Balbiani ring nucleotide sequences in cytoplasmic 75 S RNA of Chironomus tentans salivary gland cells

The giant puffs, the Balbiani rings (BR) 1 and 2 of Chironomus tentans polytene chromosomes synthesize large RNA molecules sedimenting at about 75S. An RNA fraction of approximately the same size is present in nuclear sap and cytoplasm. In situ hybridization of cytoplasmic 75S RNA and other electrophoretically defined cytoplasmic RNA fractions showed BR RNA to be confined to the 75S RNA, and absent in other high molecular-weight cytoplasmic RNA fractions, which indiates that BR RNA is transferred from the nucleus into the cytoplasm without an appreciable size reduction.     

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.     

Balbiani ring RNA content and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells

The content of RNA with an origin in the Balbiani rings 1 and 2 (BR 1+2) has been determined in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. Together with information on rate and completeness of export this permits an estimation of half-life of this RNA in cytoplasm and its residence time in the nucleus. The quantities in the BR, nuclear sap, and cytoplasm are roughly related as 110200. The 75 S RNA in the nuclear sap with an origin in the BR 1+2 must to a high extent be a precursor to the cytoplasmic 75 S RNA in vivo. The half-life of the cytoplasmic component is about 20 h and the half-life (residence time) for BR 1+2 RNA in the nuclear sap around one hour. The presence of a large pool of BR RNA in the sap explains the previously observed delay in its cytoplasmic appearance in vivo.     

Identification of the spI products of Balbiani ring genes in Chironomus thummi

The spI fraction of high molecular weight secretory proteins was analysed in Chironomus thummi. These proteins are encoded by giant Balbiani ring (BR) genes which develop specifically in salivary gland cells. Each component of the spI fraction was studied electrophoretically from early and middle 4th instar larvae and prepupae, as well from galactose-treated larvae where changes in the relative puffing pattern of BR1 and BR2 are known to occur. The spI fraction consists of at least two bands with electrophoretic mobilities slower than those of the spI components of Camptochironomus. The slow migrating component remains throughout the 4th larval instar, while the amount of the faster component changes, being abundant in early 4th instar and prepupae, but not present (or very weak) in middle 4th instar. The correlated shifts in BR puffing pattern during these developmental stages suggest that the slow and fast components are encoded by BR2 and BR1. The spI fraction is modified by galactose treatment, the fast component being induced in parallel with a decrease in the slow component. These changes are correlated with changes in the steady-state levels of RNA: an increase in BR1 RNA and a decrease in BR2 RNA, and of proteins. These proteins could correspond to the spIb and spIa fractions allocated to BR2 and BR1, respectively, in Camptochironomus. After galactose treatment a new faster band sometimes appears, that could correspond to the spIc fraction of Camptochironomus. A possible spId equivalent was also identified. In conclusion the main features of the spI family in C. thummi are similar to those of spI in Camptochironomus.Abbreviations BR Balbiani ring - spI family of M_r=10⁶ secretory polypeptides     

Cell-Free translation of Balbiani ring RNA (75S) ofChironomus tentans salivary glands into high molecular weight products

Summary Cell-free translation of salivary gland RNA or of purified Balbiani ring RNA (75S) in a reticulocyte lysate system gives rise to high molecular weight translational products (HMTP). In addition to a common size (approx. 1×10⁶ daltons) HMTP share imunogenic determinants with the giant secretory proteins of salivary glands. This suggests that HMTP correspond to in vivo secreted proteins and thus, corroborates the notion that 75S-RNA is the messenger for these proteins. The time course of HMTP synthesis and the lack of appearance of lower molecular weight components as translational products of 75S-RNA indicate that the synthesis of HMTP (and of secretory proteins) occurs in one piece by an uninterrupted process. HMTP are regarded the largest polypeptides so far synthesized in a cell-free system.     

Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75S RNA

Polysomes from the salivary glands of Chironomus tentans were investigated to determine whether Balbiani ring 75S RNA is incorporated into polysomal structures, and thus probably acts as messenger RNA. A new extraction technique for obtaining ribonucleoproteins was applied that gives a high yield of polysomes with only moderate degradation of the cytoplasmic, high molecular weight RNA. The polysomes sedimented in a broad region (200-2,000S) with a peak value of about 700S, which suggested that they were partly of very large sizes. This was confirmed by visualization of the polysomes in the electron microscope: 400S polysomes contained mainly 11-16 ribosomes, and 1,500S polysomes about 60 ribosomes per polysome. However, polysomes containing 100 or more ribosomes were also observed. It was further established that most of the cytoplasmic 75S RNA was located in polysomes, preferentially in the most rapidly sedimenting ones. From the available information on Balbiani ring RNA in cytoplasm and the present demonstration of 75S RNA molecules in polysomes, it was concluded that at least some Balbiani ring RNA, generated as 75S RNA within the Balbiani rings, eventually enters polysomes without being measurably changed in size. The present information on the potential amino acid coding sequences in 75S RNA is discussed in relation to the large size of the polysomes observed.     

Cell-specific synthesis and glycosylation of secretory proteins in larval salivary glands of Chironomus thummi

Synthesis and glycosylation of larval salivary gland secretory proteins of Chironomus thummi were analyzed with respect to cell specific differences in the Balbiani ring (BR) pattern and glycoprotein composition of secretion formerly detected by histochemical staining procedures. In the secretion of a special cell type in salivary glands, which is characterized by the appearance of an additional BR, an additional polypeptide with a relative molecular weight (M_r) of 160 kD was found differing in its antigenic properties and tryptic fingerprint pattern from main cell secretion proteins. This so-called ssp-160 component is preferentially synthesized and glycosylated in the special cells. In the same cells, both the synthesis and glycosylation of all other major secretory proteins was found to be diminished or even repressed. In contrast to the conspicuous cell-specific differences at the level of protein synthesis, RNA analyses show the prominent synthesis of 75 S RNA in both main and special cells and gave no clear indication of the synthesis of a smaller RNA fraction as expected from the size of ssp-160 component. — These and further data on synthesis and properties of secretory proteins as well as expression of BR DNA are discussed with regard to the assumption that at least some of the eight major secretory polypeptides are coded for by BR DNA. The BR gene(s) might have originated by manifold duplications and modifications of short repetitive prototype DNA sequences, which are coordinatively expressed.On the occasion of the 60th anniversary of his birth-day we wish to dedicate this paper to Professor Wolfgang Beermann who was the first to detect, by the discovery of cell specific expression of BR 4 of Chironomus pallidivittatus salivary gland chromosomes and the concomitant occurrence of cell specific secretion granules, a causual relationship between the activity of a Balbiani ring and the appearance of a secretion component (Beermann, 1961)addressee for reprint requests     

Dependence of Balbiani ring induction in Chironomus salivary glands on inorganic phosphate

Galactose or certain other monosaccharides, administered for several days in the culture medium to larvae of Chironomus pallidivittatus, induce a new Balbiani ring, BR6, in their salivary gland chromosomes (W. Beermann, 1973, Chromosoma, 7, 198–259). This also applies to ethanol (Beermann, personal communication) and as found here, to glycerol. Induction of BR6 has previously been found to be paralleled by the appearance of one or two giant proteins (Ic₁ and Ic₂) probably deriving from allelic genes. We record here that the induction also includes the production of a new giant RNA species similar in size to the RNA from the Balbiani rings normally present, BR1 and BR2. Administration of inorganic phosphate together with glycerol prevented the appearance of BR6, as well as of the new RNA and component Ic protein(s); by contrast chloride and sulfate at similar concentrations did not prevent these effects. Administration of inorganic phosphate several days after the inducer and its continued presence reversed the effect of induction. Glycerol caused a marked depression in the level of inorganic phosphate in the hemolymph which persisted throughout its administration; the phosphate level in the glands was, however, unaffected. Inorganic phosphate administered together with the inducer at equimolar concentrations largely prevented the decrease in phosphate levels. It is concluded that a decrease in phosphate level is required for BR6 induction by glycerol. The two other inducers, galactose and ethanol, which were studied in less detail, seem to have a similar action.