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Using polytene chromosomes of salivary gland cells of Chironomus tentans, phosphorylation state-sensitive antibodies and the transcription and protein kinase inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol II0) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol II0 is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of II0: there is a time-dependent clearance from chromosomes of phosphoCTD (PCTD) after addition of DRB, which coincides in time with the completion and release of preinitiated transcripts. Furthermore, the staining of smaller transcription units is abolished before that of larger ones. The staining pattern of chromosomes with anti-CTD antibodies is not detectably influenced by the DRB treatment, indicating that hypophosphorylated pol IIA is unaffected by the transcription inhibitor. Microinjection of synthetic heptapeptide repeats, anti-CTD and anti-PCTD antibodies into salivary gland nuclei hampered the transcription of BR2 genes, indicating the requirement for CTD and PCTD in transcription in living cells. The results demonstrate that in vivo the protein kinase effector DRB shows parallel effects on an early step in gene transcription and the process of pol II hyperphosphorylation. Our observations are consistent with the proposal that the initiation of productive RNA synthesis is CTD-phosphorylation dependent and also with the idea that the gradual dephosphorylation of transcribing pol II0 is coupled to the completion of nascent pol II gene transcripts.

Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.

Using indirect immunofluorescence visualization techniques we investigated the in situ distribution of RNA polymerase B on Drosophila melanogaster polytene chromosomes. The enzyme was found at many sites distributed throughout the genome in a pattern clearly distinct from that observed for histone H1, but it was especially concentrated in puffs induced by heat shock.

We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the beta' subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the alpha-amanitin-resistant allele, RpII215C4, includes all sequences required to produce amanitin-resistant transformants.