one end while, A and T ending ones are clustered at the other end. We have also found a significant positive correlation between the expressivities of genes and GC contents at the synonymous third codon positions. In the second major trend, it is seen that the genes are clustered into three distinct parts. A comparative analyses of codon usage data of T. acidophilum and Sulfolobus solfataricus reveals that one of the three clusters of genes of T. acidophilum is very similar to a considerable number of S. solfataricus genes, suggesting possible occurrences of lateral gene transfer between these two microorganisms as reported by earlier workers.

Codon↗
PubMed2004

Evolutionary forces in shaping the codon and amino acid usages in Blochmannia floridanus.

Endosymbiotic relationship has great effect on ecological system. Codon and amino acid usages bias of endosymbiotic bacteria Blochmannia floridanus (whose host is an ant Camponotus floridanus) was investigated using experimentally known genes of this organism. Correspondence Analysis on RSCU values show that there exists only one single explanatory major axis that is linked to the strand specific mutational biases. Majority of the genes have a tendency to concentrate on the leading strand, which may be related to the adaptive property related to the replication mechanisms. Amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analyses of the orthologous genes of Escherichia coli and Blochmannia floridanus show that highly expressed genes are significantly more conserved than lowly expressed genes. Based on our results we concluded that strand specific mutational bias is strongly operational in selecting the codon usage in this organism. Replicational-transcriptional selection can be invoked from the presence of majority of highly expressed genes in the leading strand. Conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism. Hydrophobicity of the genes is the second major source in differentiating the genes according to their amino acid usages in this organism.

Amino Acid Sequence↗
PubMed2004

Investigation on the causes of codon and amino acid usages variation between thermophilic Aquifex aeolicus and mesophilic Bacillus subtilis.

Base composition, codon usages and amino acid usages have been analyzed by taking 529 orthologous sequences of Aquifex aeolicus and Bacillus subtilis, having different optimal growth temperatures. These two bacteria do not have significant difference in overall GC composition, but GC(1+2) and GC3 levels were found to vary significantly. Significant increments in purine content and GC3 composition have been observed in the coding sequences of Aquifex aeolicus than its Bacillus subtilis counterparts. Correspondence analyses on codon and amino acid usages reveal that variation in base composition actually influences their codon and amino acid usages. Two selection pressures acting on the nucleotide level (GC3 and purine enrichment), causes variation in the amino acid usage differently in different protein secondary structures. Our results suggest that adaptation of amino acid usages in coil structure of Aquifex aeolicus proteins is under the control of both purine increment and GC3 composition, whereas the adaptation of the amino acids in the helical region of thermophilic bacteria is strongly influenced by the purine content. Evolutionary perspectives concerning the temperature adaptation of DNA and protein molecules of these two bacteria have been discussed on the basis of these results.

Amino Acids↗
PubMed2004

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.).

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.

Amino Acid Sequence↗
PubMed2001

A versatile in vivo footprinting technique using 1,10-phenanthroline-copper complex to study important cellular processes.

A number of reagents have been used to define the sequence-specific protein-DNA contacts by footprinting analysis. We report a new in vivo technique using the complex of 1,10-phenanthroline and copper [(OP(2))Cu] as a probe to study various intracellular DNA-protein interactions in whole cells. The versatility of the protocol is demonstrated by applying the technique to address various processes. The protocol is applied to (i) detect structural alterations in DNA as a result of single base substitution, (ii) footprint site-specific DNA-binding proteins, (iii) analyze promoter occupancy by RNA polymerase and (iv) analyze molecular interactions during transcription initiation. The results demonstrate that in vivo (OP)(2)Cu probing is a useful tool in studying important cellular processes involving DNA-protein interactions and has potential applications in post-genomic research.

Base Sequence↗
PubMed2001

DNA unwinding mechanism for the transcriptional activation of momP1 promoter by the transactivator protein C of bacteriophage Mu.

Transcription factor-induced conformational changes in DNA are one of the mechanisms of transcription activation. C protein of bacteriophage Mu appears to transactivate the mom gene of the phage by this mode. DNA binding by C to its site leads to torsional changes that seem to compensate for a weak momP1 promoter having a suboptimal spacing of 19 bp between the poor -35 and -10 elements. The C-mediated unwinding could realign the promoter elements for RNA polymerase recruitment to the reoriented promoter. In this study, the model has been tested by mutational analysis of the spacer region of momP1 and by assessing the strength of the mutant promoters. The response to C-mediated transactivation was dependent on the spacer length of the promoters. Mutants with 17-bp spacing between the two promoter elements showed reduced activity in the presence of the transactivator as compared with their basal level. A synthetic promoter with near consensus promoter elements and optimal 17-bp spacing was also tested to evaluate the model. The results imply a role for C-mediated unwinding in mom transcription activation.

Antigens, Bacterial↗
PubMed2001

Effect of osmolytes and chaperone-like action of P-protein on folding of nucleocapsid protein of Chandipura virus.

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such as d-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells.

Light↗
PubMed2001

Effect of cholesterol on interaction of dibucaine with phospholipid vesicles: a fluorescence study.

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid membranes.

Anesthetics, Local↗
PubMed2001

Intrinsic DNA distortion of the bacteriophage Mu momP1 promoter is a negative regulator of its transcription. A novel mode of regulation of toxic gene expression.

The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19-base pair suboptimal spacer between the -35 (ACCACA) and -10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its own. DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5' end of the -10 element might prevent RNA polymerase from binding to momP1. To investigate the influence of the T(6) run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline ((OP)(2)Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription. This is a novel mechanism of regulation of toxic gene expression.

Bacteriophage mu↗
PubMed2001

Power Doppler ultrasonography for the evaluation of skin tumors other than malignant melanoma.

The purpose of this prospective study was to evaluate the value of power Doppler ultrasonography (PDUS) in assessing the vascularity of skin tumors other than malignant melanoma, and to investigate possible diagnostic criteria to help to distinguish malignant from benign tumors preoperatively. Seventy-one patients with the clinical diagnosis of a skin tumor were initially evaluated by ultrasonography. Then PDUS was performed and the presence and type of vascularity (peripheral or mixed) were investigated. Of the 19 benign and 52 malignant lesions, 3 could not be visualized with US. Forty-nine lesions were found to have vascularity (21 peripheral, 28 mixed-type) on PDUS. Of the malignant lesions, 26 showed mixed and 17 showed peripheral-type vascularity; for benign lesions these numbers were 2 and 4 respectively. The presence of vascularity correlated well with pathological behavior (malignant versus benign) (P = 0.00002), width (P = 0.0001), thickness (P = 0.001), dermal disruption (P = 0.0018), and subdermal extension (P = 0.002) of the tumor; however, the type of vascularity correlated only with thickness (P = 0.014). In relation to malignancy, the sensitivity of the presence of vascularity on PDUS was 88% and the specificity was 63%. These values were 93% and 40% respectively, for the mixed type of vascularity when it was accepted as a feature of malignancy. PDUS may help to distinguish malignant from benign skin tumors during preoperative evaluation.

Adolescent↗
PubMed2000

Inhibition of Mycobacterium smegmatis topoisomerase I by specific oligonucleotides.

DNA topoisomerase I from Mycobacterium smegmatis unlike many other type I topoisomerases is a site specific DNA binding protein. We have investigated the sequence specific DNA binding characteristics of the enzyme using specific oligonucleotides of varied length. DNA binding, oligonucleotide competition and covalent complex assays show that the substrate length requirement for interaction is much longer ( approximately 20 nucleotides) in contrast to short length substrates (eight nucleotides) reported for Escherichia coli topoisomerase I and III. P1 nuclease and KMnO(4) footprinting experiments indicate a large protected region spanning about 20 nucleotides upstream and 2-3 nucleotides downstream of the cleavage site. Binding characteristics indicate that the enzyme interacts efficiently with both single-stranded and double-stranded substrates containing strong topoisomerase I sites (STS), a unique property not shared by any other type I topoisomerase. The oligonucleotides containing STS effectively inhibit the M. smegmatis topoisomerase I DNA relaxation activity.

Binding, Competitive↗
PubMed2000

N-terminal region of P protein of Chandipura virus is responsible for phosphorylation-mediated homodimerization.

The phosphoprotein P of Chandipura (CHP) virus, an Indian isolate of rhabdovirus, was found to support transcription upon phosphorylation by casein kinase II (CKII). A phosphorylation-induced change in the protein conformation was found to occur at the N-terminal region of the protein. Biochemical studies for further characterization of this phosphorylation-based conformational alteration demonstrated that phosphorylation leads to the transition from an 'open' to 'closed' structure of the protein. The phosphate group introduced by CKII was found to be resistant to phosphatases. This phosphorylation-based structural alteration changes the accessible hydrophobic surface area of the protein and also the available digestion sites of different proteases. The phosphorylated form of P protein was found to be a dimer by His-tag dilution assay. Using the same approach it was found that the N-terminal 46 amino acids are responsible for P-P dimerization, only after phosphorylation.

Binding Sites↗
PubMed2000

Colorectal cancer vaccines: antiidiotypic antibody, recombinant protein, and viral vector.

The colorectal cancer antigen GA733 (also termed CO17-1A, KSI-4, Ep-CAM, KSA) has proved to be a useful target in passive immunotherapy with monoclonal antibody and in active immunotherapy with antiidiotypic antibodies in cancer patients. The GA733 antigen was molecularly cloned and expressed in baculovirus (BV), adenovirus (AV), and vaccinia virus (VV). Recombinant BV-, VV-, and AV-GA733 induced antigen-specific cytotoxic antibodies and proliferative and delayed-type hypersensitive lymphocytes. However, only the AV recombinant induced antigen-specific cytolytic T lymphocytes and regression of established tumors. Cured mice were protected against challenge with antigen-negative tumors, indicating antigen spreading of immune responses. In a model of active immunotherapy against the murine homologue of the human GA733 antigen, murine epithelial glycoprotein (mEGP), BV-derived mEGP protein in various adjuvants did not protect mice against a challenge with mEGP-positive tumors. AV mEGP, only when combined with interleukin-2, significantly inhibited growth of established mEGP-positive tumors. This is in contrast to the same vaccine expressing the human antigen that was effective without interleukin-2. AV GA733, in combination with interleukin-2, is a candidate vaccine for colorectal cancer patients.

Animals↗
PubMed1999

Structural changes of horseradish peroxidase in presence of low concentrations of urea.

The presence of very low concentrations of the widely used denaturant urea induces structural changes in the monomeric heme-containing enzyme, horseradish peroxidase (HRP). Structural alterations in the protein were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of urea, even in concentrations below 100 mm, were higher than those measured in absence of the denaturant. The fluorescence emission maximum of 1, 8-ANS, used as a probe for monitoring conformational changes in the enzyme, was blue-shifted from 530 nm in aqueous buffer to 518 nm when incorporated in native HRP. This blue shift increased further by 3 nm in presence of HRP preincubated with 100 mm urea, whereupon it steadily decreased with increasing urea concentration to become zero at 8 m urea. The mean fluorescence lifetime of 1,8-ANS incorporated in HRP was much higher than that of ANS in aqueous buffer, and showed continuous variation with the concentration of urea in which the enzyme was incubated. Systematic changes in the microenvironment of the heme moiety in HRP were also reflected in the visible CD spectra of the enzyme incubated with low concentrations of urea. These results are consistent with those of our earlier studies performed with the denaturant guanidinium chloride and indicate structural relaxation of HRP, with retention of enzymatic activity and native-like secondary structure, in presence of millimolar concentrations of urea.

Anilino Naphthalenesulfonates↗
PubMed1998

Sensorineural hearing loss associated with otitis media with effusion in children.

Sensorineural hearing loss (SNHL) is known to occur in various types of otitis media. Although the mechanism by which SNHL develops in association with otitis media with effusion (OME) is unknown, several hypotheses have been advocated up to now. We reviewed the clinical records of children with otitis media with effusion (OME) to reveal the association with sensorineural hearing loss. The material consisted of 71 children (119 ears) who were diagnosed as having OME and gave reliable audiograms in our clinic during an 11 month period from February 1997 through January 1998. From these cases those which showed bone conduction loss of 25 dB or higher at any one of the frequencies of 250 through 4 kHz were selected and considered to be cases of SNHL. Eight cases (9%) which had temporary threshold shift (TTS) or permanent threshold shift (PTS) were considered to be etiologically related to OME. The clinical course in each of these cases with SNHL was reviewed and evaluated in detail. We noted that all children with TTS improved completely. The result of this study indicates that we have to be aware of a possible development of SNHL during the course of OME.

Adolescent↗
PubMed1998

Transcriptional activator C protein-mediated unwinding of DNA as a possible mechanism for mom gene activation.

The bacteriophage Mu mom gene encodes the unique DNA-modification function of the phage. Regulation of the mom gene at the transcriptional level is brought about by the transactivator protein C of the phage. The mom promoter is an activator-dependent weak promoter having poor -10 and -35 elements separated by a 19 bp suboptimal spacer region. These features could constrain RNA polymerase occupancy at the promoter. Here, we have probed into the mechanism by which C protein acts as a transcriptional activator at Pmom. In vivo dimethyl sulfate footprinting studies demonstrate C protein-mediated asymmetric distortion of its specific site at the mom regulatory region. Using a coupled topoisomerase assay, we demonstrate that C protein induces the unwinding of DNA. This C-mediated unwinding seems to be localised to the 3' flanking region of the C binding site located adjacent to and overlapping the -35 element of Pmom. These results suggest that C protein-mediated torsional changes could be reorienting the -10 and -35 elements to a favorable conformation for RNA polymerase occupancy at the mom promoter.

Bacteriophage mu↗
PubMed1998

Conformation induction in melanotropic peptides by trifluoroethanol: fluorescence and circular dichroism study.

Conformation induction in the two related peptides, alpha-melanocyte stimulating hormone (alpha-MSH) and delta-melanocyte stimulating hormone (delta-MSH), have been studied in solvent media containing varying percentages of the membrane-mimetic solvent 2,2,2-trifluoroethanol (TFE) using fluorescence and circular dichroism (CD) spectroscopy. Singular value decomposition (SVD) analysis of the CD spectra at different TFE concentrations showed that these spectra can be described as linear combinations of only two distinct basis spectra, corresponding to the peptides in the random-coil and 'folded' conformations. For alpha-MSH the spectrum of the folded state is very similar to the standard spectrum of the alpha-helix, while that for delta-MSH has partial resemblance to the helical spectrum. Fitting the data on ellipticity (at 222 nm) as a function of TFE volume fraction to an equation based on a two-state model describing TFE-induced conformation induction in the peptides gave values of (1.1 +/- 0.4) and (4.2 +/- 0.5) kcal mol-1 for alpha-MSH and delta-MSH, respectively, for the free energy of equilibrium between the helix and coil forms in water. Measurement of fluorescence emission parameters (emission maximum, quantum yield, steady-state anisotropy and mean excited-state lifetime) indicated that the microenvironment around the single tryptophan residues of both peptides changes in like manner with increasing concentration of TFE in the solvent. The similarity of fluorescence behaviour of the peptides suggests that their Trp fluorophores do not participate in secondary structure formation in TFE.

Circular Dichroism↗