Stage dependent synthesis of heat shock induced proteins in early embryos of Drosophila melanogaster

Summary The synthesis of heat shock proteins (hsp) has been examined during the early embryogenesis of Drosophila melanogaster. Normal protein synthesis stops after heat shock at all developmental stages, while hsp synthesis is induced only after treatment at blastoderm and later stages. The small hsps continue to be synthesised after heat shock for a longer period than the larger ones. Heat shocks at 35°C, 37°C and 40°C were compared for their effect on hsp synthesis and the effect of heat shock on the normal course of development was analysed.     

Conservation of the 93D puff of Drosophila melanogaster in different species of Drosophila

Temperature shock (TS) results in activation of a specific set of puffs in polytene nuclei of D. melanogaster. Earlier studies in this species from several laboratories revealed certain unique features of the major TS puff at 93D locus, which is also specifically induced by benzamide (BM) and by incubation of glands in heat shocked glands' homogenate (HSGH). We have now extended studies on TS response to several other species of Drosophila to ascertain whether loci homologous to 93D puff of D. melanogaster are present in other species. In polytene nuclei of two closely related (D. ananassae, D. kikkawai) and in two distantly related species (D. hydei, D. nasuta), six to nine puffs are induced by TS. Interestingly, in each species one of the major TS puffs, viz., 2L-2C in D. ananassae, E-11BC in D. kikkawai, 2R-48A in D. nasuta and 2-48C in D. hydei, is also specifically induced by BM, autologous species' HSGH and vitamine-B₆ (vit-B₆) treatment. HSGH of a different species fails to induce these puffs. These puffs thus resemble the 93D locus of D. melanogaster, although the 93D puff does not respond to vit-B₆. These observations are discussed in relation to the conservation of 93D puff locus in different species of Drosophila.     

Heat-shock DNA homology in distantly related species of Drosophila

Polytene chromosomes of D. melanogaster and D. virilis were hybridized in situ with ¹²⁵I labeled mRNA isolated from polysomes of D. melanogaster tissue culture cells incubated at 37° C. ¹²⁵I mRNA hybridized preferentially with subdivisions 87A and 87Cl of the D. melanogaster 3R chromosome; grains were also observed at regions 93D, 95D and over the chromocenter. A considerable cross hybridization of this mRNA with D. virilis polytene chromosomes was observed. The 29C region of the D. virilis second chromosome was the main site of hybridization. Significant grain numbers also appeared in region 20F of the same chromosome. The two regions mentioned belong to heat shock loci in the latter species. Based on label intensity we conclude that region 29C of D. virilis contains DNA sequences retaining molecular homology with those at subdivisions 87A and 87Cl of D. melanogaster. SDS-polyacrylamide gel electrophoresis revealed similar distributions of heat shock proteins in the two species studied.     

Immunofluorescence localization of a small heat shock protein (hsp 23) in salivary gland cells of Drosophila melanogaster

Summary An aggregate present in cell-free extracts of Drosophila melanogaster tissue culture cells, sedimenting at 20 to 30S, contains hsps 23, 26 and 27. Hsp 23 was purified from this aggregate and a monospecific antibody was raised against it. Immunofluorescence microscopy showed the presence of hsp 23 preferentially in nuclei after heat shock, while on return to 25° C, hsp 23 was reduced in nuclei and increased in the cytoplasm. Thus the immunofluorescence observations reported here unambignously confirm for hsp 23 earlier reports that heat shock proteins are mainly found in nuclei after heat shock and that upon return to 25° C, they move to the cytoplasm.Abbreviations NP-40 Nonidet P40 - PMSF Phenylmethylsulfonyl-fluoride - SDS Sodium dodecyl sulfate - EDTA Ethylene diamine tetraacetic acid - TCA Trichloroacetic acid. hsp 22, hsp 23 etc.: heat shock proteins of 22,000, 23,000 daltons etc. molecular weight     

Dosage compensation of X-linked heat-shock puffs in Drophila pseudoobscura

Four major puffs are inducible by heat shock in the larval salivary gland chromosomes of D. pseudoobscura. Two of these puffs are present at 23 and 39–40 on the right arm of the X chromosome and two are present at 53 and 58 on chromosome 2. By means of in situ hybridization, residual homologies were demonstrated between the puffs at 23 in D. pseudoobscura and at 63C in D. melanogaster, and between the two chromosome 2 puffs of D. pseudoobscura and 87A and 87C of D. melanogaster. RNA synthesis was monitored as a function of ³H-uridine incorporation in the major heat-induced puffs of D. pseudoobscura and was found to be equivalent in males and females indicating dosage compensation of the two X-linked loci. The evolution of the regulatory controls of these genes is discussed.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence

Using indirect immunofluorescence visualization techniques we investigated the in situ distribution of RNA polymerase B on Drosophila melanogaster polytene chromosomes. The enzyme was found at many sites distributed throughout the genome in a pattern clearly distinct from that observed for histone H1, but it was especially concentrated in puffs induced by heat shock.     

Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopic (EM) analysis was performed on regions of Drosophila melanogaster polytene chromosomes that contain inserted DNA segments of 19 and 8 kb. These segments had been inserted by P-elementmediated transformation. The 19 kb segment includes both the Drosophila hsp70 gene fused to the Escherichia coli -galactosidase gene and the rosy gene (Lis et al. 1983). This insert generates a new moderate-size band at the 9D4-9E1-2 region in polytene chromosomes. Upon heat shock, a puff originates from a portion of the new band. The 8 kb segment includes the Sgs7 and Sgs3 genes (Richards et al. 1983). This insert generates very diffuse thin bands that decondense at the stage of activation of the Sgs genes to produce wide interbands or small puffs. In all of the above cases, the insertion appears to occur at interband regions, and the genetically complex DNA segments that are inserted generate only a single detectable band.     

Expression of 93D heat shock puff of Drosophila melanogaster in deficiency genotypes and its influence on activity of the 87C puff

Using the overlapping deficiencies Df(3R)GC14 and Df(3R)e ^Gp 4 of the 93D region of Drosophila melanogaster, the benzamide (BM)-inducible site in polytene chromsomes was localised to the 93D6-7 region, which had earlier been identified as heat inducible. The normal developmental and BM-induced 93D6-7 puff was found to be dosage compensated since in larvae heterozygotus for a deficiency, with one dose of 93D6-7, the rate of ³H-uridine incorporation in this puff was the same as in the wild type with two doses. Curiously, however, heat shock (37° C) caused regression of the 93D6-7 puff on the normal chromosome in heterozygotes. In agreement with earlier results from our laboratory, the non-inducibility of the single-dose 93D locus by heat shock in the heterozygotes, caused the 87C puff to be nearly half as active as the 87A puff at 37° C. However, in e ^Gp 4/GC14 larvae, lacking the 93D6-7 locus on both homologues, the 87C puff was less active than 87A in some heat-shocked larvae but in other larvae 87A and 87C were equally active. Possible reasons for this inter-larval variability are discussed.     

RNA Polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes

Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.     

Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA

Fluorochrome-labeled RNA allows the rapid detection of in situ hybrids without the need for long exposure times as in the autoradiographical hybridisation methods. Resolution is high because of the high resolving power of fluorescence microscopy. The application of a previously reported method for the hybrido-cytochemical detection of DNA sequences to polytene chromosomes of Drosophilia is described. — The specificity and sensitivity of the method are demonstrated by the hybridisation with polytene chromosomes of 1) rhodamine-labeled 5S RNA, to the 5S rRNA sites of D. melanogaster (56F) and D. hydei (23 B), 2) rhodamine-labeled RNA complementary to a plasmid containing histone genes, to the 39DE region of D. melanogaster, 3) rhodamine-labeled D. melanogaster tRNA species (Gly-3 and Arg-2), to their respective loci in D. melanogaster, 4) rhodamine-labeled RNA complementary to the insert of plasmid 232.1 containing part of a D. melanogaster heat shock gene from locus 87 C, to D. hydei heat shock locus 2-32A. In the latter instance it was possible to demonstrate the labeling of a double band which escaped unambiguous detection by autoradiography in the radioactive cytochemical hybridisation procedure because of the low topological resolution of autoradiograms. — The sensitivity of the fluorochrome-labeled RNA method is compared with the radioactive methods which use ³H- or ¹²⁵I-labeled RNAs. The factors governing the sensitivity and the number of bound fluorochrome molecules to be expected are discussed.Dedicated to Professor W. Beermann in honour of his 60th birthday