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.     

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.     

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.     

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.     

Correlated changes in steady-state levels of Balbiani ring mRNAs and secretory polypeptides in salivary glands of Chironomus tentans

Balbiani rings (BRs) on polytenized chromosomes in Chironomid salivary glands contain members of a homologous multigene family that encodes a family (the sp-I family) of high M _r secretory polypeptides. Each of these BR genes is comprised largely of tandemly duplicated core repeat sequences consisting of related constant (C) regions and intergenically divergent subrepeat (SR) regions. A set of oligodeoxyribonucleotide probes were synthesized that correspond to the transcribed strand of the SR region of BR1, BR2, BR2/, and BR6 core repeats. Under a defined set of conditions, it was possible to show that each oligonucleotide probe hybridized exclusively to its cognate repeat type without hybridization to other repeat types in cloned DNA templates. These BR probes were then used in dotblot hybridization experiments to simultaneously follow alterations in the steady-state level of BR mRNAs in response to prolonged exposure of larvae to galactose. The results indicated that the relative amounts of these four BR mRNAs may change in a noncoordinate manner. These BR probes were also used in experiments to compare simultaneously the salivary gland content of sp-I components and specific BR mRNAs in larvae that exhibited naturally occurring or induced alterations in BR gene expression. A correlation was found which suggested that sp-Ia is encoded in a gene comprised of BR1 repeats, sp-Ib is encoded by BR2 repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2 repeats.     

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.     

Effects of ligation on ethanol-induced Balbiani ring puffing in salivary glands of Chironomus

Treatment of Chironomus larvae with dilute (0.5%–1.0%) ethanol results in puffing changes similar to those obtained with galactose in the Balbiani rings (BRs) of the salivary gland chromosomes. A shift in the relative size of BR1 and BR2 in chromosome 4 of C. pallidivittatus or C. tentans was observed within 1–2 days after ethanol treatment. The exceptional Balbiani ring, BR6 in chromosome 3, began to appear within 1 day after ethanol treatment of C. pallidivittatus and was fully developed after 3–4 days. Prepupae appeared to be refractory to the treatment. To localize possible controls of BR puffing in Chironomus, ligatures were made at various positions along the thorax and the anterior abdominal segments of the ethanoltreated larvae. In surviving larvae, ligated anterior to the brain or posterior to the salivary glands, induction of BR6 could be detected. In contrast, little or no BR6 puff induction was found in animals ligated in the middle of the second segment approximately between the brain and the salivary glands. No shift in the BR1/BR2 relation occurred with any of the ligations combined with ethanol treatment.     

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.     

Balbiani ring 6 gene in Chironomus tentans: a diverged member of the Balbiani ring gene family

We describe the internal organization of a large part of the Balbiani ring (BR) 6 gene in Chironomus tentans. The BR6 gene is a diverged member of the BR gene family. It displays the characteristic hierarchic organization of repetitive sequences, but in the constant region of the repeat units the overall sequence homology is only 49% when compared to other BR genes. All four cysteines are among the few amino acids conserved in the constant region. In the subrepeat region the central part is built from a repeated tripeptide, Pro-Glu--Arg+. A similar charge distribution adjacent to prolines is found in other BR gene subrepeat regions, most pronouncedly in the BR2-encoded protein. These conserved properties of the BR gene products are relevant to the issue how the various BR gene products interact to form a supramolecular structure, the larval tube, and how functional demands influence the evolution of a eucaryotic gene family.     

Correlated changes of Balbiani ring expansion and secretory protein synthesis in larval salivary glands ofChironomus tentans

Salivary glands of various stages of the last larval instar ofChironomus tentans were quantitatively analyzed with respect to the expansion of their Balbiani rings (B1, B2, B3) by a fast green staining procedure as well as to the rate of synthesis of their secretory proteins (S1, S2, S3) by a scintillation counting procedure of electrophoretic fractions. The extent of expansion of B1, B2 and B3 correlates positively with the rate of synthesis of S3, S2 and S1, respectively. With B1 and S3 these parameters undergo a parallel and developmentally specific change being rather depressed in intermolt, and particularly in diapausing animals.The material published in this paper is taken from the unpublished Doctorate Thesis of W. Pankow (1973): Entwicklungsspezifische Balbianiring-Aktivität und Sekretproteinsynthese in Speicheldrüsen von Chironomus tentans. Diss-Nr. 5166. Eidgenöss. Techn. Hochschule, Zürich; pp. 1–60. However, parts of it have been evaluated or presented in a different form     

Demonstration of Balbiani ring RNA sequences in polysomes

A polysome extract from salivary glands of C. tentans was sedimented in a 15-60% sucrose gradient. Fractions from the heavy polysome region (1,000-2,000S) and fractions from the light polysome region (200- 1,000S) were pooled separately, and the long-term labeled RNA was released by Sarkosyl/pronase and analysed by in situ hybridization. The results showed that BR 1 and BR 2 sequences were present in the heavy and the light polysome regions of the sucrose gradient. From control experiments with EDTA-treated extracts, it was concluded that most of the recorded BR 1 and BR 2 sequences were in fact located in polysomes. The finding that BR products enter polysomes suggests that they act as messenger RNA molecules. This study therefore strongly supports the concept that chromosome puffs represent active genes.