The in situ structure of the active 75 S RNA genes in Balbiani rings of Chironomus tentans

Abstract

Balbiani rings 1 and 2 in the salivary glands of Chironomus tentans are known to contain transcriptionally active 75S RNA genes. These have earlier been characterized in the electron microscope when spread on a surface according to Miller. In the present study the 75S RNA genes are studied as they appear as loops within the cell. Serial sections through Balbiani rings have been analysed, and both proximal and distal segments of active 75S RNA genes have been reconstructed. The growing ribonucleoprotein particles were visualized as well as the putative RNA polymerases at the bases of the ribonucleoprotein (RNP) fibres and a thin (5 nm) chromosomal axis. In the proximal portion of the active gene (about one-quarter of the gene) the RNP fibres appear as 20 nm thick fibers, gradually increasing in length. In the distal part of the gene no further increase in length of the 20 nm RNP fibres takes place. Instead the peripheral parts of the RNP fibres are packed into globular structures, which increase in size along the gene. In the terminal part of the active gene the globules attain about the same diameter (50 nm) as the large nuclear sap granules, which are likely to represent the completed and released RNP particles from the 75S RNA genes. Information on the density in situ of the growing RNP fibres (36 fibres/μm chromosome fibre axis) was used to estimate the DNA compaction of the chromosome fibre during transcription. Assuming the same fibre density as in the spread genes (10/μm DNA), we calculated that the DNA compaction of the chromosome fibre amounts to 3.6. The thin chromosome axis as well as the low DNA compaction figure imply that the nucleofilament is not supercoiled in the intensively transcribed 75S RNA genes. The chromosome fibre is in fact considerably more extended than a nucleofilament in non-transcribed chromatin (DNA compaction 5–7; micrococcal nuclease digestion experiments show a 189 base pair repeat for Chironomus polytene chromosomes). Our data on the properties of the chromosome fibre in situ are discussed in relation to the transcription process.