Endre Egyházi

Electrophoretic characterization of nucleolar RNA from Chironomus tentans salivary gland cells

Abstract RNA has been extracted from micro-isolated nucleoli from salivary gland cells of Chironomus tentans and analysed by electrophoresis on agarose-acrylamide composite gel. The glands were labelled with tritiated nucleosides in vitro or in vivo. After a short period of labelling, heterodisperse RNA fractions of nucleolar origin were observed between 20 and 30 s. With increased incorporation time a monodisperse 38 s RNA component appeared, which was subsequently converted to one 30 and 23 s component. The fractions designated 38, 30 and 23 s incorporated methyl groups, indicating a relationship with ribosomal RNA. Any further transformation of 30 and 23 s did not take place within the nucleolus, during in vitro or in vivo incorporation. The 30 and 23 s RNA fractions are likely to be precursors to 28 and 18 s ribosomal cytoplasmic RNA. The pattern of formation of RNA compounds was closely similar in the two nucleolar organizers.

A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis

Abstract The nucleoside analogue 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole inhibits labelling of chromosomal, high molecular weight RNA in the salivary gland cells of Chironomus tentans but does not interfere with the synthesis of ribosomal RNA and chromosomal low molecular weight RNA. When DRB† was added after an initial labelling period (pulse-chase experiment) the radioactivity diminished preferentially in the lower molecular weight region of the HnRNA spectrum. After short chase periods the activity decreased moderately, or even increased, in the higher molecular weight region of the spectrum (75–100 S). After prolonged chases there was an overall and similar reduction in the activity in the whole HnRNA distribution. If the glands were preincubated in DRB for a short period before exposure to radioactive precursors, the label was again diminished more in HnRNA of low molecular weight than in that of higher molecular weight. When α-amanitin or actinomycin D, both known to be inhibitors of RNA chain elongation, replaced DRB in pulse-chase experiments, labelling of HnRNA was depressed in all size classes to the same extent. The accumulated data suggest that DRB acts, in explanted salivary gland cells, at the polymerase level by interfering with the initiation of chromosomal HnRNA synthesis.

A majority of casein kinase II α subunit is tightly bound to intranuclear components but not to the β subunit

Nuclear casein kinase II (CK II) was purified from an epithelial cell line ofChironomus tentans and characterized. The intracellular distribution of CK II and its two intracellular subunits (α and β) was analysed by immunoblotting. The apparent molecular weights of the α and β subunits were estimated to be 36 and 28 kDa, respectively. Like other purified CK II preparations, CK II fromChironomus tentans is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and by the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual α and β subunit pools could be detected. More than 85% of the total immunostainable α subunit and essentially all immunoreactive individual β subunit and heterooligomeric enzyme molecules were localised to the nucleus. Unexpectedly, more than 80% of this nuclear α subunit was insoluble in 0.35 M NaCl, while all individual β subunit and heterooligomeric enzyme molecules were solubilized under the same conditions. Of the 0.35 M NaCl soluble kinase fractions, the active multisubunit form of CK II precipitated in 50% (NH4)2SO4 and could thus be separated from the free β subunit, which precipitated at 60% and 80% (NH4)2SO4. These results suggest that a major portion of the nuclear CK II α subunit does not form heterooligomeric structures with the β subunit, but binds tightly to nuclear components.

Evidence for transport of preribosomal RNA from the nucleolus to the chromosomes in Chironomus tentans salivary gland cells

Abstract Chromosomal RNA from the salivary gland cells of Chironomus tentans has been analysed by gel electrophoresis on agarose-acrylamide composite gels. After short times of labelling (45 min), only heterodisperse high molecular weight RNA was observed. After increased incorporation times (90 to 180 min) first a 23 s and later also a 30 s monodisperse RNA fraction appeared in the chromosomes. The electrophoretic mobility and the kinetics of labelling indicate a nucleolar origin of these fractions, known to be formed from a 38 s RNA precursor in the nucleolus and likely to be precursors to ribosomal RNA.

Chromosomal RNA synthesis in polytene chromosomes of Chironomus tentans

The presence of heterogeneous RNA of high molecular weight has been demonstrated on the giant chromosomes, in the nuclear sap and in the cytoplasm of the salivary glands in Chironomus tentans. The kinetic properties of this heterogeneous RNA have also been outlined in some detail. — Salivary glands were incubated for different time intervals (20, 45 and 180 min) in haemolymph, supplemented with tritiated cytidine and uridine. The different cellular components were isolated by micromanipulation and RNA extracted with an SDS-pronase solution and analysed with electrophoresis in agarose. — Heterogeneous, high molecular weight RNA with a peak around 35 S was saturated with label on chromosome I, II and III in 45 min, although the synthetic capacity was unchanged during at least 180 min incubation. This indicated a complete turnover of heterogeneous RNA on the chromosomes in less than 45 min. The turnover time in the giant puffs (the so called Balbiani rings) on the fourth chromosome, was even shorter and estimated to less than 30 min. No shift in the electrophoretic pattern of this heterogeneous RNA was found to occur on the chromosomes during long incubation times or during actinomycin D experiments. These labelling characteristics of heterogeneous RNA on the chromosomes indicate that all the different molecules in the heterogeneous RNA have a similar and rapid turnover. A conversion to smaller, stable molecules was excluded. — Heterogeneous RNA of a distribution corresponding to that on the chromosomes was found in the nuclear sap and also in the cytoplasm. The activity in both these cellular compartments increased between 45 and 180 min incubation. The distribution pattern for high molecular weight RNA was in all experiments similar on the chromosomes, in the nuclear sap and in the cytoplasm. It appears that at least a considerable part of the high molecular weight RNA leaves the chromosomes to enter the nuclear sap and lateron to some extent the cytoplasm in this high molecular form. Stable molecules of smaller size (6–15 S) did not appear during 180 min incubation. The data indicate, however, also a substantial breakdown of heterogeneous RNA to acid soluble products during this time.

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.

Effects of anti-C23 (Nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells☆

Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with 32P-labeled RNA precorsors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of 32P incorporation into 38S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of 32P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of 32P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. When protein extracts of Chironomus tentans salivary gland nuclei were probed on Western blots with anti-C23 antibody the predominant cross-reacting species was a 110-kDa polypeptide which had an electrophoretic mobility similar to that of protein C23. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

Low molecular weight RNA in cell components of Chironomus tentans salivary glands

Abstract The labelled low molecular weight RNA of isolated nuclear components (nucleoli, chromosomes, nuclear sap) and cytoplasm of Chironomus tentans salivary gland cells has been characterized by electrophoresis in high percentage polyacrylamide gel. 4 s RNA as well as 5 s RNA was recovered from the three nuclear components. The results suggest that 4 s RNA is formed from RNA of slower migration rate in chromosomes, nuclear sap and cytoplasm. In the nucleolus, on the other hand, label appears immediately in the 4 s position and the profile does not show any evidence of shifts with aging in the migration properties of the fractions. The life-time for 4 s RNA was found to be relatively short in the nucleolus as compared to other nucleolar RNA fractions as well as to 4 s RNA in other cellular locations. Incorporation studies with l -[methyl-3H]methionine as methyl donor for the polynucleotides showed that the larger part of the label of the low molecular weight chromosomal, nucleolar and cytoplasmic RNA was in a 4 s position, the material in the 5 s and intermediate regions remaining unlabelled. In all the nuclear components there is a fraction of 5 s RNA which does not shift position with aging and which does not undergo methylation, possibly identical to the ribosomal 5 s component. Both chromosomes and nucleoli contain fractions in the range between 5 and 16 s, but these are lacking in the cytoplasm.

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.

Phosphorylation dependence of the initiation of productive transcription of Balbiani ring 2 genes in living cells.

Using polytene chromosomes of salivary gland cells of Chironomus tentans, phosphorylation state-sensitive antibodies and the transcription and protein kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol IIO) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol IIO is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of IIO: there is a time-dependent clearance from chromosomes of phosphoCTD (PCTD) after addition of DRB, which coincides in time with the completion and release of preinitiated transcripts. Furthermore, the staining of smaller transcription units is abolished before that of larger ones. The staining pattern of chromosomes with anti-CTD antibodies is not detectably influenced by the DRB treatment, indicating that hypophosphorylated pol IIA is unaffected by the transcription inhibitor. Microinjection of synthetic heptapeptide repeats, anti-CTD and anti-PCTD antibodies into salivary gland nuclei hampered the transcription of BR2 genes, indicating the requirement for CTD and PCTD in transcription in living cells. The results demonstrate that in vivo the protein kinase effector DRB shows parallel effects on an early step in gene transcription and the process of pol II hyperphosphorylation. Our observations are consistent with the proposal that the initiation of productive RNA synthesis is CTD-phosphorylation dependent and also with the idea that the gradual dephosphorylation of transcribing pol IIO is coupled to the completion of nascent pol II gene transcripts.