Effects of ecological and developmental factors on the heat shock response

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The vicious circle of poor science, poor journals and poor recognition

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Forty years of the 93D puff of Drosophila melanogaster

The 93D puff of Drosophila melanogaster became attractive in 1970 because of its singular inducibility by benzamide and has since then remained a major point of focus in my laboratory. Studies on this locus in my and several other laboratories during the past four decades have revealed that (i) this locus is developmentally active, (ii) it is a member of the heat shock gene family but selectively inducible by amides, (iii) the 93D or heat shock RNA omega (hsrω) gene produces multiple nuclear and cytoplasmic large non-coding RNAs (hsrω-n, hsrω-pre-c and hsrω-c), (iv) a variety of RNA-processing proteins, especially the hnRNPs, associate with its >10 kb nuclear (hsrω-n) transcript to form the nucleoplasmic omega speckles, (v) its genomic architecture and hnRNP-binding properties with the nuclear transcript are conserved in different species although the primary base sequence has diverged rapidly, (vi) heat shock causes the omega speckles to disappear and all the omega speckle associated proteins and the hsrω-n transcript to accumulate at the 93D locus, (vii) the hsrω-n transcript directly or indirectly affects the localization/stability/activity of a variety of proteins including hnRNPs, Sxl, Hsp83, CBP, DIAP1, JNK-signalling members, proteasome constituents, lamin C, ISWI, HP1 and poly(ADP)-ribose polymerase and (viii) a balanced level of its transcripts is essential for the orderly relocation of various proteins, including hnRNPs, RNA pol II and HP1, to developmentally active chromosome regions during recovery from heat stress. In view of such multitudes of interactions, it appears that large non-coding RNAs like those produced by the hsrω gene may function as hubs to coordinate multiple cellular networks and thus play important roles in maintenance of cellular homeostasis

India's ambitions to be a world leader in S&T depend upon a drastic overhaul of the university system

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Functional organization of polytene X-chromosome in two X-chromosome inversion carrying larvae of Drosophila melanogaster reared at 24°C or 10°C

Larvae of D. melanogasler carrying either the In(I)BM1 or In(I)BM2 inversion have been reared at 24° or at 10°C to study the morphology, transcription and replication of the X-chromosome in the salivary gland polytene nuclei. These two inversions share a similar left-hand breakpoint in euchromatin (16A 2-5 region in polytene X-chromosome) but have different right-hand breakpoints in the proximal heterochromatin. In 10°C reared BM1 male larvae, the polytene X appears somewhat more diffused than in 24°C reared larvae. On an average, in 36% of the polytene nuclei of 10°C reared BM2 male larvae the single X-chromosome appears considerably shortened in length, enlarged in width and has very indistinct band, while the other nucIei show "normal-looking" X as in BM1 larvae. This highly disorganized form of X-chromosome, referred to as "pompon-Iike" X, is never seen in cold-reared female or in warm-reared female as well as male BM2 or BM1 larvae. Hoechst 33258 fluorescence reveals that the "pompon-like" morphology of the X is due to loose packing of chromatin in different band regions. It is suggested that the "pompon-like" morphology is due to position effect variegation associated with the particular heterochromatin breakpoint in the BM2 inversion, 3H-uridine and 3H-thymidine labelling and autoradiography of polytene nuclei from cold-reared male and femal larvae of the two genotypes shows that the "pompon-like" or the "normal-looking" X in male nuclei continues its hyperactive transcription and faster replication as in 24°C reared wild-type larvae. It appears that the hyperactive organization of the hemizygous X in larval male polytene nuclei predisposes its pattern of chromatin condensation to be specifically affected by a variety of genetic, chemical and physical factors. However, the type of chromatin dispersion seen in "pompon-like" X in cold-reared BM2 male larvae does not seem to affect the basic functional (hyperactive) organization of the hemizygous X in male polytene nuclei

Specific induction of the 93D puff in polytene nuclei of Drosophila melanogaster by colchicine

When salivary glands of late 3rd instar larvae of D. melanogaster are exposed in vitro to colchicine (100 μg/ml) or colcemid (I to 100 Iμg/ml) for 40 min at 24°C, 3H-uridine incorporation in polytene chromosomes is severely inhibited while that in nucleolus remains nearly unaffected. At the same time, the 93D puff, which is one of the major heat shock locus, is specifically induced without a concomittant induction of other heat shock loci. These effects of colchicine or colcemid treatment are strikingly similar to those of benzamide [Lakhotia & Mukherjee, Chromosome, 81 (1980) 125]. It appears that the colchicine-induced activity of 93D is not due to the effects of this alkaloid on cellular rnicrotubules. The 93D puff is much less induced when the glands are heat shocked in presence of colchicine. Presence of colchicine during heat shock also causes the 87C puff to be induced to a greater degree (nearly 2.5 x) than its duplicate locus at 87A

Molecular rhythms that regulate rhythm genes in Drosophila

Almost all living organisms display rhythms in their activities coinciding with the day-night cycles. Our current understanding of the molecular regulation of circadian rhythmicity in Drosophila comes from studies integrating genetics and molecular biology, and Drosophila is perhaps one of the best understood models in the field of circadian rhythm research. Following the initial discovery of the per (period) gene some decades ago, several other genes, viz. timeless, dclock, cycle, and double-time, that function in the generation of circadian rhythms, have been identified during the past three years: Molecular genetic studies have provided exciting insights into the regulation of the body clocks. Heterodimeric complexes of positive elements (dCLOCK and CYCLE) and their interactions with feedback loops and negative elements of per and tim genes and their products have been identified and these are providing clues to the general layout of the molecular loops that generate circadian rhythms. The lark gene, which encodes an RNA-binding protein, might function as a regulatory element in the circadian clock output pathway controlling pupal eclosion rhythms. However, a clear picture of the output pathways or downstream processes through which the clock regulates the circadian rhythmic events is yet to be understood

Heat shock but not benzamide and colchicine response elements are present within the-844 bp upstream region of the hrsω gene of Drosophila melanogaster

The selective inducibility of hsrω 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 horbouring hsrω 5' promoter deletion variants tagged to the lacZ reporter gene were used. Three different assays, viz., staining for β-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 the hsrω 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 the hsrω 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 the hsrω promoter region) showed any response of the transgene to benzamide or colchicine treatments. These results showed that while the heat shock response elements of the hsrω are included within the - 844 bp region the response elements for benzamide and colchicine treatments are outside this region

hsp83 mutation is a dominant enhancer of lethality associated with absence of the non-protein coding hsrω locus in Drosophila melanogaster

The hrsω 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) and Df(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 the hsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of the hsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when one hsp83 mutant allele (hsp83e4A) is introduced in eGp4/GC14 trans-heterozygotes, practically none of the hsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction between hsp83 and hsrω genes was further confirmed by examining the effect of the hsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that the hsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for the hsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development of Drosophila reveals novel aspects of their biological functions

Heat shock puff activity in salivary glands of Drosophila melanogaster larvae during recovery from anoxia at two different temperatures

All heat shock puff sites are induced in salivary gland polytene nuclei of D. melanogaster during recovery from anoxia at 24°C. 3H-uridine autoradiographic analysis reveals that the puff at 93D locus is most active in glands recovering from anoxia at 24°C but in glands recovering at 37°C this puff is completely repressed. During recovery from anoxia at 24°C as well as 37°C, the 87C puff is nearly twice as active as its duplicated locus at 87A