Chromosomal arrangement of heat shock locus 2-48B in Drosophila hydei

cDNA, copied from nuclear RNA isolated from heat shocked Drosophila hydei cells, has been cloned. From this collection of clones a clone, N09-15, with a 450 bp insert has been isolated that hybridizes in situ to the heat shock locus 2-48B of Drosophila hydei. The N09-15 sequence is present in two different genomic arrangements, as shown by restriction mapping, in our wild type D. hydei population. These genomic arrangements are allelic. Both alleles contain multiple copies of the N09-15 sequence but differ in their lengths and in the distribution of Msp I and Taq I sites.     

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.     

Cloning of heat-shock locus 93D from Drosophila melanogaster.

Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.     

Heat shock proteins and aging in Drosophila melanogaster

Heat shock proteins (Hsps) are conserved molecular chaperones that are upregulated following exposure to environmental stress and during aging. The mechanisms underlying the aging process are only beginning to be understood. The beneficial effects of Hsps on aging revealed in mild stress and overexpression experiments suggest that these proteins are part of an important cell protection system rather than being unspecific molecular chaperones. Among the Hsps families, small Hsps have the greatest influence on aging and the modulation of their expression during aging in Drosophila suggest that they are involved in pathways of longevity determination.     

Heat shock protection against cold stress of Drosophila melanogaster.

Heat shock protein synthesis can be induced during recovery from cold treatment of Drosophila melanogaster larvae. Survival of larvae after a cold treatment is dramatically improved by a mild heat shock just before the cold shock. The conditions which induce tolerance to cold are similar to those which confer tolerance to heat.     

Frequency-dependent competitive abilities and differential resource use in competition between Drosophila hydei and D. melanogaster

Several experiments, each involving competition between Drosophila melanogaster and D. hydei in population cages, were set up and allowed to run for up to 50 weeks. The population sizes of both species, and hence the species frequencies, were monitored once a fortnight, i.e. approximately once per generation. Coexistence of the two species was observed in cages containing resource bottles with 5 g of food medium; cages whose resource bottles contained only 1.5 g resulted in competitive exclusion of D. hydei. Competitive abilities were frequency-dependent in the former case but not in the latter. Tests of larval depth distributions revealed that D. hydei larvae feed at a deeper level in the food medium than larvae of D. melanogaster. The explanation of the contrasting results of competition when bottles contained 5 g and 1.5 g of resources lies in the production of frequency-dependent competitive abilities by larval resource partitioning in the bottles with 5 g, and the preclusion of such partitioning in the 1.5 g bottles because of the very limited depth of food medium then available. The relevance of these results to a model of competition is discussed, and the potential generality of differential resource use as a stabilizing mechanism in both interspecific and intergenotypic competition is noted.     

Identification of the heat shock band 2-48B of Drosophila hydei and determination of its haploid DNA content

The heat shock locus 2-48B situated at the tip of the second chromosome of Drosophila hydei has been studied by various cytologic methods. Both from puffing behaviour and in situ hybridization on both wildtype animals and heterozygotes for the mutant chromosome Df(2)e20 the actual site is localized in band 2-48B8. Electron microscopically this band appears to be a medium size, dotted band. From cytophotometric measurements on Feulgen stained chromosomes and electron microscopic observations the DNA content of band 2-48B8 is calculated to be approximately 40 kb on the haploid level, with a compaction of the DNA of about 160 times. The interbands 2-48B7-8 and 2-48B8-9, flanking the heat shock band, were calculated to contain on the haploid level 1.5 kb and 3.0 kb, respectively. The results are discussed also in relation to the present data on the molecular organization of this locus.     

Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.     

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.     

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.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.