Adenosine selectively inhibits labeling of chromosomal RNA, especially hnRNA, probably by acting at or near the site of chain initiation.

The effects of adenosine on labeling of nucleolar preribosomal RNA, chromosomal plus nuclear sap hnRNA, and 4-5S RNA in explanted salivary gland cells of chironomus tentans has been studied. Of chromosomal transcripts it is the labeling of polymerase II-promoted RNA that is interrupted preferentially, but 4-5S RNA is influenced as well. The labeling of hnRNA and 4-5S RNA is diminished by 70-90 percent and 45-60 percent, repectively, while the incorporation into the nucleolar preribosomal RNA remains essentially unchanged. Labeled adenosine is transported efficiently across the plasma membrane and becomes phosphorylated to AMP, ADP, and ATP, of which ATP predominates at noninhibitory concentrations. The rate of the formation of [(3)H]AMP is, however, enhanced in response to the increase in external adenosine doses, whereas that of [(3)H]ATP increases only slowly or remains essentially unaltered. A rise in exogenous [(3)H] adenosine concentration to 200 muM yields a [(3)H]ATP/[(3)H]AMP ratio that is about one order of magnitude lower than that at 20 muM of the nucleoside. In parallel with this, there is a gradual repression of the labeling of chromosomal RNA. A similar treatment with guanosine produces only minor reduction in GTP/GMP quotient and does not influence significantly the labeling of any sizable RNA fraction. Thus the experimental data strongly indicate that the purine ribonucleoside adenosine, but not guanosine, gives rise to a markedly diminished triphosphate/monophosphate quotient simultaneously with a selective suppression of the labeling of chromosomal RNA, especially hnRNA, when applied in overdoses. The sequence of hnRNA events during inhibition by adenosine resembles the effect of the purine nucleoside analogue 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole, indicating that the site of inhibitory action is at or close to the initiation of transcription.

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

The effect of alpha-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 alpha-amanitin on RNA labeling in these cells is similar but not identical to that of the substituted benzimidazole 5,6-dichloro-1(beta-D-ribofuranosyl) benzimidazole (DRB).

Phosphorylation of some chromosomal nonhistone proteins in active genes is blocked by the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The distribution of rapidly phosphorylated chromosomal proteins between chromosome I, chromosome II + III, chromosome IV, and nuclear sap including the matrix was investigated in salivary gland cells of Chironomus tentans. Chromosome IV, which carries most active nonribosomal genes in the cell, was found to be enriched in four rapidly phosphorylated nonhistone polypeptides (Mr = 25,000, 30,000, 33,000, and 42,000) in parallel with the transcriptional activity rather than with the DNA content of the chromosome. Also the histones H2A and H4 are rapidly phosphorylated but the phosphorylation is proportional to the DNA content of each chromosome sample. The 32P-labeled Mr = 42,000 polypeptide immunologically cross-reacted with an antibody elicited against the transcription stimulatory factor S-II isolated from Ehrlich ascites tumor cells (Sekimizu, K., D. Mizuno, and S. Natori, 1979, Exp. Cell Res., 124:63-72). In addition, indirect immunofluorescence studies on chromosome IV with antisera against the stimulatory factor II revealed a selective staining of the active gene loci. The incorporation of 32P into three chromosome IV nonhistone polypeptides, especially into the Mr = 42,000 polypeptide, was lowered by 70-85% shortly after administration of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a likely inhibitor of heterogeneous nuclear RNA transcription at initiation level. The possibility of a causal relationship between inhibited phosphorylation of chromosomal proteins and blocked transcription of heterogeneous nuclear RNA genes by DRB is discussed.

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.

Intracellular distribution of low molecular weight RNA in Chironomus tentans.

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4-5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10(5) and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10(5) and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10(4) and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (beta-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4-5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

Purified anti-topoisomerase I immunoglobulin G (IgG) was microinjected into nuclei of Chironomus tentans salivary gland cells, and the effect on DNA transcription was investigated. Synthesis of nucleolar preribosomal 38S RNA by RNA polymerase I and of chromosomal Balbiani ring RNA by RNA polymerase II was inhibited by about 80%. The inhibitory action of anti-topoisomerase I IgG could be reversed by the addition of exogenous topoisomerase I. Anti-topoisomerase I IgG had less effect on RNA polymerase II-promoted activity of other less efficiently transcribing heterogeneous nuclear RNA genes. The pattern of inhibition of growing nascent Balbiani ring chains indicated that the transcriptional process was interrupted at the level of chain elongation. The highly decondensed state of active Balbiani ring chromatin, however, remained unaffected after injection of topoisomerase I antibodies. These data are consistent with the interpretation that topoisomerase I is an essential component in the transcriptional process but not in the maintenance of the decondensed state of active chromatin.

The inhibition of nuclear RNA synthesis by the rifampicin derivative AF/013 in living cells.

The rifampicin derivative, AF/013, completely inhibits synthesis of the nucleolar and chromosomal RNA in explanted salivary gland cells of Chironomus tentans. When the glands are preincubated in rifampicin AF/013 for a short period before the addition of the radioactive precursors, labelling of RNA is depressed in all size classes to the same extent. In contrast, if rifampicin is replaced by the nucleoside analogue, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a tentative initiation inhibitor of heterogeneous nuclear RNA, the label is reduced preferentially in the lower molecular weight region of the heterogeneous nuclear RNA spectrum. In chase type experiments, when rifampicin AF/013 is added after an initial labelling period, the synthesis of heterogeneous nuclear RNA is suppressed equally in all size classes, a result analogous to that obtained with the elongation inhibitor, alpha-amanitin. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole, under similar chase conditions, preferentially inhibits the labelling of smaller heterogeneous nuclear RNA molecules, but later on abolishes labelling of molecules with higher S values, also. Rifampicin AF/013 prevents or affects seriously the normal processing of the prelabelled preribosomal RNA in the nucleolus. It further interferes with the export of nuclear RNA to the cytoplasm, and/or promotes a non-physiological breakdown of cytoplasmic RNA. The experimental data suggest that rifampicin AF/013 acts on RNA synthesis in living cells by interference with chain elongation.

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins.

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).

Specific inhibition of hnRNA synthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Requirement of a free 3'-hydroxyl group, but not 2'- or 5'-hydroxyls.

Five structural analogues of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), all with modified sugar moieties, have been examined for their inhibitory activities on RNA transcription in salivary glands of Chironomus tentans. The well-known ability of the parent DRB at 65 microM concentration to selectively inhibit hnRNA/mRNA synthesis by approx. 90% was essentially abolished on methylation of the 3'-OH; but, at an overdose the analogue suppressed labeling of all RNA classes examined (hnRNA/mRNA, rRNA, 4-5 S RNA) by 70-80%. By contrast, the 2'-O-methyl derivative of DRB was almost as effective as DRB itself in blocking transcription of hnRNA/mRNA genes. Blocking of both the 2' and 3' hydroxyls (2',3'-O-isopropylidene-DRB) completely abolished inhibitory activity, irrespective of the concentration employed. The 5'-deoxy-5'-chloro derivative of DRB was only slightly less effective than the parent DRB. An unusual aspect of the activities of 2'-O-methyl-DRB and 5'-deoxy-5'-chloro-DRB was their ability to stimulate synthesis of 4-5 S RNA by 25-45%. Also investigated was the influence of the various analogues on the rate of formation of [3H]UTP from [3H]uridine used as an RNA precursor. The rate of such formation of [3H]UTP was suppressed 2-6-fold by treatment with 2'-O-methyl or 3'-O-methyl-DRB, but was unaffected by 5'-deoxy-5'-chloro-DRB or 5,6-dichloro-1-alpha-D-arabinofuranosylbenzimidazole. The overall data point to the importance of a free 3'-OH in the ribose moiety of DRB for selective inhibitory activity. The alpha-D-arabinofuranosyl analogue, although less selective in inhibition of RNA transcription, still exhibits about 50% of the activity of DRB.

Rapidly phosphorylated histone-like proteins are modulated in the course of transcription block by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

A putative histone H2A and H4 protein with posttranslationally added and covalently linked phosphate group(s) have been found in salivary gland cells of Chironomus tentans. The phosphate moieties possess a rapid turnover rate and the incorporation of 32P reaches steady-state level within 5 to 10 min of incubation. The H2A-like protein incorporates twice as much label as the H4-like one. The core histones H2B and H3 are not measurably phosphorylated under identical experimental conditions. The administration of the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), a likely inhibitor of transcription initiation, rapidly modulates the phosphorylation of H2A- and H4-like proteins. The incorporation of 32P into the H2A-like phosphoprotein is enhanced by up to 180% after 3 to 10 min of preincubation with DRB but the rise in phosphorylation is of a transient character. The phosphorylation of H4-like protein is affected somewhat later than that of H2A but the stimulatory effect persists even after a longer pretreatment with DRB. If the elongation inhibitor alpha-amanitin replaces DRB in similar experiments no significant effect on histone phosphorylation can be registered. The results are discussed in relation to the possibilities that there is a cause and effect relation between the rapid modulation of phosphorylated putative histone proteins and the repression of gene activity or condensation of active chromatin.

Effects of a DNA helix-destabilizing protein on transcription in living cells.

The effects of microinjected rat DNA helix-destabilizing protein (HDP) and anti-HDP sera on the transcription of various RNAs in nuclei of Chironomus tentans salivary gland cells were investigated. The results showed that injected antisera have the greatest inhibitory effect on the RNA polymerase II-based transcription of Balbiani ring puffs, about 80%. The inhibition of RNA polymerase I-based transcription of nucleolar preribosomal RNA was about 70%, while the effect on the heterogenous nuclear RNA (hnRNA) from chromosome I to III was about 40%. In all cases, the antiserum against the denatured subunit HDP was more inhibitory than that against the native HDP. In correlative experiments, microinjection of the HDP itself showed stimulated transcription of all RNAs. Indirect localization by immunofluorescence showed immunoreactive HDP to be preferentially concentrated on transcriptionally active Balbiani rings 1 and 2. Western blot analysis of the protein extract from isolated Chironomus tentans salivary gland nuclei with anti-HDP (rat) sera showed cross-reactive protein bands with molecular masses of about 33,000, 42,000 and 65,000 daltons. These results suggest that a homologue of rat HDP and other C. tentans proteins immunologically related to it play an important role in transcription in vivo.

A novel nuclear 42-kDa casein kinase identified in Chironomus tentans.

We have purified and characterised an apparently novel nuclear 42-kDa casein kinase from epithelial cells of Chironomus tentans which comigrates with a phosphoprotein associated with transcriptionally active salivary gland genes. The protein kinase promotes phosphorylation of casein and phosvitin, using either ATP or GTP as phosphate donors, and undergoes autophosphorylation. The casein kinase activity of the 42-kDa protein is sensitive to heparin, 5,6-dichloro-1-beta-D-ribofuranosylbezimidazole (DRB), spermine and spermidine indicating that it is a novel enzyme with similar but not identical properties to casein kinase II or nuclear protein kinase NII.