Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.     

The complexity of 75S premessenger RNA in balbiani ring granules studied by a new RNA band retardation assay.

Under normal growth conditions, Balbiani ring granules constitute premessenger ribonucleoprotein (RNP) particles synthesized in two chromosomal puffs, Balbiani ring (BR) 1 and 2, in the larval salivary glands of Chironomus tentans. At least three genes encoding 75S RNA are present in these two BRs: one in BR1 and two in BR2 (BR2.1 and BR2.2). The complexity of BR granule 75S RNA was studied by agarose gel electrophoresis under non-denaturing conditions. We recorded three main bands, designated I, II and III. Experiments with denaturing gels demonstrated that the differences in migration reflected mainly, but not exclusively, conformational differences. Northern blotting experiments showed that band I contained BR1 sequences, band II contained BR2.1 sequences, and band III contained BR2.2 sequences. To study whether additional genes contributed to the BR granule 75S RNA, an RNA band shift assay was developed. When an oligodeoxyribonucleotide complementary to repetitive BR1 and BR2.2 sequences was hybridized to 75S RNA prior to electrophoresis, bands I and III were retarded but not band II. An oligonucleotide complementary to a repetitive BR2.1 sequence only shifted band II. Since no detectable 75S RNA remained unchanged in these experiments, and all bands were identified by Northern blotting, all the BR granules are likely to originate from the BR1, BR2.1 and BR2.2 genes; no additional genes have to be invoked. Possible applications of the new RNA band shift assay are discussed.     

The distribution of DNA and RNA in salivary gland chromosomes of Chironomus tentans as revealed by fluorescence microscopy

Summary In acridine orange stained chromosomes of Chironomus tentans, ribonuclease-sensitive reddish-orange fluorescence is found in all bands and in all interband regions as well as in nucleoli and Balbiani rings.Following ribonuclease digestion, deoxyribonuclease-sensitive yellowish-green fluorescence is found in all bands and in all interband regions. Banded fibres, apparent in Balbiani rings and in nucleoli, and formed by the splitting of the chromosome axis, also show no evidence of discontinuities in their yellowish-green fluorescence. From these results it is concluded that DNA is present in interbands and (at least at the level of the light microscope), is continuous through these regions of polytene chromosomes.     

RNA synthesis in a Balbiani ring in Chironomus tentans salivary gland cells

Rapidly labelled RNA in Balbiani ring 2 on chromosome IV in the salivary glands of Chironomus tentans was investigated. This RNA is likely to be transcribed from only one chromosomal band, supposed to be a single operational unit in these polytenic cells (Beermann, 1966).Salivary glands were incubated in larval haemolymph, supplemented with tritiated RNA precursors and fixed afterwards. Balbiani rings 2 (in some experiments also Balbiani ring 1 and 3) were isolated with micromanipulation. The labelled RNA was extracted with SDS-pronase and analysed with electrophoresis in agarose.The rapidly labelled RNA in Balbiani ring 2 was as heterogeneous as RNA from the remainder of the chromosome set (10–90 S) but the peak of the distribution of label in BR 2 corresponded to molecules of about 50 S as compared to that of RNA from the rest of the chromosome set which was about 35 S. When the synthetic activity in Balbiani ring 2 was very high, relatively more molecules with very high molecular weights were produced compared with the state when the synthetic activity was moderate or low. The synthetic activity in Balbiani ring 2 compared to that in Balbiani ring 1 was well correlated to the relative sizes of the two Balbiani rings. The results on Balbiani ring 2 are discussed in relation to the size and structure of the chromomere.     

Heat-shock induced puffing changes in Balbiani rings

A 5 minutes exposure of Chironomus larvae to near-lethal temperatures (39–40° C) produces a characteristic sequence of puffing changes in the Balbiani rings of the 4th salivary gland chromosomes: An initial phase of complete puff regression is followed, after a variable time lag, by a phase of rapid recovery to overnormal puff sizes. This is accompanied by RNP droplet formation. RNA synthesis at Balbiani rings during the initial puff regression still occurs. Regression is inhibited by 2,4-dinitrophenol, while recovery can be prevented by both 2.4dinitrophenol and actinomycin D. Regression and recovery are insensitive to cycloheximide. RNP droplets, as observed in Balbiani rings, in the nuclear sap and at the nucleolar rim, are composed of a fine fibrillar matrix which is covered by Balbiani ring granules in various phases of assembly. The results are discussed in terms of a model of puffing based on an equilibrium between RNA synthesis, RNA processing and RNP release from the puff.

     

Heat-shock induced puffing changes in Balbiani rings

A 5 minutes exposure of Chironomus larvae to near-lethal temperatures (39–40° C) produces a characteristic sequence of puffing changes in the Balbiani rings of the 4th salivary gland chromosomes: An initial phase of complete puff regression is followed, after a variable time lag, by a phase of rapid recovery to overnormal puff sizes. This is accompanied by RNP droplet formation. RNA synthesis at Balbiani rings during the initial puff regression still occurs. Regression is inhibited by 2,4-dinitrophenol, while recovery can be prevented by both 2.4dinitrophenol and actinomycin D. Regression and recovery are insensitive to cycloheximide. RNP droplets, as observed in Balbiani rings, in the nuclear sap and at the nucleolar rim, are composed of a fine fibrillar matrix which is covered by Balbiani ring granules in various phases of assembly. The results are discussed in terms of a model of puffing based on an equilibrium between RNA synthesis, RNA processing and RNP release from the puff.