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.     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.     

Sequences translated by Balbiani ring 75S RNA in vitro are present in giant secretory protein from Chironomus tentans

The 75S RNA originating in the large Balbiani rings 1 and 2 (BR1 and 2) was isolated and used for in vitro translation in the mRNA dependent reticulocyte lysate. Conditions (K⁺-concentration, temperature, time etc), were optimized for obtaining translation products of maximal size. Polypeptide chains up to about 500,000 D were obtained but no complete translation products. Tryptic fingerprints were performed on the in vitro products as well as on the secretory protein components nos. I and II+III labelled with ³⁵S-methionine. There was a large degree of correspondence between the fingerprint of the in vitro product and that of component I but less to that of component II+III. The results suggest that 75S RNA with an origin in the BR1 and BR2 codes for the giant secretory protein component I.     

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.     

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.     

Delayed flow-through cytoplasm of newly synthesized Balbiani ring 75S RNA.

With a nonaqeous microdissection technique, the cytoplasm of Chironomus salivary gland cells can be separated into concentric zones situated at increasing distances from the nuclear envelope. This dissection technique is used here to investigate the cytoplasmic distribution of 75S RNA of Balbiani ring origin. The Balbiani ring 75S RNA has properties of a messenger RNA coding for secretory proteins. After a pulse of RNA precursor to the living animal, labeled Balbiani ring 75S RNA is found mainly in the cytoplasm located closer to the nuclear envelope, with smaller amounts toward the periphery of the cell. This gradient, initially very steep, lasts for a least 2 days, but less than 6 days. Experiments with 5-fluorouridine indicate that the formation of the gradient does not depend upon simultaneous export of ribosomal subunits. After a pretreatment of the animals with the protein synthesis inhibitor cycloheximide, however, newly synthesized 75S RNA distributes evenly in the cytoplasm-that is, this treatment prevents the formation of the 75S RNA gradient. The gradient in salivary glands of normally cultured animals is therefore likely to be the result of diffusion restriction of the labeled 75S RNA. Thus the 75S RNA located closer to the nuclear envelope is the most recently exported 75S RNA. An explanation of these results is the the 75S RNA associates with the membranes of the endoplasmic reticulum early or immediately after nuclear release. This association should occur in the cytoplasm surrounding the nucleus and may occur either as single particles and/or as parts of polysomes.     

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.     

Nuclear localization of a DNA-binding C-terminal domain from Balbiani ring coded secretory protein.

All known Balbiani ring (BR) genes in Chironomus tentans, coding for giant secretory proteins, the sp-I family, end with a short (110 codons) 3'-end exon which is highly conserved in evolution and is structurally unrelated to the sequences characterizing the core of these proteins. We find that the expressed product, the C-terminal domain, shows sequence-specific DNA binding and that it is likely to be absent in one of the sp-I components, sp-Ib, believed to be coded by the BR2.2 gene. Immunohistochemistry shows that material with reactivity towards antibody against the C-terminal domain is present in the nuclei, and specifically enriched in Balbiani ring 1 and 2. Western blotting of extracts from isolated nuclei demonstrates a component with the same antibody reactivity and of an apparent size somewhat larger than that of the domain. The possibility is discussed that the C-terminal part, which is part of the secretion when derived from some of the BR genes, might be cleaved off and function as a feedback signal to control BR gene activity when derived from the BR2.2 gene.     

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.     

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.