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.     

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.     

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.     

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.     

Effects of -amanitin on in vitro labeling of RNA from defined nuclear components in salivary gland cells from Chironomus tentans

The effect of α-amanitin on nucleoside labeling of RNA in nucleoli, chromosomes, nuclear sap, and cytoplasm from Chironomus tentans salivary gland cells was investigated by radioautography and gel electrophoresis. Preribosomal RNA formation and processing in the nucleolus was not measurably influenced by the drug, and both 28 S and 18 S ribosomal RNA were transferred to the cytoplasm. In the chromosomes the heterogeneous RNA labeling was completely inhibited for the large size range (above 45–50 S) and partially for the low range. The labeling of 4–5 S chromosomal RNA was only moderately reduced. Most of the chromosomes showed radioautographically a disappearance of the normal band pattern, but some retained a pattern of weakly labeled bands. The electrophoretic results for the nuclear sap paralleled those for the chromosomes. The effect of α-amanitin on RNA labeling in these cells is similar but not identical to that of the substituted benzimidazole 5,6-dichloro-1(β-D-ribofuranosyl) benzimidazole (DRB).     

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.     

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.     

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.     

Electron microscopic visualization of a discrete class of giant translation units in salivary gland cells of Chironomus tentans

Lysates were made of whole salivary glands from Chironomus tentans and electron microscopic spreads prepared according to Miller and Beatty (1969). The sedimented material consisted mainly of ribosomes, most of which were present in giant polysomes which showed gradients of material protruding laterally from the ribosomes, interpreted as nascent polypeptide chains. Each giant polysome contained one such gradient. On the basis of the presence of a small group of terminal ribosomes without nascent chains at the 3' end, 19 apparently complete polysomes were selected, representing a discrete class of polysomes containing between 66 and 92 ribosomes (79 +/- 7, m +/- s.d.). Arguments are presented that they constitute the translation units for giant secretory proteins coded by RNA from the large Balbiani rings, BR1 and BR2.     

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.     

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.