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E Egyházi

Publications and source records attributed to E Egyházi.

At least 19 recordsLinked to original sources
PubMed1999

The binding of the alpha subunit of protein kinase CK2 and RAP74 subunit of TFIIF to protein-coding genes in living cells is DRB sensitive.

In a previous report, we documented that a major portion of the nuclear protein kinase CK2alpha (CK2alpha) subunit does not form heterooligomeric structures with the beta subunit, but it binds tightly to nuclear structures in an epithelial Chironomus cell line. We report here that the CK2alpha, but not beta, subunit is co-localized with productively transcribing RNA polymerase II (pol II) on polytene chromosomes of Chironomus salivary gland cells. Likewise, the RAP74 subunit ofTFIIF, a potential substrate for CK2, is co-localized with pol II. The occupancies of chromosomes with the CK2alpha and RAP74 subunits are sensitive to DRB, an inhibitor of pol II-based transcription and the activity of CK2 and pol II carboxyl-terminal kinases. DRB alters the chromosomal distribution of the CK2alpha and RAP74 subunits: there is a time-dependent clearance from the chromosomes of CK2alpha and RAP74 subunits, which coincides in time the completion and release of preinitiated transcripts after addition of DRB. The results suggest that both the CK2alpha and RAP74 subunits travel with the elongating pol II molecules along the DNA template during the entire transcription cycle. No detectable re-association of CK2alpha and RAP74 with the promoters takes, however, place after the completion of the preinitiated transcripts in the presence of DRB. In contrast, the binding of hypophosporylated pol II and TFIIH to the active gene loci is not abolished by the DRB regimen. Our data are consistent with the possibility that in living Chironomus salivary gland cells, DRB interferes with the recruitment of TFIIF, but not of TFIIH, to the promoter by interference with the activity of the CK2alpha subunit enzyme and phosphorylation of RAP74 and thereby DRB blocks transcription initiation.

Animals↗
PubMed1998

Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.

Animals↗
PubMed1996

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-beta-D-ribofuranosylbenzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol II0) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol II0 is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of II0: 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 II0 is coupled to the completion of nascent pol II gene transcripts.

Amanitins↗
PubMed1994

The salivary gland 42-kDa phosphoprotein is a single-stranded DNA-binding protein with characteristics of the epithelial casein kinase N42 in Chironomus tentans.

The DNA-binding and phosphorylation properties of a rapidly phosphorylated nuclear 42-kDa phosphoprotein and of its two structurally related proteins, pp43 and pp44 in Chironomus tentans salivary glands were investigated. pp42, pp43 and pp44 bind promoter probes of the ecdysterone controlled I-18C gene and of the joint histone H2A/H2B genes in a sequence-selective and single-stranded DNA (ssDNA) specific manner. Rapid phosphorylation appears to give pp42 and pp43 uniquely hydrophilic characters making them soluble in the aqueous phase during phenol treatment. Dephosphorylation of the nuclear proteins markedly stimulates the ssDNA-binding activity of pp42 but not of pp43 and pp44. All three phosphoproteins are sensitive to heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) in vitro, but their sensitivity to heparin is more than one order of magnitude lower than that of casein kinase II. The heparin sensitivity of pp42 and pp43 is, however, similar to that described for a previously identified nuclear 42-kDa phosphoprotein in a Chironomus tentans epithelial cell line, casein kinase N42 (CKN42). pp42 and pp43 bind with high affinity to a Phosvitin-Sepharose matrix, like casein kinase I, II and N42, and can be eluted with high salt buffers from the affinity column. In intact salivary gland cells, microinjected (gamma-32P)GTP labels pp42 in a heparin sensitive manner, and this GTP-phosphorylation of pp42 could be competed out by a large excess of phosvitin. (gamma-32P)ATP-based phosphorylation of pp42 was uninfluenced by phosvitin in intact cells. The experimental data suggest that the salivary gland 42-kDa phosphoprotein, pp42, is a ssDNA-binding protein with characteristics of the epithelial CKN42.

Adenosine Triphosphate↗
PubMed1994

Analysis of a novel DNA-binding protein kinase CKII-like enzyme of Chironomus cells.

We have previously described a Chironomus tentans nuclear 42 kDa phosphoprotein preferentially associated with transcriptionally active chromatin. In an attempt to purify and identify the kinase responsible for the phosphorylation of the 42 kDa protein, a casein-phosvitin affinity chromatography was used. Unexpectedly, in the eluted kinase fraction, a novel 42 kDa casein kinase, designated protein kinase CK42, with a kinase activity similar, but not identical, to protein kinase CKII, could be identified. In other studies, a nuclear protein that comigrates with protein kinase CK42 in electrophoresis and is capable to bind different gene promoters in single-stranded forms in a sequence-selective manner was found. The observations that both protein kinase and ssDNA-binding activities could be ascribed to a 42 kDa protein raised the possibility that the ssDNA-binding 42 kDa phosphoprotein is a protein kinase. By specific ssDNA-binding affinity chromatography, using a biotinylated oligodeoxyribonucleotide promoter probe and Streptavidine-agarose matrix, evidence that both activities arise from the same protein molecules was obtained. The similarity in the enzyme activities between protein kinase CK42 and CKII raised the question of whether the former was an alpha subunit of the latter. To provide an answer to this issue, CKII, isolated and purified from an epithelial cell line of C. tentans, was characterized and compared with protein kinase CK42 purified from the same cell system. Like other purified CKII preparations, CKII from Chironomus is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). However, the heparin and DRB sensitivities of protein kinase CKII were substantially higher than those of the protein kinase CK42. Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools of CKII could be detected. More than 80% of the nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta subunit were solubilized under the same conditions suggesting that a major portion of the nuclear CKII alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components, probably to chromatin. The biochemical and immunological data taken together strongly suggest that CK42 is a novel DNA-binding protein kinase that is not the alpha subunit of CKII.

Adenosine Triphosphate↗
PubMed1989

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.

Animals↗
PubMed1987

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).

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

Selective repression of RNA polymerase II by microinjected phosvitin.

We have used a microinjection technique to examine whether injected phosvitin, in its capacity as substrate for casein kinase NII, could compete out the endogenous phosphorylation of some nuclear phosphoproteins with regulatory potential and thereby interfere with the activity of RNA polymerase II. Phosphorylation, which utilizes ATP as phosphate donor, was separated from phosphorylation which uses GTP. Phosvitin introduced into nuclei of salivary gland cells becomes phosphorylated by the endogenous nuclear protein kinase(s) and incorporates phosphates from ATP as well as from GTP. The phosphorylation of nuclear proteins and phosvitin is heparin-sensitive, indicating that they are phosphorylated by casein kinase NII. Microinjected phosvitin does not seem to affect the incorporation of phosphate groups from ATP into nuclear proteins, but protein phosphorylation by GTP is influenced. Apart from a minor overall reduction of 32P-incorporation, the phosphorylation of a 42 kDa nuclear protein, a putative transcription stimulatory factor, and of a 115 kDa nuclear protein was competed out by 70%-80% compared with the control value obtained in the absence of phosvitin. Parallel analyses of DNA transcription in phosvitin-injected nuclei showed that the RNA polymerase II-mediated synthesis of hnRNA and Balbiani ring RNA was diminished by 80% and 90%, respectively. In contrast, the transcription of nucleolar pre-ribosomal 38 S RNA by RNA polymerase I remained unaffected. The inhibitory effect of injected phosvitin could be reversed by in vitro phosphorylation of phosvitin prior to injection, using isolated nuclei as source of protein kinase(s). Taken together, the results suggest a causal relationship between the modification of the GTP-dependent phosphorylation of specific non-histone proteins and the activity of RNA polymerase II.

Animals↗
PubMed1986

Transcriptional inhibition in early chick embryos as a result of polyamine depletion.

In the early chick embryo, inhibition of polyamine synthesis by alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, blocks development at gastrulation. This effect was paralleled by a marked suppression of RNA and protein synthesis. There was no major change in cell cycle distribution in DFMO-treated embryos. Nevertheless, analysis of DNA synthesis and mitotic index indicated a prolongation of the cell cycle, possibly affecting all the phases. The inhibition of RNA synthesis in polyamine-depleted embryos, as evaluated by [3H]uridine incorporation, was not a result of reduced uptake or expansion of the UTP pool, and there was no deficiency or major imbalance among the ATP, GTP, and CTP pools. On the basis of agarose gel electrophoretic analyses of the various RNA species, and experiments using RNA synthesis inhibitors with different modes of action (actinomycin D, alpha-amanitin, and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole), it was concluded that the DFMO-induced gastrular arrest was due to general inhibition of transcription.

Amanitins↗
PubMed1985

Transport and metabolism of adenosine in relation to the transcriptional activity of hnRNA genes in Chironomus salivary gland cells.

The transport and metabolism of adenosine in explanted salivary gland cells of Chironomus tentans have been investigated. The adenosine transport is rapid and reaches a maximum velocity within seconds after administration. Nevertheless, a transmembrane equilibrium in adenosine concentrations could never be attained because of the efficiency of the intracellular trapping reaction. Only about 10% or less of the extracellular adenosine concentration could be maintained intracellularly. The rapidity of adenosine phosphorylation did not allow us the assessment of transport kinetics with any degree of accuracy. At lower external [3H]adenosine doses, [3H]ATP was the predominating metabolite, yielding a [3H]ATP/[3H]AMP ratio of 2.5-3.5, while at higher concentrations the [3H]ATP/[3H]AMP ratio was lowered to below 0.9. The [3H]AMP fraction derived from [3H]adenosine-treated cells was not uniform, but rather it consisted of 3H-labeled 5'AMP, 3'AMP and 2'AMP isomers. Whereas the accumulation of 3H-labeled 5'AMP and ATP attained steady-state levels after 30-60 min of incubation at higher exogenous adenosine concentrations, the content of 3H-labeled 3'AMP and 2'AMP continuously and linearly increased. The data indicate that the metabolism of adenosine to 2'AMP and 3'AMP represents a salvage pathway operating at unphysiological adenosine levels and that the well-known inhibitory effect of adenosine on polymerase-II-promoted RNA transcription is not exerted by its phosphorylated metabolites.

Adenosine↗
PubMed1985

Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: retention of in vivo sensitivity to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The rapidly turning over phosphorylation of specific nuclear nonhistone proteins, especially 42-, 33-, and 30-kDa polypeptides, and its relation to the transcriptional activity of hnRNA genes was investigated in isolated nuclei from salivary gland cells of Chironomus tentans. Incubation conditions promoting the phosphorylation of nonhistone proteins as well as the transcriptional activity of RNA polymerase II were established. The pattern of 32P incorporation into the nonhistone proteins found in isolated nuclei resembled that obtained in experiments with intact cells, and the endogenous RNA polymerase II retained its ability to reinitiate the transcription under in vitro assay conditions. In addition, the in vivo sensitivity of the phosphorylation of 42-, 33-, and 30-kDa polypeptides, like the sensitivity of the initiation of hnRNA transcription to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were preserved in the nuclear preparation. The experimental data taken together provide further support for the idea that the activation of hnRNA genes is causally related to the phosphorylation of specific nonhistone proteins.

Animals↗
PubMed1984

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.

Amanitins↗
PubMed1983

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.

Amanitins↗
PubMed1982

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.

Amanitins↗
PubMed1981

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.

Adenosine↗