• Alok Bhattacharya

      Articles written in Journal of Biosciences

    • Expression ofMycobacterium tuberculosis genes inEscherichia coli

      Sudha Bhattacharya Satishn Ranadive Alok Bhattacharya

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      TwoEscherichia coli clones expressingMycobacterium tuberculosis antigens were isolated from a gene-bank in the plasmid vector pBR 325. ‘Western blot’ analysis revealed the presence of a unique protein band of molecular weight 68,000 and 38,000, respectively in cellextracts from each clone. The 68,000 dalton antigen was found to be expressed onEscherichia coli outer surface. Plasmid DNA from a third clone could confer leucine independence on two differentleu B mutants ofEscherichia coli but not on mutants in otherleu genes, pointing to the possibility ofgenetic complementation. Thus,Mycobacterium tuberculosis DNA is capable of expression inEscherichia coli.

    • Pathogenesis of infection byEntamoeba histolytica

      Devinder Sehgal Alok Bhattacharya Sudha Bhattacharya

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      Entamoeba histolytica, a protozoan parasite, is the etiologic agent of amoebiasis in humans. It exists in two forms—the trophozoite which is the active, dividing form, and the cyst which is dormant and can survive for prolonged periods outside the host. In most infected individuals the trophozoites exist as commensals. In a small percentage of infections, the trophozoites become invasive and penetrate the intestinal mucosa, causing ulcers. The trophozoites may reach other parts of the body—mainly liver, where they cause tissue necrosis, leading to lifethreatening abscesses. It is thought that pathogenesis of infection byEntamoeba histolytica is governed at several levels, chief among them are (i) adherence of trophozoite to the target cell, (ii) lysis of target cell, and (iii) phagocytosis of target cell. Several molecules which may be involved in these processes have been identified. A lectin inhibitable by galactose and N-acetyl-D-galactosamine is present on the trophozoite surface. This is implicated in adherence of trophozoite to the target cell. Various amoebic poreforming proteins are known, of which 5kDa protein (amoebapore) has been extensively studied. These can insert into the lipid bilayers of target cells, forming ion-channels. The phagocytic potential of trophozoites is directly linked to virulence as measured in animal models. Factors like association of bacteria with trophozoites also influence virulence. Thus, pathogenesis is determined by multiple factors and a unifying picture taking into account the relative contributions of each factor is sought. Recent technical advances, which includes the development of a transfection system to introduce genes into trophozoites, should help to understand the mechanism of pathogenesis in amoebiasis.

    • Preface

      Alok Bhattacharya

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    • Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

      Jaishree Paul Alok Bhattacharya Sudha Bhattacharya

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      Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.

    • Foreword

      N Srinivasan R Sowdhamini Alok Bhattacharya

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    • Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

      Girdhar K Pandey Amita Pandey Vanga Siva Reddy Renu Deswal Alok Bhattacharya Kailash C Upadhyaya Sudhir K Sopory

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      Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants.

    • Foreword

      Alok Bhattacharya

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    • Single-nucleotide variations associated with Mycobacterium tuberculosis KwaZulu-Natal strains

      Sarbashis Das Ragothaman M Yennamalli Anchal Vishnoi Parul Gupta Alok Bhattacharya

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      The occurrence of drug resistance in Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB), is hampering the management and control of TB in the world. Here we present a computational analysis of recently sequenced drug-sensitive (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. Single-nucleotide variations (SNVs) were identified in a pair-wise manner using the anchor-based whole genome comparison (ABWGC) tool and its modified version. For this analysis, four fully sequenced genomes of different strains of M. tuberculosis were taken along with three KwaZulu-Natal (KZN) strains isolated from South Africa including one XDR and one MDR strain. KZN strains were compared with other fully sequenced strains and also among each other. The variations were analysed with respect to their biological influence as a result of either altered structure or synthesis. The results suggest that the DR phenotype may be due to changes in a number of genes. The database on KZN strains can be accessed through the website http://mirna.jnu.ac.in/mgdd/.

    • Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA

      Ankita Srivastava Alok Bhattacharya Sudha Bhattacharya Gagan Deep Jhingan

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      Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

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