Articles written in Journal of Biosciences

    • Generation of bacteriophage T3 mRNAs by post-transcriptional processing by RNase III

      Hemanta K Majumder Samit ADhya Umadas Maitra

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      The primary transcripts synthesizedin vitro from a T3 DNA template byEscherichia coli RNA polymerase and by T3 phage-specific RNA polymerase have been characterized with regard to cleavage by RNase III and the size of the products of the cleavage reaction have been compared with those ofin vivo T3 RNAs. It has been observed that the large RNA molecule synthesized invitro byEscherichia coli RNA polymerase from the early region of T3 DNA are cleaved at specific sites byEscherichia coli RNase III to produce all the early mRNAs normally observed in T3-infected cells. In contrast, evidence presented here shows that some of the late T3 mRNAs are generated as direct products of transcription of late regions of T3 DNA by T3 RNA polymerase without mediation of RNase III, while many other late T3 mRNAs are formed by RNase III cleavage of two of the high molecular weight T3 RNA polymerase transcripts. Thesein vitro data appear to be in good agreement with the observed sizes of late T3 mRNAs formedin vivo in T3-infected RNase III-deficient and RNaseIII+Escherichia coli cells.

    • Organization and chromosomal localization ofβ-tubulin genes inLeishmania donovani

      Saumitra Das Samit Adhya

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      The genomic organization and chromosomal location of theβ-tubulin isogenes inLeishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned β-tubulin gene. We have cloned aβ-tubulin gene fragment, 3.3 kbp long, from genomic DNA ofLeishmania donovani using a heterologousβ-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11–15 copies of theβ-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3.5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of theβ-tubulin clone and is found to be nearly identical to that ofLeishmania mexicana amazonensis.

    • Transcription and processing ofβ-tubulin messenger RNA inLeishmania donovani promastigotes

      Samit Adhya Saumitra Das Mantu Bhaumik

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      Transcription of the multicopyβ-tubulin locus inLeishmania donovani promastigotes was examined by nucleic acid hybridization techniques. By northern analysis of promastigote RNA multipleβ-tubulin mRNAs were detected. The major species of 2.2 kb RNA is derived from the tandem repeat cluster ofβ-tubulin genes, the other two (2.4 and 2.6 kb) are presumably derived from dispersed genomic loci. Combined S1-nuclease and primer extension mapping experiments demonstrated the presence of a single 5′-terminus with a 35 nucleotide spliced-leader sequence. The 3′-termini are heterogeneous. The development of a nuclear run-on system inLeishmania for studying transcription of individual genes is reported. Active but transient RNA polymerase II activity was observed in this system. Using specific DNA probes for labelled run-on RNA it was shown thatβ-tubulin transcription occurs asymmetrically (i.e., on one strand of the DNA template) in anα-amanitin sensitive manner. The significance of these results for the life cycle of the parasite is discussed.

    • Mini-exon derived RNA gene ofLeishmania donovani: structure, organization and expression

      Md Quamarul Hassan Saumitra Das Samit Adhya

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      Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such asLeishmania which donates its 5′-terminal 39 nucleotides to the 5’-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene fromLeishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5′-terminus of a cellular mRNA (Β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5′-upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed.

    • Chromosome profile ofLeishmania donovani: Interstrain and interspecific variations

      Siddhartha Sankar Ghosh Sandeep Mukerjee Samit Adhya

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      The genome ofLeishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies ofβ-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). Aβ-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rnini-exon-derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain ofLeishmania donovani for coordinated genome mapping of this clinically important organism.

    • Involvement ofLeishmania donovani major surface glycoprotein gp63 in promastigote multiplication

      Sanjeev Pandey Phuljhuri Chakraborti Rakhi Sharma Santu Bandyopadhyay Dwijen Sarkar Samit Adhya

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      The major surface glycoprotein gp63 of the kinetoplastid protozoal parasiteLeishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector inL. donovani promastigotes, gp63-deficient transfectants were obtained. Reduction of the gp63 level resulted in increased generation times, altered cell morphology, accumulation of cells in the G2/M phase of the cell cycle, and increased numbers of binucleate cells with one or two kinetoplasts. Growth was stimulated, and the number of binucleate cells reduced, by addition to the culture of a bacterially expressed fusion protein containing the fibronectin-like SRYD motif and the zinc-binding (metalloprotease) domain of gp63. These observations support an additional role of gp63 in promastigote multiplication; the fibronectin-like properties of gp63 may be important in this process

    • Posttranscriptional regulation of cyclin D1 by ARE-binding proteins AUF1 and HuR in cycling myoblasts


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      RNA binding proteins (RBPs) can regulate the stability and/or translatability of messengerRNAs (mRNAs) throughinteractions with their 30-untranslated regions. However, individual mRNAs may be regulated simultaneously or successivelyby more than one RBP, as well as by Argonaute (AGO)-bound miRNAs; the coordination of these various influenceson an individual mRNA is therefore complex and not well studied. In this report we examine the roles of two RBPs thatbind to AU-rich elements (ARE) – AUF1 and HuR – in the stability and translation of cyclin D1 (Ccnd1) mRNA in ratmyoblasts transiting the G phase of the cell cycle, and their interactions with miRNAs. Knockdown (KD) of AUF1 resultedin (1) transient upregulation of the mRNA level as well as an advancement of translation onset time (TOT) from 6 to 5 hpost-serum addition, (2) loss of miRNA loading on AGO1 and AGO2 and (3) reduction in the level of AGO-1 and AGO-2bound mRNA. In contrast, KD of HuR had no effect on the mRNA level, or on the AGO–mRNA complexes, but delayedTOT by 1 h independent of miRNA let-7. Thus the dynamics of RBP–mRNA binding and –RBP–AGO–miRNA interactionsare coordinated to fine tune the expression of Ccnd1 in the G1 phase.

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