Genotypic control of chromosome pairing in Avena longiglumis × A. sativa hybrids

In normal fourth larval instar Chironomus larvae, the secretory protein component I (or 1, according to Grossbach, 1969) consists of two subfractions, Ia and Ib, with an average molecular weight of 850.000 D (Rydlander and Edström, 1980). Data in the preceding paper suggest that component I is coded for by 75S RNA derived from the two large Balbiani rings, BR1 and BR2 (Rydlander et al., 1980). If Chironomus pallidivittatus larvae are exposed to galactose, the size relations between BR1 and BR2, which are usually in favour of BR2, are inverted and upon prolonged exposure a new BR, BR6, appears (Beermann, 1973). Here we describe how one or two new subfractions within component I, Ic₁ and Ic₂, appear during treatment with galactose, in parallel with the development of BR6. During the treatment there is also a change in the ratio between Ia and Ib proteins so that Ia becomes dominant, whereas in controls Ib is more pronounced. Fractions Ia, Ib and Ic are at least partially immunologically different but Ic₁ and Ic₂ cannot be distinguished from each other. Since the relative amounts of Ic₁ and Ic₂ do not vary in extracts from single animals, we have assumed that they represent alle lic products. —Fraction Ic can become the dominating protein within component I during galactose treatment. Since component I accounts for about 50% of the total protein synthesis, the sugar treatment is accompanied by major quantitative changes in genetic expression. —The correlation between the occurrence of particular Balbiani rings and protein fractions, evident from measurements of either protein mass or amino acid incorporation remains in agreement with the general relation earlier shown to exist between the large Balbiani rings and the total component I. Our data support the hypothesis that BR1 codes for fraction Ia, BR2 for Ib and BR6 for Ic. Conclusive evidence will, however, have to be provided by molecular techniques.     

Induction of balbiani ring puffing changes by sugars and alcohols in Chironomus thummi

The capacity of several substances to induce in Chironomus thummi larvae the repression of Balbiani ring 2 (BR2) and the coordinated activation of Balbiani ring 1 (BR1) has been evaluated. Hexoses as well as some disaccharides, except lactose, were efficient inducers. The pentoses tested failed to provoke puffing changes. Alcohols, such as ethanol and glycerol, showed a moderate capacity as inducers, whereas methanol and isopropanol were ineffective. When a mixture of galactose and glycerol was used, a significant acceleration of the response, at the puffing as well as the translational level, was observed.To search for common effects of different inducers, an attempt was first made to test whether the lowering of the inorganic phosphate level in the haemolymph could play a causal role in the shift of BR1/BR2 puffing as it does in the response of the Camptochironomus group. In C. thummi, we have found effective inducers which increased, decreased or did not modify the inorganic phosphate concentration in the haemolymph thus suggesting the independence of the induced puffing changes and phosphate level modifications.     

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 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-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     

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     

The repetition frequency of DNA in Balbiani ring 2 of Chironomus thummi

The RNA of Balbiani ring BR2 of polytene chromosomes from Chironomus thummi salivary glands was microisolated and reassociated in the presence of an excess of total larval DNA. BR2 RNA reacts as a single component with a C₀t_1/2 of 8.6. Ribosomal precursor RNA from microisolated nucleoli reassociates under identical conditions with a C₀t_1/2 of 12.3. These C₀t_1/2-values suggest repetition frequencies in the range of 35 and 50 for ribosomal DNA and Balbiani ring 2 DNA, respectively. The data presented here favour the view that the gene for BR2 RNA of C. thummi is internally repeated and contains only one type of DNA sequence.     

Galactose-induced puffing changes inChironomus thummi Balbiani rings and their dependence on protein synthesis

InChironomus thummi, puffing changes induced by galactose treatment (sugar effect) are restricted to the Br1/BR2 (Balbiani ring) system. No obvious induction of additional BRs such as BR6 inCamptochironomus pallidivittatus occurs. The response to feeding galactose (or other sugars), i.e. BR2 regression and concomitant BR1 activation, usually takes 24–48 h but can be accelerated somewhat by the application of two 6 h galactose treatments separated by an 18 h interval without sugar. In the special cells composing the lateral lobe of the salivary gland galactose causes regression on BR2 without concomitant BR1 activation which, however, appears delayed. The autonomous collapse of BR2 therefore could be considered as the primary effect of galactose at the puffing level. On the other hand, inhibition experiments performed with cycloheximide (CHM) emphasize the relevance of translational events in the control of the sugar effect. At highly inhibitory doses, CHM prevents the induction or causes reactivation of galactose-repressed BR2, suggesting that both induction and maintenance of the galactose effect are dependent on newly synthesized proteins. Present address: Departamento de Biología Cellular y Fisiología, Universidad Autónoma de Barcelona, E-08193, Barcelona, Spain     

Phosphate incorporation into secretory protein of Chironomus salivary glands occurs during translation

The Balbiani rings (BR) in Chironomus salivary gland cells code for giant secretory proteins, the sp-I family. During normal growth conditions the phosphorylated proteins sp-Ia and sp-Ib are formed with most phosphate present as phosphoserine. We can show that most if not all incorporation of ³²P into sp-I occurs in parallel with the incorporation of [³⁵S]-methionine in the giant polysomes that form sp-I and contain BR-derived mRNA. We suggest that the main function of phosphorylation of sp-Ia and sp-Ib is to provide charge neutralization of an excess of lysine and arginine residues and is therefore required during early stages of protein folding. This view is supported by the previous observation that glutamic (and aspartic) acid largely substitute for phosphoserine in a non-phosphorylated member of the sp-I family, sp-Ic, which is produced during phosphate starvation.