Transcription of heat shock loci of Drosophila in a nuclear system

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Abstract
Transcription in nuclei isolated from 6-18 h D. melanogaster embryos was investigated. Kinetic aspects and specific products of the reaction were analyzed: in particular, whether or not a faithful representation of an induced state, in this case the heat shock response, is maintained in the isolated nuclei. RNA polymerase II is active for at least 30 min. The combined RNA polymerase I and III activities continue synthesis for 60 min. Maximal synthesis by all enzymes is obtained in the absence of Mn2+. Transcripts from the 5S DNA were characterized. These consist of 2 species, of 135 and 120 nucleotides, apparently the precursor and mature forms of 5S RNA. Extensive initiation by RNA polymerase III occurs on 5S DNA in this system. Transcription from the heat shock loci at 87A and 87Cl were analyzed by hybridization of the newly synthesized RNA (selected by using 5''-mercuri-UTP) to plasmids containing the hsp70 gene and adjacent regions. Only those segments of the DNA to which transcripts were mapped in vivo hybridized with the in vitro synthesized RNA, and this transcription was observed only in nuclei isolated from heat-shocked embryos; no transcripts are detected in nuclei isolated from control embryos. These RNA species are synthesized by RNA polymerase II. Further analysis of transcription of the hsp68 gene (at locus 95D) was carried out. In this system RNA polymerases II and III transcribe the chromatin template accurately. The changes related to gene activation by heat shock are stable during nuclear isolation.
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