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).     

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.     

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.     

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.     

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.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

RNA synthesis in isolated polytene nuclei from Chironomus tentans

An RNA synthesizing system with isolated polytene nuclei from Chironomus tentans is described. This system allows one to monitor the effect of salt concentration on chromosome structure and to assign in vitro RNA synthesis to structural modifications of the chromosome (i.e. nucleoli, Balbiani rings and puffs).-At a salt concentration of 0.15 M monovalent cations (standard salt medium=SSM) chromosomal structure appears to be best preserved during in vitro incubation. At low and high ionic strength the bands decondense and the microscopically visible chromosomal structure is lost completely. These three states of condensation and decondensation are distinguished with respect to RNA synthesis: (1) in low salt overall RNA synthesis is depressed, (2) in SSM ribosomal RNA synthesis predominates and continues for 30 min, (3) in high salt RNA synthesis is stimulated 3–4 fold again. This stimulation is due solely to chromosomal, non-ribosomal RNA synthesis, which proceeds in high salt for more than 10 h, though new initiation of RNA chains is prevented. Molecular weight determinations of the RNA synthesized demonstrate a time dependent increase in size of the newly synthesized molecules under these conditions. — Autoradiographs of nuclei incubated in SSM reveal prominent label in nucleoli, significant label in Balbiani rings and rather reduced activity at other sites. Addition of various exogenous RNA polymerases does not markedly alter this pattern. Autoradiographs of nuclei incubated in high salt exhibit extensive RNA synthesis spread over the chromosomes. Preparations of autoradiographs from isolated chromosomes show that the high salt induced label is localized in single bands. Though the majority of bands is still unlabelled, the actual number of bands exhibiting incorporation in high salt is higher than in any individual functional state in vivo. These results are discussed in terms of activated and preactivated genes.     

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.     

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.     

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.     

The distribution of DNA and RNA in salivary gland chromosomes of Chironomus tentans as revealed by fluorescence microscopy

Summary In acridine orange stained chromosomes of Chironomus tentans, ribonuclease-sensitive reddish-orange fluorescence is found in all bands and in all interband regions as well as in nucleoli and Balbiani rings.Following ribonuclease digestion, deoxyribonuclease-sensitive yellowish-green fluorescence is found in all bands and in all interband regions. Banded fibres, apparent in Balbiani rings and in nucleoli, and formed by the splitting of the chromosome axis, also show no evidence of discontinuities in their yellowish-green fluorescence. From these results it is concluded that DNA is present in interbands and (at least at the level of the light microscope), is continuous through these regions of polytene chromosomes.     

Physico-chemical properties of chromosomal RNA in Chironomus tentans polytene chromosomes

Labelled chromosomal RNA of the dipteran Chironomus tentans was studied with respect to its migration properties during electrophoresis in agarose. The RNA was isolated from polytene chromosomes which had been microdissected from fixed salivary glands and obtained free from nucleoli and nuclear sap. Labelled material migrates as 4–5 S RNA and as polydisperse material in a range where the lower limit corresponds to 10–15 S, the upper limit to 80–90 S RNA and the maximum in the distribution to 30–40 S RNA. The data indicate that the latter fractions are formed by unbroken, single-stranded RNA molecules, partly of very high molecular weights. It is shown in a number of tests that the distribution is not a consequence of formation of complexes or aggregates between RNA molecules on one hand and DNA, proteins or other RNA molecules on the other.     

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.