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.     

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.     

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.     

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.     

IN SITU DEMONSTRATION OF DNA HYBRIDIZING WITH CHROMOSOMAL AND NUCLEAR SAP RNA IN CHIRONOMUS TENTANS

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chromosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2). Salivary glands were incubated with tritiated nucleosides. The labeled RNA was extracted from microdissected nuclei and hybridized to denatured squash preparations of salivary gland cells under conditions which primarily allow repeated sequences to interact. The bound RNA, resistant to ribonuclease treatment, was detected radioautographically. It was found that BR 2 RNA hybridizes specifically with the BR 2 region of chromosome IV. Nuclear sap RNA was fractionated into high and low molecular-weight RNA; the former hybridizes with the BR 2 region of chromosome IV, the latter in a diffuse distribution over the whole chromosome set. RNA from chromosome I hybridizes diffusely with all chromosomes. Nucleolar RNA hybridizes specifically with the nucleolar organizers, contained in chromosomes II and III. It is concluded that the BR 2 region of chromosome IV contains repeated DNA sequences and that nuclear sap contains BR 2 RNA.     

Large sized nascent protein as dominating component during protein synthesis in Chironomus salivary glands

The main secretory protein fractions from Chironomus tentans have been investigated with particular emphasis on the dominant fraction, component 1, here designated I (Grossbach, 1969). This polypeptide was suggested to be the translatory product of 75 S RNA from Balbiani ring 2 (BR2) because of its size and quantitative prominence. Its molecular weight was estimated by gel filtration in 8 M urea at 850,000+101,000 D. During short pulses with radioactive amino acids a large fraction of the label was found in a population of polypeptide chains suggestive of molecules continuously growing to the size of component I. Populations of nascent large protein chains of similar size distribution were dominant in the polysomes and constituted the only population present in the largest polysomes, known to contain 75S RNA from BR2 (and BR1) as predominant or only component (Daneholt et al., 1977; Wieslander and Daneholt, 1977). These data indicate strongly that the large size of component I is not a result of posttranslational modifications. No sequence similarities, using limited proteolysis, were found between component I and component II, both of which have been considered to be BR2 products. There was, furthermore, no detectable immunological identity between component I and smaller secretory protein fractions. The data support Grossbach's and Daneholt's suggestion that component I is closely related to the primary translation product of 75S RNA from the large Balbiani rings.     

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.     

The activity of Balbiani rings 1 and 2 in salivary glands of Chironomus tentans larvae under different modes of development and after pilocarpine treatment

The activity of the Balbiani rings 1 and 2 (BR1 and BR2) in the salivary gland was followed during development of fourth instar larvae of Chironomus tentans under different modes of development, with or without a previous pilocarpine treatment. The activity was determined in parallel by two different methods, by incorporation of [3H]uridine into BR-RNA (75 S) and by morphometry of BR1 and of BR2. In glands of untreated larvae BR2 does not change dramatically except for a depression of activity during oligopause (resting phase). BR1 is completely inactive during this phase but exhibits a pronounced activity maximum in the middle of the prepupal period, in subitaneously developing (i.e., uninterrupted) as well as in postoligopause cultures. After pilocarpine treatment the activity of BR2 (rather than of BR1) is generally increased. The extent of this stimulation, however, is strongly development dependent. A most striking activity difference is observed in postoligopause between animals of stage 5 and of stage 6. The relationship between BR2 activity and degree of emptying of the salivary gland lumen was investigated. A model is proposed in which BR2 activity is conceived as being regulated by two parameters: by the degree of filling of the gland lumen and by the stage and mode of development of the larva.