• S C Lakhotia

      Articles written in Journal of Biosciences

    • Heat shock response in ovarian nurse cells ofAnopheles stephensi

      B B Nath S C Lakhotia

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      Transcriptional and translational changes following temperature shock at 37, 39 or 41°C to ovarian cells ofAnopheles stephensi were studied. Temperature shock at 39°C induced 6 puffs on polytene chromosomes in the nurse cells as revealed by [3H] uridine incorporation studies. Only the 2R-19B puff was induced at 37°C and was found to be a major temperature shock locus remaining most active at all the 3 temperatures tested. Other temperature shock loci were activated only at 39°C. There was progressive inhibi tion of general chromosomal transcription with the rise of temperature. Transcription was drastically inhibited at 41°C but all the temperature shock loci still remained relatively active. Examination of [35S]methionine labelled newly synthesized ovarian proteins using sodium dodecyl sulphate-polyacrylamide slab gels revealed that all the heat shock polypeptides except the HSP 70 were synthesized in ovarian cells even at control temperature (29°C). Temperature shock induced the synthesis of HSP 70 and elevated the levels of other heat shock polypeptides (82, 30, 29, 23 and 17 KD). Present results suggest that the threshold level for induction of a complete heat shock response in mosquitos is higher (39°C) than the other dipteran insects studied and that a 41°C treatment is not lethal as in the case ofDrosophila, Chironomus etc. These features reflect the adaptations of mosquitos to tropical climate and their dietary habit of warm blood meal.

    • In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

      P K Tiwapi S C Lakhotia

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      The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [3H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [3H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.

    • Restriction enzyme digestion of heterochromatin inDrosophila nasuta

      P K Tiwari S C Lakhotia

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      In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.

    • hsp 83 mutation is a dominant enhancer of lethality associated with absence of the non-protein codinghsrω locus inDrosophila melanogaster

      S C Lakhotia Pritha Ray

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      Thehrsω or the 93D heat shock locus ofDrosophila melanogaster, which does not code for any protein, has an important role in development since nullosomy of this locus in transheterozygotes for two overlapping deficiencies, viz.,Df(3R)eGp4 (eGp4) andDf(3R)GC14 (GC14), is known to cause a high (∼ 80%) mortality with the small number of escapee nullosomic flies being sterile, weak and surviving for only a few days. We now show that a majority of thehsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of thehsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when onehsp83 mutant allele (hsp83e4A) is introduced ineGp4/GC14 trans-heterozygotes, practically none of thehsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction betweenhsp83 andhsrω genes was further confirmed by examining the effect of thehsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that thehsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for thehsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development ofDrosophila reveals novel aspects of their biological functions.

    • Heat shock but not benzamide and colchicine response elements are present within the — 844 bp upstream region of thehrsω gene ofDrosophila melanogaster

      S C Lakhotia Mousumi Mutsuddi

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      The selective inducibility ofhsrω gene by heat shock and several chemical agents and its selective non-inducibility by heat shock under certain conditions led to suggestion that this locus is subject to multiple controls at the level of transcription. With a view to delimit these different control elements, transgenic lines horbouringhsrω 5’ promoter deletion variants tagged to thelacZ reporter gene were used. Three different assays, viz., staining forβ-galactosidase activity in different larval tissues using chromogenic X-gal substrate, [3H] uridine labelling of polytene nuclei andin situ DNA-DNA hybridization with a non-radioactive probe to polytene chrmosome spreads for checking the puffing status of the resident and the transgene in larval salivary glands, were applied to monitor the activiy of the reporter gene following different treatments. Our results showed that the − 844 bp to +107 bp sequence was sufficient for heat shock induction of the transgene in all tissues. An analysis of the base sequence of thehsrω promoter revealed the presence of three consensus heat shock elements at − 466, − 250 and at − 57 bp and of two GAGA factor binding sites at − 496 and at − 68bp within the − 844 bp region. Germline transformants carrying the − 346 bp to − 844 bp region of thehsrω promoter showed only a very weak heat shock inducibility of the reporter gene in agreement with the presence of only one of the three putative heat shock elements and one of the two GAGA factor binding sites in this region. Interestingly, neither of the transformed lines (carrying the − 844 bp to + 107 bp or the − 844 bp to −346 bp of thehsrω promoter region) showed any response of the transgene to benzamide or colchicine treatments. These results showed that while the heat shock response elements of thehsrω are included within the − 844 bp region the response elements for benzamide and colchicine treatments are outside this region.

    • Regulation of HSP70 in excitatory neurons: Possible implications for neuronal functioning

      AnoopKumar Thekkuveettil S C Lakhotia

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      Neurons maintain an intricate organization of cytoplasmic and membrane proteins for their integrity, quick communication across synapses and for other complex activities. Molecular chaperones such as members of the 70 kDa heat shock protein (HSP70) family may play very important roles in these functions. However, in spite of a recent report suggesting the presence of HSP70 related proteins in the synaptic vesicle docking complex at presynaptic sites and the known significant roles for HSP70 in excitotoxicity, there are remarkably few studies that have explored the potential role of HSP70 family proteins in physiological functions of neurons. Here we bring together direct and indirect evidences which suggest that several different pathways involved in long-term potentiation can influence the HSP70 levels at the synapse and hypothesize on possible physiological significance of this family of proteins in neuronal functions.

    • Developmental regulation and complex organization of the promoter of the non-codinghsrω gene ofDrosophila melanogaster

      S C Lakhotia T K Rajendra K V Prasanth

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      The nucleus-limited large non-coding hsrω-n RNA product of the93D or thehsrω gene ofDrosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene byin situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying thehsrω05241 allele due to insertion of aP-LacZ-rosy+ transposon at — 130 bp position of thehsrω promoter. We also examinedLacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of thehsrω promoter upstream of theLacZ reporter. ThehsrΩ gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of thehsrω05241 allele in the enhancer-trap line, as revealed byin situ hybridization to hsrω transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of theLacZ gene in this enhancer-trap line was similar to that of thehsrω RNA in all diploid cell types in embryos and larvae but in the polytene cells, theLacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns ofhsrω gene and those of theLacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types.

    • Stress biology — from molecules to populations and environment

      S C Lakhotia

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