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.     

Localized heat-shock induction in Drosophila melanogaster

We describe a technique for inducing localized expression of genes fused to heat-shock gene promoters. We demonstrate that a localized heat-shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for beta-galactosidase activity. Induced beta-galactosidase activity is confined to the cells in the region of heating; the beta-galactosidase activity can still be detected 48 hr after the heat shock. Given the heat inducibility of Drosophila heat-shock promoters in heterologous systems, we suggest that this technique will be useful for allowing spatially controlled induction of a gene of interest in any organism into which fusion genes can be introduced. Additional uses of the technique for following cell movements during development are discussed.     

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.     

The puff inducible in region 93D is responsible for the synthesis of the major “heat-shock” polypeptide in Drosophila melanogaster

In Drosophila, temperature shocks lead to the activation of definite puffs and to the appearance of definite new polypeptides. The effects of deletions and triplications of region 93D, the site of one of the largest inducible puffs, on the induced pattern of polypeptides has been studied. A direct correlation between the dose of this chromosomal region and the relative amount of the major inducible polypeptide (about 72,000 MW) has been observed. Uninduced embryos show a low basal level of synthesis of a 72,000 MW polypeptide and this synthesis sharply increases after temperature shocks. Out of a cross which segregates zygotes completely lacking the inducible site of region 93D, embryos were found which show no increased synthesis of the 72,000 MW polypeptide after temperature shocks. The inducible 72,000 MW polypeptide is distinctly larger than the larger subunit (about 66,000 MW) of glutamine synthetase 1, which is also induced by temperature shocks and whose structural gene was shown to map also in this region. Possible explanations are discussed.     

Ecdysteroid-regulated heat-shock gene expression during Drosophila melanogaster development

Peaks in hsp 26, 28, and 83 RNA levels are correlated with peaks in ecdysteroid titers during mid-embryogenesis, pupariation, and mid-pupation, and with a peak in the level of RNA from the 74EF ecdysone puff at pupariation. Inhibition of the ecdysteroid peak at pupariation by temperature shift of the conditionally ecdysteroid-deficient strain ecd-1 was followed by a disappearance of hsp 26 RNA and a decline in hsp 83 RNA level; subsequent addition of exogeneous 20-OH-ecdysone to the temperature-shifted strain resulted in a severalfold increase in hsp 83 RNA level, and a dramatic increase in that of hsp 26. These results are consistent with the induction of the hsp 83, 28, and 26 genes by ecdysteroid at several developmental stages.     

Polymorphism and divergence at the prune locus in Drosophila melanogaster and D. simulans

The prune locus of Drosophila melanogaster lies at the tip of the X chromosome, in a region of reduced recombination in which nearby loci show reduced variation relative to evolutionary divergence from D. simulans. DNA sequencing of prune alleles from D. melanogaster and D. simulans reveals extremely low variation in D. melanogaster but greater variation in D. simulans. Divergence between the two species is not reduced. This pattern may be explained by either positive selection leading to hitchhiking of neutral variation or background selection against deleterious mutations. The pattern of silent versus replacement polymorphism and divergence at prune is consistent with either a model of weakly deleterious selection against amino acid substitutions or balancing selection.      

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

Polymorphism of yellow locus in Drosophila melanogaster from natural populations

We studied molecular characteristics of yellow (y; 1-0.0) locus, which determines the body coloration of phenotypically wild-type and mutant alleles isolated from geographically distant populations of Drosophila melanogaster in different years. According to Southern data, restrictions map of yellow locus of all studied strains differ from each other as well as from that of Oregon stock. FISH analysis shows that in the neighborhood of yellow locus in X chromosome neither P nor hobo elements are found in y1-775 stock, while only hobo is found there in y1-859 and y1-866 stocks, only P element in y+sn849 stock, and both elements in y1-719 stock. Thus, all studied mutant variants of yellow are of independent origin. Yellow locus residing at the very end of X chromosome (region 1A5-8 of cytologic map) carries significantly more transposon than retrotransposon-induced mutations, as compared to white locus (regions 3C2). It is possible that transposons are more active than retrotransposons at the chromosomal ends of D. melanogaster.