Rapid sequence divergence in a heat shock locus of Drosophila

In situ hybridization of cRNA transcribed from cloned D. melanogaster heat shock sequences to D. hydei chromosomes has shown that the D. hydei locus 2–32 A corresponds to the D. melanogaster locus 87 A/C and the D. hydei locus 2–36 A to the D. melanogaster locus 95 D, while the D. hydei locus 4–81 B corresponds to the D. melanogaster locus 63 BC. No hybridization to D. hydei chromosomes was found with cRNA transcribed from a clone containing the sequences encoded by the D. melanogaster locus 87 C. Neither D. melanogaster heat shock RNA nor D. virilis heat shock RNA hybridized significantly to the D. hydei heat shock locus 2–48 B. Furthermore, D. hydei heat shock RNA did not hybridize to the cytological homologs of locus 2–48 B found in D. repleta or in D. virilis. D, hydei heat shock. RNA did hybridize to the cytological homologs of locus 2–48 B in D. neohydei and D. eohydei, both of which belong to the hydei subgroup.     

In situ hybridization of nuclear and cytoplasmic RNA to locus 2-48BC in Drosophila hydei

The maximum grain density over the heat-shock locus 2-48BC of Drosophila hydei polytene chromosomes obtained after in situ hybridization of nuclear RNA extracted from tissue culture cells labelled during incubation at 37° C is five times higher than that obtainable by using polysomal RNA isolated from the same cells. Furthermore, the addition of a large excess of unlabelled polysomal RNA reduced the amount of in situ hybridization of nuclear RNA by only 20% showing that nuclear 2-48BC RNA contains sequences not present in polysomal 2-48BC RNA. — The polysomal 2-48BC RNA is polyadenylated, as are the RNA sequences present in the polysomes complementary to the other two major heat shock loci 2-32A and 2-36A. Polyadenylated RNA, with an apparent size of 15S, complementary to locus 2-48BC is also found in the cytoplasm of D. hydei salivary glands.     

Induction of a heat shock gene (hsp 70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue

Elevation of body temperature by 2–3°C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A new genetic model system for studying position effect variegation in Drosophila melanogaster was found. It allows the analysis of genetic inactivation and changes in chromosome morphology in the same cells. In T(1;2)dor ^var7 strains the 2B5 early ecdysone puff, and the ecs locus which maps in this puff are translocated into the vicinity of centromeric heterochromatin. The ecs locus plays a key role in the system of ecdysone puffs: genetic damage to this locus results in loss of sensitivity of cells to the hormone and, as a consequence, ecdysone-induced puffs do not develop. In the T(1;2)dor ^var7 chromosome the ecs and at least five adjoining loci are inactivated in a variegated fashion. In the salivary gland cells of T(1;2)dor ^var7/ ecs^lt435 0 h prepupae which do not show the ecdysone puffs, the morphology of the 2B region was analysed. In all cases where the ecs locus was inactivated, a dense block of chromatin reminiscent of a solid band was found in the 2B region instead of the four bands 2B1–2, 3–4, 5 and 6. Sometimes compaction of the chromatin reached the 2A1–2 or even 1E1–4 bands. Formation of the compact block of chromatin coincided with late replication in this region. In situ hybridization of polytene chromosomes with a DNA clone from the ecs locus showed that when the dense chromatin block was present, no DNA was accessible for hybridization in 2B5. Hybridization of DNA of another clone located in the region of the translocation breakpoint (2B7–8) was found only in polytene chromosomes of larvae grown at 25° C, and never in those grown at 18° C, independently of the morphology of the 2B5 puff. The possibility that in the case of block formation both late replication and, as a consequence, underreplication of chromosome DNA take place, is discussed.Dedicated to Professor W. Beermann on the occasion of his 65th birthday     

Deficiency mapping of the 93D heat-shock locus in Drosophila melanogaster

The 93D heat shock locus was mapped relative to an overlapping series of deficiencies of the 93D region by three criteria: the ability of the deleted chromosomes to puff at 93D, the ability of the deleted chromosomes to synthesize RNA from the 93D region after a temperature shift and the presence of heat shock RNA sequences at 93D as assayed by in situ hybridization. The results are essentially the same by all three criteria. Chromosomes with deficiencies that did not extend distal to 93D4 puffed and incorporated ³H-uridine after a temperature shift, and were labelled at 93D following in situ hybridization of heat shock RNA from tissue culture cells. All the other deficiency chromosomes tested failed to puff and to incorporate ³H-uridine following a temperature shift and did not show hybridization in this region after in situ hybridization with heat shock RNA. The heat shock locus was mapped to the overlapping region of Df(3R)e ^Gp4and Df(3R)GC14 just outside the inverted region of In(3R)GC23.     

Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster

Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

The location of 5S (ribosomal) RNA genes in Drosophila hydei

The location of the 5S ribosomal RNA cistrons in band 2-23B1,2 of the polytene (salivary gland) chromosomes of Drosophila hydei was indicated by in situ hybridization of tritiated low molecular weight RNA fractionated from total in vivo synthesized larval RNA or from in vitro synthesized salivary gland RNA and competition of the hybridization of this RNA by 5S RNA obtained from calf lens ribosomes. -- At the submicroscopic level, band 2-23B1,2 in salivary gland chromosomes shows a compact organization. The adjacent region, 23B2, is slightly puffed and displays typical RNP particles, some of which may be observed close to band 2-23B1,2.     

Heat shock phenomena in Chironomus tentans

Incubation of 4th instar larvae of Chironomus tentans at elevated temperatures leads in salivary and Malpighian chromosomes to the appearance of 4–5 new puffs. Previously present puffs, particularly Balbiani rings in salivary chromosomes, become drastically reduced. The reactions of region IV-5C and Balbiani ring 1 and 2 in salivary glands are quantitatively analyzed. Statistically significant heat shock effects are observed already after 5 min and reach a maximum between 30 and 60 min. The effective temperature range is small (between 33 to 40 ° C) with an optimum at 37 ° C. Above 40 ° C, i.e., at overheat shock temperatures, heat shock reactions are suppressed. Larvae heat or overheat shocked for 1–7 h or 15–30 min, respectively, survive when returned to normal culturing temperatures. The recovery from heat shock of the puffing pattern occurs in two phases: a fast one (10–20 min) and a slow one (up to 5 h) sometimes separated by a period of backlash. Quenching of overheat shocked larvae does not result in a delayed heat shock reaction.     

Molecular analysis of the ecdysterone-inducible 2B5 "early'' puff in Drosophila melanogaster.

The 2B5 region of the X-chromosome in Drosophila melanogaster plays a developmentally important role in the ecdysterone-triggered response of the late third instar salivary gland. Using a combination of transposon-tagging and chromosomal walking techniques, we have isolated 231 kb of contiguous genomic DNA sequences corresponding to this region. We have more precisely aligned this DNA to the 2B1,2 to 2B5-6 interval of the cytogenetic map by locating the position of three well-characterized chromosomal breakpoints by in situ hybridization and genomic DNA blotting experiments. Labeled cDNA, synthesized from poly(A)+ RNA isolated from hormone-induced salivary gland and imaginal disc tissues and hybridized to the cloned DNA, demonstrated that the ecdysterone-inducible sequences mapped to DNA segments corresponding to the 2B3,4 to 2B5-6 interval. Although some of these sequences were inducible in only one tissue type, many were found to be inducible in both salivary glands and imaginal discs. RNA blotting experiments have detected a major 4.5-kb RNA which is hormone inducible in the larval salivary gland and whose quantitative induction is not inhibited by cycloheximide. Thus, the 4.5-kb RNA represents at least one product from the ecdysterone-responsive 2B5 "early' puff.     

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.     

The products of the “heat-shock” loci of Drosophila hydei

Antibody was raised against total Drosophila hydei embryonic cellular protein with a molecular weight between 65,000 and 70,000 dalton. This antiserum reacted with the 70,000 MW heat-shock peptide found, in ³⁵S labelled cell extracts of heat-shocked D. hydei tissue culture cells or salivary glands. — The antibody was coupled to Sepharose 4B and this material was used to absorb polysomes obtained from tissue culture cells incubated at 37° C in the presence of tritiated RNA precursors. The relative concentrations of various RNA species complementary to the heat-shock loci 2-32A, 2-36A, and 2-48C in either bound, non-bound, or total polysomal material was then determined by in situ hybridization. The RNA species complementary to locus 2-36A was found to be enriched in the bound polysomal material.     

TMV protein synthesis is not translationally regulated by heat shock

Summary Tobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.     

Reduction of nucleic acid content in cell biomass of methanol-utilizing mixed bacterial culture by heat treatment

Summary A heat treatment method to reduce nucleic acid content in cell biomass of a mixed methanol-utilizing bacterial culture was studied. Maximum nucleic acid reduction in the bacterial cells was achieved by using heat shock at 65°C for 5–10 min followed by 2 h incubation at 55°C and 7.2±0.2 pH. In this treatment, 81–85% nucleic acid content was removed from the cells without affecting their true protein content and essential amino acids profile.