Properties of a 110000 molecular weight protein in rat liver hnRNP particles

Particles carrying heterogeneous nuclear RNA (30–40 S-particles) were isolated from rat liver nuclei and the particle proteins separated by sodium dodecylsulfate gel electrophoresis. Some properties of a 110000 molecular weight component (P 110/103) were studied in detail: (i) P 110/103 was labeled to a 4–5 times higher specific activity than the major particle proteins in the presence of [¹⁴C]-amino acidsin vivo. (ii) In nuclei incubated with [³H]- or [³²P]-nicotinamide adenine dinucleotide P 110/103 was labeled presumably by ADP-ribosylation. (iii) A protein with the same molecular weight as P 110/103 and isolated from the nuclear extract by affinity chromatography was phosphorylated in vitro.Abbreviations hnRNA heterogeneous nuclear RNA - hnRNP and mRNP ribonucleoproteins which contain hnRNA respectively mRNA     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.     

Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with [3H]leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.     

Guanylylation and adenylylation of the alpha regulatory proteins of herpes simplex virus require a viral beta or gamma function.

Herpes simplex virus genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Most of the products of the first group, the alpha genes, appear to have regulatory functions. We report that the alpha proteins, infected cell proteins 4, 0, 22, and 27 of herpes simplex virus 1 and 4, 0, and 27 of herpes simplex virus 2, were labeled in the isolated nuclei of infected HeLa cells with [alpha-32P]GTP or [alpha-32P]ATP late in infection and that these proteins represent the largest group of virus-specific proteins labeled in this fashion. Studies with [2-3H]ATP, in which the label is in the purine ring, showed that a portion of the label in alpha proteins and in at least one other infected cell protein is due to nucleotidylylation. Analyses of the labeling reactions in nuclei of (i) cells infected with temperature-sensitive mutants at nonpermissive temperatures, (ii) cells infected with wild-type virus and harvested at different times postinfection, and (iii) cells treated with inhibitors of protein synthesis or of synthesis of viral DNA led to the conclusion that viral gene functions expressed after the synthesis of alpha proteins are required for the labeling of the alpha proteins with [alpha-32P]GTP. We conclude that several of the alpha proteins are extensively posttranslationally modified and that these modifications include nucleotidylylation.     

A lysophosphatidic acid-binding cytosolic protein stimulates mitochondrial glycerophosphate acyltransferase

Rat liver cytosolic fraction caused up to five fold stimulation of mitochondrial glycerophosphate acyltransferase apparently by removing the lysophosphatidic acid formed by the acyltransferase. When mitochondria were incubated with palmityl-CoA, [2-3H]-sn-glycerol 3-phosphate and the cytosolic fraction and the supernatant fluid of the incubated mixture was passed through a Sephadex G-100 column, labeled lysophosphatidic acid eluted in three peaks with Mrs (i) 60-70 kDa, (ii) 10-20 kDa, and (iii) less than 5 kDa. Proteins, responsible for binding of lysophosphatidic acid in peaks (i) and (ii), were purified to near homogeneity as judged by electrophoretic analysis. The lysophosphatidic acid binding protein in peak (i) appears to be serum albumin and peak (iii) represents largely unbound lysophosphatidic acid. The 15 kDa protein, purified from peak (ii), bound lysophosphatidic acid, stimulated the acyltransferase and export of lysophosphatidic acid from mitochondria.     

Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.     

Nuclear protein synthesis in a temperature-sensitive Chinese hamster ovary cell line at 40 degrees C

We examined the incorporation of radioactive amino acids into nuclear proteins occurring at nonpermissive conditions in tsH1 Chinese hamster ovary cells with a temperature-sensitive defect in cytosol nonmitochondrial protein synthesis. In leucine-free medium at 40 degrees C, total cellular protein synthesis declined by 1-1.5%/min. As reported by others, preincubating these cells at 42 degrees C for 5-10 min sharply increased the rate of decline. The synthesis of acidic nuclear proteins at nonpermissive conditions (40 degrees C + 300 micrograms/ml cycloheximide) was demonstrated by the nuclear incorporation of 3H-tryptophan. Radioactivity, seen by autoradiography to be associated with these isolated Triton-X-100-washed nuclei, was released after incubating labelled nuclei with proteolytic enzymes. During incubation of tsH1 cells at nonpermissive conditions, pulse/chase experiments were consistent with the loss of some nuclear radioactivity into the cytoplasm. The distribution of cytosol and nuclear proteins, labelled at permissive or nonpermissive conditions and separated by isoelectric focusing, differed quantitatively and probably qualitatively, confirming the residual synthesis of acidic nuclear proteins at 40 degrees C in the presence of cycloheximide. Most newly synthesized acidic proteins retained by nuclei from cells labeled at nonpermissive conditions were present in a transciptionally active chromatin fraction. Although under these conditions the apparent rate of cellular RNA synthesis was unchanged, inhibiting residual cycloheximide-resistant nuclear protein synthesis with puromycin proportionately reduced RNA synthesis. Preincubating cells with 20 micrograms/ml of actinomycin D did not inhibit residual labelling of nuclear proteins; effects on residual nuclear labelling of impaired mitochondrial respiration were ambiguous. Nuclear proteins labelled under nonpermissive conditions probably included some of the 'prompt' heat shock proteins recently described. Provided certain assumptions are correct, our results are consistent with very limited protein synthesis associated with and even intrinsic to cell nuclei. They also suggest that this residual cycloheximide-resistant protein synthesis could be concerned with optimum synthesis or processing of certain nuclear RNA species.     

Induction of stress proteins by electromagnetic fields in cultured HL-60 cells.

HL-60 cells in culture were exposed for 2 h to a sinusoidal 0.1 or 1 mT (1 or 10 Gauss) magnetic field at 60 Hz and pulse labeled after exposure with radioactive isotopes by incubation by using either [(35)S]methionine, [(3)H]leucine, or [(33)P]phosphate. The radioactive labels were incorporated into cellular proteins through synthesis or phosphorylation. Proteins were extracted from electrostatically sorted nuclei, and the heat shock/stress proteins (sp) were analyzed for synthesis and phosphorylation by two-dimensional polyacrylamide gel electrophoresis. In the control cultures (no exposure to the magnetic field), sp 72c (cognate form) was faintly observed. A 0.1 mT exposure did not show sp metabolism to be different from that of the controls; however, after a 1 mT exposure of the HL-60 cells, sp 70i (inducible form) was synthesized ([(35)S]methionine incorporation). Sp 90 was not synthesized at either field level, but was phosphorylated ([(33)P]phosphate incorporation) in the 1 mT exposure. Sp 27 (isoforms a and b) was induced after a 1 mT exposure as reflected by labeling with [(3)H]leucine. These sps were not detected after a 0.1 mT exposure. After a 1 mT exposure and labeling with [(33)P], sp 27 isoforms b and c were phosphorylated whereas isoform 'a' was not observed. Sps 70i, 72c, and 90 were identified by commercial sp antibodies. Likewise, polypeptides a, b, and c were verified as sp 27 isoforms by Western blotting. Statistical evaluation of sp areas and densities, determined from fluorographs by Western-blot analysis, revealed a significant increase in sps 90 and 27a after a 1 mT magnetic field exposure. The 1 mT magnetic field interacts at the cellular level to induce a variety of sp species. Bioelectromagnetics 20:347-357, 1999. Published 1999 Wiley-Liss, Inc.     

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.     

Protein cross-migration during isolation of nuclei from mixtures of heated and unheated HeLa cells

To investigate the cross-migration of proteins during nuclear isolation heated and control cells were mixed prior to nuclear isolation. These nuclei were stained with fluorescein isothiocyanate (FITC, a protein-specific stain) and with propidium iodide (PI, a DNA-specific stain). Flow cytometric (FCM) analysis showed two populations distinguishable on the basis of protein content. The protein content of the nuclei in the upper population was identical to that for nuclei isolated from heated cells while that for the lower population had a protein content identical to the protein content of nuclei from control cells. This result shows that the heat-induced increase in nuclear protein content occurred throughout the entire population of nuclei (i.e., in G1, S, and G2 nuclei) and that the measured protein content of nuclei was not affected by the presence of the other population during isolation. The capability of the FCM to sort subpopulations from different regions of a histogram was used to separate the subpopulations after analysis. When control cells were prelabeled with [3H]leucine and mixed with unlabeled heated cells, 11% of the radioactivity was found to be associated with the nuclei from heated cells. Autoradiographs showed grains over approximately 99% of the nuclei from heated cells. When [3H]TdR was used as a label in a similar experiment, only 0-3% of the label was observed to become associated with the population of nuclei from heated cells and autoradiography showed that 97% of these nuclei were not labeled. Comparable results were obtained when the labeled cells were heated and the control cells were left unlabeled. These results show that a small amount of protein (approximately 10% of the nuclear protein) will cross-migrate during nuclear isolation without affecting the net amount of protein in either population.     

Content of nonhistone protein in nuclei after hyperthermic treatment

When nuclei were isolated from Chinese hamster ovary cells after being heated, there was a large increase in the amount of ³H-tryptophan labeled nonhistone protein in the nucleus relative to the whole cell. After 15 min or 30 min of heating at 45.5°C, the nuclear nonhistone protein content increased by 1.6 or 1.8, respectively. In contrast, when the nuclear nonhistone protein content was determined in the intact cell by using autoradiography to quantify ³H-tryptophan labeled protein in the nucleus and cytoplasm in sections of fixed cells, the nuclear nonhistone protein content increased by only 1.14 or 1.28 for 15 or 30 min at 45.5°C, respectively. Therefore, heat does not induce a massive movement of cytoplasmic protein into the nucleus. © 1993 Wiley-Liss, Inc.     

Phosphohistidine is found in basic nuclear proteins of Physarum polycephalum

Nuclear extracts of the true slime mold, Physarum polycephalum, show protein histidine kinase activity towards exogenous histones [(1985) J. Biol. Chem. 260, 16106-16113]. Physarum microplasmodia were labeled with [32P]phosphate in vivo and two basic proteins containing alkali-stable phosphate were detected. The labeled proteins comigrated with Physarum histones H1 (approximately) and H2A and phosphoamino acid analysis showed that each protein contained [32P]-phosphohistidine. The H2A-like protein was also labeled in isolated nuclei incubated with [35S]thio-ATP. We conclude that some Physarum nuclear proteins contain phosphohistidine.     

Effects of calcium buffering on the synthesis of the 26-kDa heat-shock protein family

We have reported on the effect of heat in C127 cells having various basal levels of the Ca(2+)-binding proteins calmodulin (CaM) or parvalbumin [Evans, Simonette, Rasmussen, Means, and Tomasovic, J. Cell. Physiol. 142, 615-627 (1990)]. These studies suggested that induction of the synthesis of 26-kDa heat-shock protein (hsp-26) depended on increased intracellular free Ca2+ [Ca2+]i and that induction was abrogated by increased Ca(2+)-binding capacity. To evaluate further the role of [Ca2+]i in mediating the response to hyperthermia and the potential for Ca(2+)-buffering to affect these processes, we loaded C127 parental cells with the Ca2+ chelators BAPTA or quin-2 (5 microM for 60 min) and then immediately heated the cells (30 min at 43 degrees C) and labeled them (3 h at 37 degrees C) with [3H]leucine. Measurements of [Ca2+]i with quin-2 and fura-2 showed that an increase in [Ca2+]i occurred with this heat dose, but that the quin-2 buffered that increase. Two-dimensional gels showed that cells loaded with BAPTA and quin-2 had a reduced rate of synthesis of the most basic (nonphosphorylated) hsp-26a isoform. The apparent synthesis of the more acidic isoforms (hsp-26b, hsp-26c) was less affected, but labeling studies with 32P showed this reflected continued accumulation of these phosphorylated isoforms, especially the most highly phosphorylated hsp-26c. Although it reduced hsp-26a synthesis, the temporary buffering of [Ca2+]i did not alter the subsequent expression of heat killing or the extent of thermotolerance significantly, possibly because phosphorylated hsp-26 was still generated. These data support the hypothesis that perturbations of [Ca2+]i directly modulate induction of hsp-26a synthesis.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.     

Association of protein C23 with rapidly labeled nucleolar RNA

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [3H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [3H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)