Characterization of active transcription units in Balbiani rings of chironomus tentans

Abstract

Specific active transcription units on chromosome IV in the salivary glands of Chironomus tentans have been visualized by the Miller spreading technique and in situ by conventional electron microscopy. These units are likely to be located in the two most conspicuous puffs on chromosome IV, Balbiani ring 1 (BR 1) and Balbiani ring 2 (BR 2). The transcription units in these Balbiani rings generate 75S RNA molecules constituting putative messenger RNA species for the predominant cellular product, the salivary polypeptides. Solitary active transcription units with a mean length of 7.7 μm were observed most frequently. The lateral ribonucleoprotein (RNP) fibers of each unit formed a single length gradient. The number of fibers per unit was 123 (± 24), or about 16 growing RNP fibers per μm of chromosome fiber. The considerable variation in the number of RNP fibers per unit suggests that transcription can be modulated at the level of the individual gene. The modulation is probably achieved via the initiation event and/or via an early pretermination step, but a change in the elongation rate could not be excluded. The number of polymerases starting to traverse the whole gene was estimated to be six per min and transcription unit, and the rate of RNA chain elongation was calculated to be 31 nucleotides per second at 18°C. The properties of the chromosome fiber within the active 75S RNA units and also within their vicinity were studied in the Miller spreads. The inactive chromosome fiber exhibited a uniform beaded conformation, while the active fiber was sparsely and irregularly beaded. Furthermore, the chromosome fiber was more extended in the active 75S RNA unit than in inactive regions (DNA packing ratios of 1.6 and 1.9, respectively). By comparing the properties of the active 75S RNA gene with those of active genes in other systems, it was inferred that the loss of beads and the extension of the fiber in the active unit is probably directly related to the level of transcriptive activity. Finally, a smooth nonbeaded segment of 0.18 μm in length was found to precede the RNP fiber gradient. This segment may have a role in the process of transcriptional regulation. On the basis of comparison with the active transcription units in spread preparations, it was possible to identify active units in the Balbiani rings in sectioned material using conventional electron microscopy. In both BR 1 and BR 2 an active unit appeared as a loop, consisting of a fiber axis and having RNP granules attached to the loop axis by stalks. The growing RNP fibers therefore seem to be organized into granular structures during the transcription process, and the final products in BR 1 and BR 2 are granules, 500 Å in diameter, each containing a 75S RNA molecule.