Chromatin structure of a P-element-transduced hsp-28 gene in Drosophila melanogaster.

Abstract

The Drosophila hsp-28 gene was heat inducible when transduced to novel chromosomal sites even when no direct selection for transduced gene expression was imposed. The pattern of DNase I-hypersensitive sites 5' to the wild type and transduced copy of hsp-28 was similar. In addition, DNase I-hypersensitive sites occurred within the P-element sequences flanking transduced loci.A diverse body of data supports a model of the eucaryotic chromosome in which active genes reside in chromatin domains that are relatively more sensitive to nucleolytic attack than those of inactive genes (reviewed in reference 3). The apparent compartmentalization of the genome into active and inactive domains raises the possibility that the domain organization of the chromosome plays a role in promoting the differential expression of genes in different domains.P-element-mediated germ line transformation in Drosophila melanogaster (21) has made possible a detailed assessment of the role of chromosome organization in eucaryotic gene expression. A variety of genes have shown a high degree of autonomy in their spatiotemporal regulation when transduced elsewhere in the euchromatin (6, 8-10, 13, 16, 20, 23-25). Such results have been interpreted as demonstrating that all of the cis-acting regulatory elements governing gene activity generally reside immediately adjacent to the transcription unit itself. However, in most cases direct selection for expression of the transduced DNA was imposed, raising the possibility that many instances of chromosomal position effects went undetected.We used P-element-mediated germ line transformation to introduce the small heat shock gene hsp-28 into novel chromosomal locations without imposing a selective screen. This gene was chosen because it is rapidly induced to high levels of expression in a variety of tissues by a brief heat shock and because the chromatin structure of the hsp-28 locus has been extensively characterized in embryos (1,11,14) and tissue culture cells (1). The plasmid used in these transformation experiments (called Ad44) is shown in Fig. 1. Insertion of 1.4 kilobases of adenovirus DNA into the coding region of hsp-28 in Ad44 permits detection of transforming DNA and RNA in an hsp-28+ background. Note that, although hsp-23 sequences are also present in the plasmid, the hsp-23 RNA cap site and TATA box sequences are missing. Thus, hsp-23 is not expected to be transcribed from the transduced loci. Ad44 DNA, at 500 jig/ml, was coinjected with pwr25.1 (at 50 ,ug/ml) into preblastoderm Oregon R Drosophila embryos as described by Rubin and Spradling (21). Injected flies were subsequently crossed to Oregon R adults, and their progeny (F1 generation) were pair mated inter se. When larvae appeared in the vials, prepared DNA from each F1 parent by the potassium acetate-sodium dodecyl sulfate method described by Lis et al. (13) RNA was prepared from heat-shocked adults of each homozygous transformed line essentially by the method of Meyerowitz and Hogness (18). Figure 2 shows the Northern blot analysis ...