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 synthesis and immunological characterization of salivary proteins from Chironomus tentans

We have isolated the salivary proteins of the larva of the harlequin fly Chironomus tentans, and characterized its constituents by gel electrophoresis and immunological techniques. The detailed composition of saliva from individual animals is shown to be very variable, but four main protein groups can be defined. The largest, Fraction A, comprises up to five species, with molecular weights of between 820,000 and 700,000 Daltons. It includes at least two distinct antigenic species. This finding is discussed in the context of the known heterogeneity of the 75S RNA fraction which is transcribed in the Balbiani rings 1 and 2. — The other prominent protein classes in isolated saliva range in size from 230,000 down to less than 20,000 Daltons. — We have also employed antiserum against salivary proteins to investigate the products of in vitro translation of salivary gland RNA in the rabbit reticulocyte lysate system. A broad spectrum of polypeptide species is obtained which are immunologically related to salivary components, including species of over 300,000 Daltons. These latter are interpreted as unfinished Fraction A polypeptides resulting from incomplete translation of 75S RNA from BR1 and BR2. Evidence is presented to demonstrate that other salivary proteins, apart from Fraction A, are faithfully translated in the reticulocyte lysate.     

Isolation and characterization of the two giant secretory proteins in salivary gland of Chironomus tentans.

The two giant secretory proteins, sp-Ia and sp-Ib, in salivary-gland cells of the larva of the fly Chironomus tentans, were isolated by preparative gel electrophoresis and characterized chemically. Their amino acid compositions are dominated by polar amino acids, with about 30% of basic amino acid residues. Crossed immunoelectrophoresis of sp-Ia and sp-Ib provided evidence that they share antigenic determinants. They also have major methionine-containing tryptic peptides in common. CNBr cleavage of sp-Ib gives a small number of low-Mr fragments, indicating that this protein has a repetitive structure.     

Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with ³²P_i. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of ³²P) and not sedimentable (supernatant) material. About 90% of ³²P in the supernatant fraction was sedimentable at 100,000 g (disperse chromatin). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of ³²P. The ³²P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the ³²P-labeled nuclear proteins into 12 major bands in the M_r range of 12,000–120,000. The incorporation of ³²P into most bands reached a steady-state within 5–10 min of incubation with ³²P_i and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an M_r 95,000 phosphoprotein. Two of the fastest moving ³²P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving ³²P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.     

Ultrastructural and cytochemical studies of salivary gland regression in Chironomus tentans

Summary The ultrastructural localization of acid phosphatase (AcPase) activity in regressing salivary gland cells of Chironomus tentans was studied with Gomori's lead method. In last instar intermolt larvae AcPase activity is restricted to Golgi vesicles, to small electrondense bodies of about 0.25 diameter, and to larger, more electron-lucid bodies which are considered to be lysosomes. The smaller bodies apparently arise from Golgi vesicles. The average frequency of lysosomes increases as development proceeds. Until the end of the pupal molt, only very few of them contain degenerating fragments of other cellular components.Overt cell regression begins in young pupae. At this stage practically all lysosomes contain degenerating cell components. In addition, cellular breakdown seems to occur outside of these organelles. Regressing cellular areas show in addition free AcPase reaction products (lead deposits), the amount of which closely parallels the degree of regression of the particular area.Possible genetic relationships between the various AcPase-containing cell organelles and the role of lysosomes in the control of gland cell breakdown are discussed.Supported by NSF Grant GB-2639 to U. Clever. The technical assistance of Mr. Hermann Bultmann in part of these studies is gratefully acknowledged.     

Protein synthesis during salivary gland development,ageing, and cell death in Chironomus tentans larvae

Salivary gland protein synthesis in Chironomus tentans larvae was analyzed from the mid-third instar to larval pupation. Correcting for stage specific variations in the specific activity of the amino acid pool revealed a 30–40% reduction in the rate of protein synthesis during the larval moult. Except for a transient increase early in the fourth instar, this low rate of protein synthesis was maintained until the pharate pupal period when protein synthesis dramatically increased: maximum synthesis occurred in mid-pharate pupae with a subsequent decline correlating with gland autolysis and cell death at pupation. Each developmental period was characterized by a particular pattern of secretory protein synthesis: high and 35,000 daltons peptides were maximally synthesized only at particular larval stages, being reduced or absent in post-ecdysis, diapause and autolysing salivary glands.Except for the ecdysone puffs, and as otherwise previously noted (Clever, 1961, 1962), puffing activity during the peri-moult period remained relatively constant and did not decrease by the 30–40% predicted from the decreased rate of protein synthesis. The nearly complete loss in synthesis of the 35,000 daltons peptide was not accompanied by regression in any puff.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.     

Protein synthesis in the Chironomus thummi salivary gland

Summary Secretory proteins isolated from the lumen of the Chironomus thummi salivary gland were labelled with radioactive amino acids in vivo and in vitro. Under both conditions all but one of the electrophoretically separated fractions became labelled, the 6 prominent polypeptides already after 10–15 min of incubation. Differences in the labelling pattern during development from early 4th instar larvae to late prepupae were not detected.After synthesis the secretory proteins are stored in the cytoplasm for different times until they are exported into the gland lumen.None of the prominent protein fractions extracted only from the cells of the gland were found to be labelled even after labelling times up to 10 hrs. Therefore, it may be concluded that the Chironomus salivary gland synthesizes predominatly secretory proteins at least after the last larval moult.Long-time treatment of whole larvae with actinomycin D has no striking effect on the protein synthesis of the gland.Some of the results together with data from the literature led us to the speculation that changes of puff patterns (Balbiani rings excluded) do not reflect subsequent changes at the translational level.     

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     

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.     

Disassembly and reassembly in vitro of complexes of secretory proteins from Chironomus tentans salivary glands

The secretory proteins of Chironomus tentans larvae form insoluble fibers that are spun into threads used to construct underwater feeding and pupation tubes. We began in vitro studies of the mechanism of assembly into fibers, the structure of the assembled proteins, and the contribution of individual proteins to the assembled structure. From measurements of turbidity and electron micrographs, we observed that the secretory proteins were isolated as complexes. These complexes are most likely at initial stages of assembly; further assembly into insoluble fibers must occur in vivo. Denaturation and reduction disrupted the complexes, and removal of the denaturing and reducing agents resulted in reassembly of the complexes. The circular dichroic spectrum of the complexes indicated that the assembled proteins had the tertiary structure alpha + beta. The largest secretory proteins were purified and shown to have both similar morphology, using electron microscopy, and a similar dichroic spectrum to that of the native complexes. We concluded that the large secretory proteins form the fibrous backbone of the complexes that we observe.     

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.