To live out your child’s dream

Often I face the question, “How come neither of your children chose science when both of you are scientists? How is it that, the choice of a non-science course, or art, visual communication, and design as a career by your children did not make you blink even once?” For us, it was simply about the pursuit of creativity. In fact, in these career options, unlike scientific research, one can contribute something which many people relate to in their own lifetime

Preparation of megabase DNA from adult insects and mammalian spleen for pulsed-field gel electrophoresis

While standard techniques for obtaining megabase-size DNA from microorganisms and cells in tissue culture are now available, new methods are needed for handling solid tissues and, in the case of small-sized animals, whole organisms. Here we describe a simple and rapid method for preparing large DNA molecules from mammalian spleen, whole insects of Drosophila and Planococcus lilacinus, a mealybug. Briefly the method involves gentle steps for preparing cell suspensions and handling of these cells within agarose blocks for preparing the DNA. In mammals like mice, the spleen, rather than the liver, is the organ of choice because, in the latter, endogenous nuclease activity is high

Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

<p>Abstract</p> <p>Background</p> <p>The <it>mce </it>operons play an important role in the entry of <it>M. tuberculosis </it>into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of <it>mce </it>mutants. Recently, <it>mce1 </it>operon was found to extend over 13 genes, <it>fadD5 </it>(Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to <it>mce1 </it>among the four <it>mce </it>operons of <it>M.tuberculosis</it>. We have examined the function of this region.</p> <p>Results</p> <p>We predicted putative promoter activity of the 200 base pairs of non-coding, intergenic region between Rv0166 and Rv0167 <it>in silico </it>using MEME software and designate it as intergenic promoter, IGPr. We demonstrate both promoter activity and a putative negative regulatory function of this fragment by reporter assays carried out in the surrogate host <it>M.smegmatis</it>. We find that the repressive elements not only control the native promoter but also repress a heterologous promoter of <it>M.smegmatis</it>. The higher activity of the intergenic promoter in a clinical isolate in comparison with the wild type sequence from <it>M.tuberculosis </it>H37Rv could be correlated with a point mutation within the negative element. We have mapped two transcription start sites for <it>mce1 </it>operon both of which are utilized in <it>M.tuberculosis </it>H37Rv as well as the clinical isolate VPCI591. Our studies show that the promoter activity in the non-coding region is relevant not only in reporter gene expression but also in the expression of <it>mce1 </it>operon in <it>M. tuberculosis </it>cells grown in synthetic medium.</p> <p>Conclusion</p> <p>The <it>mce </it>operon of <it>M.tuberculosis </it>H37Rv potentially can be transcribed from two promoters P1 and P2, former mapping upstream of Rv0166 and the latter in the non-coding intergenic region between Rv0166 and Rv0167. The transcription initiation from P1 results in a transcript with Rv0166 while that from P2 will be without it. The sequences between the translation start site of Rv0167 and the promoter P2 have a negative regulatory role, as point mutation within the sequence leads to enhanced activity of P2 as well as a heterologous promoter from <it>M.smegmatis</it>. The mutation detected in the clinical isolate VPCI591 therefore behaves like a gain-of-function mutation.</p

Comparative analysis of chromatin landscape in regulatory regions of human housekeeping and tissue specific genes

BACKGROUND: Global regulatory mechanisms involving chromatin assembly and remodelling in the promoter regions of genes is implicated in eukaryotic transcription control especially for genes subjected to spatial and temporal regulation. The potential to utilise global regulatory mechanisms for controlling gene expression might depend upon the architecture of the chromatin in and around the gene. In-silico analysis can yield important insights into this aspect, facilitating comparison of two or more classes of genes comprising of a large number of genes within each group. RESULTS: In the present study, we carried out a comparative analysis of chromatin characteristics in terms of the scaffold/matrix attachment regions, nucleosome formation potential and the occurrence of repetitive sequences, in the upstream regulatory regions of housekeeping and tissue specific genes. Our data show that putative scaffold/matrix attachment regions are more abundant and nucleosome formation potential is higher in the 5' regions of tissue specific genes as compared to the housekeeping genes. CONCLUSION: The differences in the chromatin features between the two groups of genes indicate the involvement of chromatin organisation in the control of gene expression. The presence of global regulatory mechanisms mediated through chromatin organisation can decrease the burden of invoking gene specific regulators for maintenance of the active/silenced state of gene expression. This could partially explain the lower number of genes estimated in the human genome

Effect of dietary supplementation of Bacillus subtilis on haematological and immunological parameters of Catla catla (Hamilton)

Effect of dietary supplementation of a Gram-positive, aerobic, probiotic bacterium Bacillus subtilis on the immunohaematological indices during pre- and post-challenge in Indian major carp, catla (Catla catla), was studied. The B. subtilis was administered orally at four different doses 1.0 × 106 (T1), 1.0 × 107 (T2), 1.0 × 108 (T3), and 1.0 × 109 (T4) cfu g−1 feed to C. catla for 90 days. The positive control (Cp) and negative control (Cn) were fed with feed without B. subtilis for the same period. On the 60th day, blood and serum were sampled to determine various haematological and serum parameters. Fish were challenged intraperitoneally with Aeromonas hydrophila after 60 days in all the treatment groups and Cp, while the Cn was challenged with phosphate-buffered saline (PBS, pH 7.2) only. Dietary supplementation of B. subtilis leads to the rise of various immunological and haematological parameters in catla during the pre- and post-challenge. During pre-challenge, the highest TEC (1.30 ± 0.02 × 106 cells mm−3), haemoglobin (7.43 ± 0.25 g %), total serum protein (3.89 ± 0.08 g dL−1), and serum lysozyme activity (8.39 ± 0.01 µg ml−1) were recorded in fishes fed feed containing B. subtilis at 1 × 109 cfu/g feed (T4). The highest survival percentage (86.33 %) was also observed in T4 group. The significantly increased survival percentage (P < 0.05) of B. subtilis-treated groups in comparison with control group (Cp) suggests that dietary supplementation of this probiotic bacterium can protect catla from A. hydrophila infection by enhancing innate immunity

Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach

Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains

<p>Abstract</p> <p>Background</p> <p>The presence of four mammalian cell entry (<it>mce</it>) operons in <it>Mycobacterium tuberculosis </it>suggests the essentiality of the functions of the genes in these operons. The differential expression of the four <it>mce </it>operons in different phases of <it>in vitro </it>growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.</p> <p>Here we investigate the extent of polymorphism in eight genes in the <it>mce1 </it>and <it>mce4 </it>operons of <it>M. tuberculosis </it>from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform.</p> <p>Results</p> <p>We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of <it>mce1 </it>and <it>mce4 </it>operons revealed that <it>yrbE1A </it>[<it>Rv0167</it>] was most polymorphic in <it>mce1 </it>operon while <it>yrbE4A </it>[<it>Rv3501c</it>] and <it>lprN </it>[<it>Rv3495c</it>] had the highest number of SNPs in the <it>mce4 </it>operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to <it>M. tuberculosis </it>using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation.</p> <p>Conclusions</p> <p>Our results show that SNPs in the coding sequences of <it>mce1 </it>and <it>mce4 </it>operons in clinical isolates can be significantly high. Moreover, <it>mce4 </it>operon is significantly more polymorphic than <it>mce1 </it>operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in <it>mce1 </it>operon and synonymous substitutions are more in <it>mce4 </it>operon. <it>In silico </it>modeling predict that nonsynonymous SNP at <it>mce1A </it>[<it>Rv0169</it>], a virulence gene could play a pivotal role in causing functional changes in <it>M. tuberculosis </it>that may reflect upon the biology of the bacteria.</p

Interaction of the chromatin remodeling protein hINO80 with DNA

The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity in vitro. Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes

To live out your child’s dream

Often I face the question, “How come neither of your children chose science when both of you are scientists? How is it that, the choice of a non-science course, or art, visual communication, and design as a career by your children did not make you blink even once?” For us, it was simply about the pursuit of creativity. In fact, in these career options, unlike scientific research, one can contribute something which many people relate to in their own lifetime