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.

Fissioning in planarians: control by the brain.

Reduced population densities lead to increased rates of fissioning in planarians whereas higher population densities suppress fissioning. This effect is not primarily due to mucus deposition or substances secreted into the water. Experiments are presented which show a system of population feedback control. In the presence of other planarians, the brain exerts an influence (probably neurohormonal) to suppress fissioning. This influence becomes attenuated with axial distance from the brain.

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.

Growth-affecting activity of cellular microexudate of Acanthamoeba.

Cells of Acanthamoeba castellanii release microexudates into the culture medium. The precursor material of the microexudates can be separated into four fractions on the basis of their solubility. One of these fractions carries a growth inhibitor, which is normally neutralized by other components of the precursor. A crude preparation of growth inhibitory material added to synthetic culture medium marks the growth of amoebae dependent on the quality of the substratum, and on the initial population density.

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.

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.

A majority of casein kinase II alpha subunit is tightly bound to intranuclear components but not to the beta subunit.

Nuclear casein kinase II (CK II) was purified from an epithelial cell line of Chironomus tentans and characterized. The intracellular distribution of CK II and its two intracellular subunits (alpha and beta) was analysed by immunoblotting. The apparent molecular weights of the alpha and beta subunits were estimated to be 36 and 28 kDa, respectively. Like other purified CK II preparations, CK II from Chironomus 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-beta-D-ribofuranosylbenzimidazole (DRB). Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools could be detected. More than 85% of the total immunostainable alpha subunit and essentially all immunoreactive individual beta subunit and heterooligomeric enzyme molecules were localised to the nucleus. Unexpectedly, more than 80% of this nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta 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 beta subunit, which precipitated at 60% and 80% (NH4)2SO4. These results suggest that a major portion of the nuclear CK II alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components.

Cephalic mechanism for social control of fissioning in planarians: III. Central nervous system centers of facilitation and inhibition.

Previous studies have indicated that asexual reproduction (fissioning) in the planarian Dugesia dorotocephala is socially controlled through a cephalic mechanism: Isolation releases fissioning; grouping inhibits it; decapitation, at the level of the auricles, releases it even in grouped subjects. The brain is not necessary for programming the actual events of fissioning; these are orchestrated by the segmental plexus fissioning (SPF) system. Various surgical cuts were made to ablate selected portions of the central nervous system of isolated and grouped planarians in order to ascertain the inhibitory or facilitatory effects of these in the physiological mediation of such control on the SPF system. These results were synthesized into a model of this control system; the anterior lobes and optic regions of the brain inhibit the SPF system, and the anterior and caudal segmental plexuses facilitate it. These influences are partially tonic and partially contingent upon social stimulation.

Sequences translated by Balbiani ring 75S RNA in vitro are present in giant secretory protein from Chironomus tentans.

The 75S RNA originating in the large Balbiani rings 1 and 2 (BRI and 2) was isolated and used for in vitro translation in the mRNA dependent reticulocyte lystate. Conditions (K+-concentration, temperature, time etc). were optimized for obtaining translation products of maximal size. Polypeptide chains up to about 500,000 D were obtained but no complete translation products. Trypic fingerprints were performed on the in vitro products as well as on the secretory protein components nos. I and II+III labelled with 35S-methionine. There was a large degree of correspondence between the fingerprint of the in vitro product and that of component I but less to that of component II+III. The results suggest that 75S RNA with an origin in the BR1 and BR2 codes for the giant secretory protein component I.

Analysis of the structural relationships between the DNA-binding phosphoproteins pp42, pp43 and pp44 by in situ peptide mapping.

A structural homology is established between three DNA-binding phosphoproteins located in the 42 to 44 kDa range, referred to as pp42, pp43 and pp44, from Chironomus tentans salivary gland cells by in situ peptide mapping. The staining patterns of pp42, pp43 and pp44 which resulted from digestion with Staphylococcus aureus V8, trypsin or papain proteases show the presence of 8 to 15 spots majority of which have identical mobility. In the patterns of the digests generated by treatments with trypsin about 10 spots appear in common between any pair of the protein substrates. In addition, each pattern includes two to three peptides of mobility not present in the other. Thus the peptide mapping of pp42, pp43 and pp44 based on the staining patterns of proteolytic digests suggest the existence of structural homology between the three unlabelled substrates. The proteolytic peptides carrying the rapidly turning over phosphate groups form markedly different electrophoretic patterns than the unlabelled peptides visualized by staining. Treatment of 32P-labelled pp42, pp43 and pp44 with V8 generates only one labelled fragment in the 30 kD range. The cleavage patterns of pp44 produced by chymotrypsin or papain contain seven to ten labelled fragments while those of pp42 and pp43 contain only two. The 32P-labelled tryptic peptides of pp42, pp43 and pp44 exhibit a ladder pattern for each substrate which probably arise by a consecutive removal of 25 to 35 amino acid residues from the primary digestion products pp29, pp29.5 and pp30 by cleavage of four to five putative interdomain regions. The possibility that these three structurally related phosphoproteins belong to the category of transcription factors is discussed.

The rapidly phosphorylated chromosomal 42-kDa protein is a subunit of larger protein complexes.

We have isolated, purified and characterized a 42-kDa phosphoprotein which has been found to be preferentially associated with active gene loci of salivary gland cells of Chironomus tentans. The rapidly phosphorylated form of this protein could be extracted with 0.2 M NaCl. Chromatographic analysis by gel filtration revealed that a significant fraction of labelled 42-kDa polypeptide elutes with an apparent molecular mass of 150 to 200 kDa. The result suggests that a portion of the phosphorylated 42-kDa polypeptide in native state forms a multisubunit protein complex consisting of rapidly phosphorylated 42-kDa polypeptide chains alone.

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.

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.

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.

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.

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.