Indole-based cyanine as a nuclear RNA-selective two-photon fluorescent probe for live cell imaging.

We have demonstrated that the subcellular targeting properties of the indole-based cyanines can be tuned by the functional substituent attached onto the indole moiety in which the first example of a highly RNA-selective and two-photon active fluorescent light-up probe for high contrast and brightness TPEF images of rRNA in the nucleolus of live cells has been developed. It is important to find that this cyanine binds much stronger toward RNA than DNA in a buffer solution as well as selectively stains and targets to rRNA in the nucleolus. Remarkably, the TPEF brightness (Φσmax) is dramatically increased with 11-fold enhancement in the presence of rRNA, leading to the record high Φσmax of 228 GM for RNA. This probe not only shows good biocompatibility and superior photostability but also offers general applicability to various live cell lines including HeLa, HepG2, MCF-7, and KB cells and excellent counterstaining compatibility with commercially available DNA or protein trackers.

Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading.

RNAi is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We include a method for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. This protocol facilitates the characterization of nuclear RNAi, and it can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4-6 d to complete.

A perspective of the dynamic structure of the nucleus explored at the single-molecule level.

Cellular life can be described as a dynamic equilibrium of a highly complex network of interacting molecules. For this reason, it is no longer sufficient to "only" know the identity of the participants in a cellular process, but questions such as where, when, and for how long also have to be addressed to understand the mechanism being investigated. Additionally, ensemble measurements may not sufficiently describe individual steps of molecular mobility, spatial-temporal resolution, kinetic parameters, and geographical mapping. It is vital to investigate where individual steps exactly occur to enhance our understanding of the living cell. The nucleus, home too many highly complex multi-order processes, such as replication, transcription, splicing, etc., provides a complicated, heterogeneous landscape. Its dynamics were studied to a new level of detail by fluorescence correlation spectroscopy (FCS). Single-molecule tracking, while still in its infancy in cell biology, is becoming a more and more attractive method to deduce key elements of this organelle. Here we discuss the potential of tracking single RNAs and proteins in the nucleus. Their dynamics, localization, and interaction rates will be vital to our understanding of cellular life. To demonstrate this, we provide a review of the HIV life cycle, which is an extremely elegant balance of nuclear and cytoplasmic functions and provides an opportunity to study mechanisms deeply integrated within the structure of the nucleus. In summary, we aim to present a specific, dynamic view of nuclear cellular life based on single molecule and FCS data and provide a prospective for the future.

Genus identification of toxic plant by real-time PCR.

Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants.

Megakaryocyte polyploidization is associated with a functional gene amplification.

It is believed that polyploidy induces an orchestrated increase in gene expression. To know whether all alleles remain functional during megakaryocyte polyploidization, we used a well-established fluorescence in situ hybridization technique which allows one to simultaneously detect pre-mRNAs and assess ploidy level in a single cell. All alleles of GPIIb, GPIIIa, VWF, beta-actin, hsp70, c-mpl, Fli-1, and FOG-1 genes are transcriptionally active in megakaryocytes from 4N to 32N. All X chromosomes in male cells are transcriptionally active but only half of them are transcriptionally active in female megakaryocytes, as revealed by the transcriptional activity of the GATA-1 gene. Nuclear untranslated XIST RNA accumulates on the inactivated X chromosomes, indicating that they are subjected to a normal inactivation process. Altogether, our results demonstrate that megakaryocyte polyploidization results in a functional gene amplification whose likely function is an increase in protein synthesis parallel with cell enlargement.

Posttransplant lymphoproliferative disorder associated with an Epstein-Barr-related virus in cynomolgus monkeys.

BACKGROUND: Human posttransplant lymphoproliferative disorder (PTLD) has been shown to be associated with Epstein-Barr virus (EBV) infection. Primate animal models of PTLD and the use of molecular markers in its diagnosis have not been reported. This study was designed to evaluate the frequency, pathology, and molecular characteristics of PTLD in cynomolgus kidney allograft recipients. METHODS: Over a 5-year period (January 1995 to November 2000), 160 primate renal transplants were performed at the Massachusetts General Hospital (MGH). Of these, all cases (n=9) that developed PTLD were included. H&E stained paraffin sections of all available tissue samples from the cases were evaluated for the presence of PTLD. Immunoperoxidase staining for T cells (CD3), B cells (CD20), kappa and lambda light chains as well as EBV nuclear antigens (EBNA2) and latent membrane proteins (EBV LMP-1) was done on paraffin sections using standard immunohistochemical (IHC) methods. In situ hybridization for EBV encoded RNA (EBER) was performed in all tissue samples with atypical lymphoid proliferations, using a novel EBER nucleotide probe based on consensus gene sequences from EBV and the related herpes lymphocryptoviruses (LCV) infecting baboons and rhesus macaques. RESULTS: Of 160 consecutive primate renal transplants performed at MGH, 5.6% developed PTLD 28-103 days after transplantation. In all cases, the lymph nodes were involved and effaced by an atypical polymorphous lymphoid proliferation of EBER+ B cells, diagnostic for PTLD. Focal staining for EBNA-2 was noted in tumor cells. In 67% (six of nine) the PTLD infiltrates were present in extra nodal sites, notably liver (56%), lung (44%), heart (44%), renal allograft (44%), and native kidney (22%). The spleen was involved by PTLD in all four animals that had not undergone a pretransplant splenectomy. The PTLD morphology was similar in all cases and predominantly of the polymorphous type, however, some of these showed areas that appeared minimally polymorphous. No cases of monomorphic PTLD were seen. CONCLUSIONS: By in situ hybridization, expression of the RNA product, homologous for EBV-encoded RNA (EBER) was identified in the PTLD tumor cells of all cases, indicating latent primate EBV- related infection. This report identifies a novel animal model of EBV associated PTLD in the setting of kidney transplantation, with valuable implications for managing and understanding human PTLD and oncogenesis.

A synthetic triterpenoid selectively inhibits the induction of matrix metalloproteinases 1 and 13 by inflammatory cytokines.

OBJECTIVE: To address the effects of a novel synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), on the induction of matrix metalloproteinases 1 and 13 (MMP-1, MMP-13) by inflammatory cytokines. METHODS: Human chondrosarcoma cells stimulated with inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha) were used to study the effects of CDDO on the induction of MMPs and the invasion of cells through a collagen matrix. RESULTS: CDDO selectively reduced the induction of MMP-1 and MMP-13 at the levels of messenger RNA and protein. Treatment with CDDO prior to cytokine stimulation enhanced this inhibition, and we demonstrated that CDDO functions at the level of transcription. Additionally, CDDO reduced IL-1beta-mediated invasion of cells through a collagen matrix. CONCLUSION: This study demonstrates that CDDO is a novel inhibitor of MMP-1 and MMP-13 gene expression mediated by inflammatory cytokines. Thus, CDDO may have therapeutic potential for the inhibition of joint degradation in osteoarthritis.

Expression of the recombination activating genes (RAG1 and RAG2) is not detectable in Epstein-Barr virus-associated human lymphomas.

The expression of the recombination activating genes (RAG1 and RAG2) is largely restricted to immature lymphoid cells. Previous studies have suggested that Epstein-Barr virus (EBV) infection may lead to a re-induction of RAG expression in mature B lymphocytes. To assess the significance of this mechanism for the pathogenesis of malignant lymphomas, we have examined the expression of RAG genes in 11 cases of EBV-associated endemic Burkitt's lymphoma (BL), 25 cases of Hodgkin's disease (HD, 17 EBV(+), 8 EBV(-)) and 10 cases of follicular non-Hodgkin's lymphoma (NHL). Using in situ hybridization, expression of the RAGs was detected in cortical thymocytes in normal thymus and in the tumor cells of 2 of 3 lymphoblastic NHL. By contrast, there was no detectable RAG expression in the BL, HD and follicular NHL cases. Our results indicate that re-induction of RAG expression does not occur in human lymphomas in vivo. Thus, it is unlikely to play a role in the development of translocations involving immunoglobulin gene loci which are characteristically found in BL and follicular NHL. Moreover, our study shows that in situ hybridization is a suitable method for the analysis of RAG expression in human tissue sections.

The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P.

The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant ( approximately 17S) very similar to that of the nuclear enzyme (nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNA(Ser(UCN)) precursor [ptRNA(Ser(UCN))] at the correct site. Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component. Furthermore, electrophoretic analysis of 3'-end-labeled nucleic acids extracted from the peak of glycerol gradient-fractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P. The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs. In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to approximately 33 to approximately 175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles. In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell. The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.

Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus.

We describe a method to image selectively the protein-based architecture in the eukaryotic cell nucleus using nitrogen and phosphorus mapping. In addition, we describe a method to determine total mass as well as stoichiometric relationships between protein and RNA. This method is illustrated using particulate structures in the nucleus called interchromatin granules. In so doing, we demonstrate that these granules contain heterogeneous nuclear RNA, and have an average protein and RNA content of 3.094 and 1.672 MDa, respectively. We also tested the sensitivity of phosphorus detection by exogenously applying purified duplex DNA to the surfaces of thin sections, and have shown that structures as small as single molecules of duplex DNA can be detected in situ using these electron spectroscopic imaging techniques.

Insulin induces upregulation of vascular AT1 receptor gene expression by posttranscriptional mechanisms.

BACKGROUND: An interaction of insulin with angiotensin II effects could be pathophysiologically important for the pathogenesis of atherosclerosis and hypertension. METHODS AND RESULTS: We examined the effect of insulin on AT1 receptor gene expression in cultured vascular smooth muscle cells (VSMCs). A 24-hour incubation with insulin (100 nmol/L) produced a 2-fold increase in AT1 receptor density on VSMCs, as assessed by radioligand binding assays. This enhanced AT1 receptor expression was caused by a time- and concentration-dependent upregulation of the AT1 receptor mRNA levels, as assessed by Northern analysis. The maximal effect was detected after a 24-hour incubation of cells with 100 nmol/L insulin (270+/-20%). AT1 receptor upregulation was caused by a stabilization of the AT1 receptor mRNA, because the AT1 receptor mRNA half-life was prolonged from 5 hours under basal conditions to 10 hours after insulin stimulation. In contrast, insulin had no influence on AT1 receptor gene transcription, as assessed by nuclear run-on assays. The insulin-induced AT1 receptor upregulation was followed by an increased functional response, because angiotensin II evoked a significantly elevated intracellular release of calcium in cells that were preincubated with 100 nmol/L insulin for 24 hours. The insulin-induced AT1 receptor upregulation was dependent on tyrosine kinases, as assessed by experiments with the tyrosine kinase inhibitor genistein. Furthermore, experiments using the intracellular calcium chelator bis(2-amino-5-methylphenoxy)ethane-N, N,N',N'-tetraacetic acid tetraacetoxymethyl ester suggest that intracellular calcium release may be involved in AT1 receptor regulation. CONCLUSIONS: Insulin-induced upregulation of the AT1 receptor by posttranscriptional mechanisms may explain the association of hyperinsulinemia with hypertension and arteriosclerosis, because activation of the AT1 receptor plays a key role in the regulation of blood pressure and fluid homeostasis.

Localization of intranuclear RNA by electron microscopy in situ hybridization using a genomic DNA probe.

BACKGROUND: The presence of RNA in the cell nucleus is well known. However, a high resolution in situ hybridization evidence for the presence of RNA in some nuclear particles is still lacking. The aim of this work is to localize RNA in subnuclear particles using a novel ultrastructural in situ hybridization procedure. In this study, biotinylated genomic mouse DNA as a probe to localize total RNA in the nuclei of mouse hepatocytes was used. METHODS: The procedure is based on paraformaldehyde fixation and embedding in lowicryl resin. Thin sections are mounted in formvar-coated gold grids. Hybridization is performed on non-denatured thin sections. DNA-RNA hybrids are detected with streptavidin-10 nm gold particles complex. By controlling the time of nick-translation during incorporation of biotin into the probe, labeling in the fibrillar portions of the nucleoplasm is obtained. More digested probes generate more labeling in the granular components. Nucleoli were similarly labeled. RESULTS: As expected, no label was observed in the compact chromatin clumps. These results indicate that granular components as perichromatin granules in the nucleus contain more processed RNA than fibrillar portions. As a comparison, viral DNA sequences on denatured RNase-treated thin sections of adenovirus-2 (Ad-2)-infected human cells were detected. As previously reported, at late stages DNA was observed in the viral particles and surrounding nucleoplasm, where Ad-2 DNA is synthesized. CONCLUSIONS: The present procedure allows the study of intranuclear RNA distribution and will be useful for the analysis of RNA processing in several types of cells.

Localization of intranuclear RNA by electron microscopy in situ hybridization using a genomic DNA probe

Background. The presence of RNA in the cell nucleus is well known. However, a high resolution in situ hybridization evidence for the presence of RNA in some nuclear particles is still lacking. The aim of this work is to localize RNA in subnuclear particles using a novel ultrastructural in situ hybridization procedure. In this study, biotinylated genomic mouse DNA as a probe to localiza total RNA in the nuclei of mouse hepatocytes was used. Methods. The procedure is based on Paraformaldehyde fixation and embedding in lowicryl resin. Thin sections are mounted in formvar-coated gold grids. Hybridization is performed on non-denatured thin sections. DNA-RNA hybrids are detected with streptavidin-10 mm gold particles complex. By controlling the time of nick-translation during incorporation of biotin into the probe, labeling in the fibrillar portions of the nucleoplasm is obtained. More digested probes generate more labeling in the granular components. Nucleoli were similarly labeled. Results. As expected, no label was observed in the compact chromatin clumps. These results indicate that granular components as perichromatin granules in the nucleus contain more processed RNA than fibrillar portions. As a comparison, viral DNA sequences on denatured RNase-treated thin sections of adenovirus-2 (Ad-2)-infected human cells were detected. As previously reported, at late stages DNA was observed in the viral particles and surrounding nucleoplasm, where Ad-2 DNA is synthesized. Conclusions. The present procedure allows the study of intranuclear RNA distribution and will be useful fo the analysis of RNA processing in several types of cells

Nuclear RNA accumulations contain released transcripts and exhibit specific distributions with respect to Sm antigen foci.

RNA polymerase II transcripts accumulate within mammalian nuclei at distinct sites and exhibit varying morphology. Certain RNA species are organized in elongated structures, whereas others appear as dot-like concentrations. To analyze the status of the RNA within these accumulations, we investigated the composition of accumulations derived from Epstein-Barr virus (EBV) genes, human papilloma virus 18 (HPV18) open reading frames E6 and E7, as well as heat shock protein 89a (hsp89alpha) and 89beta (hsp89beta) genes. No differential distribution of exon and intron sequences within concentrations of EBV RNA could be observed. Whereas accumulations of hsp89alpha and hsp89beta always coincided with Sm antigen foci, the RNA of EBV and HPV18 never co-localized with these foci. This excludes Sm antigen foci as the only sites of splicing and suggests gene-specific variation in the nuclear localization of transcripts. Two sets of experiments were performed to assess whether transcripts in the RNA accumulations are in statu nascendi or products released from a discrete gene locus. Because RNA transcripts derived from EBV genes, which are located on both ends of the genome, were all distributed along the entire length of the RNA signals, they cannot be derived from a highly decondensed genomic DNA extending throughout elongated RNA accumulations. Furthermore, removal of labeled RNA sequences and subsequent visualization of DNA confirmed the confinement of the genomic sequences to a small subregion of the area occupied by accumulated RNA. Therefore, this study supports the view of RNA accumulations as a stream of molecules that delineate a path from a dot-like gene locus toward the nuclear envelope for export into the cytoplasm.

Transcription of mitochondrial and mitochondria-related nuclear genes in rabbit bladder following partial outlet obstruction.

Using the rabbit model, we showed that partial outlet obstruction of the urinary bladder causes significant changes in the status and expression of the mitochondrial (mt) genetic system in bladder smooth muscle immediately after obstruction is initiated. Here we investigate quantitatively the severity of the mt genetic response to partial outlet obstruction in both short- and long-term obstructed rabbits. Based on previous functional studies, bladders with mass < 6 fold greater than control were considered compensated; bladders with mass > 6 fold that of control were considered decompensated. Analyses of DNA from compensated rabbit bladders showed that relative mt genome copy number decreased to 30% of control values. Transcript analyses for these samples showed that mt RNA levels increased 3 fold to compensate for lower template copy number. Analysis of decompensated bladders demonstrated that mt genome copy number increased to approximately 90% of control levels; mt transcripts progressively decreased in these samples by as much as 30 fold. In contrast, transcription of a mt-related nuclear gene decreased 3-9 fold in compensated bladders but increased 10-30 fold in decompensated bladders. Activity for the cytochrome oxidase complex, and for the mt enzyme citrate synthase, decreased steadily with increasing bladder hypertrophy. These data suggest that bladder dysfunction following partial outlet obstruction is mediated partly by a significant loss in mt and mt-related nuclear gene coordination.

Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus.

The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,000-2,000 clusters in the nucleoplasm. This distribution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.

Measurement of gene-specific transcription by nuclease protection of pulse-labeled nuclear RNA.

A gene-specific transcription assay was developed that is based on pulse-labeled incorporation of [3H]uridine into nuclear RNA. Transcription is quantified by scintillation counting of [3H]uridine incorporated into nuclear RNA that is protected from S1 nuclease digestion by hybridization with cold gene probes. This assay was dependent upon partial degradation of nuclear RNA and optimization of hybridization and nuclease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell lines using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in serum-stimulated fibrosarcoma HT-1080 cells determined by [3H]uridine incorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in transcription rate within two hours of stimulation that declined to basal levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greater at 6-9-fold. The relative transcriptional activities of the beta-actin gene to that of the c-myc gene were virtually identical using the two assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were measured in log-phase HL-60 leukemia cells.

[Comparative study of nuclear RNA and polysomal mRNA in sunflower seedlings]. / Sravnitel'noe issledovanie iadernoi RNK i polisomnoi mRNK prorostkov podsolnechnika.

A comparative study of nRNA and polysomal mRNA complexity in the sunflower seedlings by molecular DNA:RNA hybridization method was carried out. It is established that nRNA complexity 4 times exceeds that of mRNA and is equal to 4.85.10(8) bp. Thus the nuclear RNA is expressed from at least 50.47% of single-copy DNA or 10.40% of genome. This data allow the presence of regulation of sunflower genome expression on the posttranscriptional level to be assumed.

Molecular approaches to the characterization of cell and blood/biomaterial interactions.

In order to address questions related to cell/biomaterial interactions with respect to cell function and production of extracellular matrix proteins that support or maintain cell/tissue specific properties, we have developed molecular approaches for analysis of in vivo implanted materials and in vitro studies. In an explant of a human left ventricular assist device (LVAD), intact total cellular RNA could be isolated in sufficient quantities for hybridization analyses with gene-specific probes to evaluate cell growth, cytoskeletal organization, and production of extracellular matrix proteins. Cells harvested from a 132-day implanted LVAD exhibited proliferative activity and expressed genes for fibronectin and collagen types I, III, and IV. In vitro studies revealed that endothelial cells cultured on two different segmented polyurethane biomaterials (Biomer and Tecoflex 60D) exhibited different patterns of gene expression that reflected differences in cell growth rates, morphology, and composition of the extracellular matrix. These methodologies provide a valuable approach for a detailed evaluation of: (1) the biocompatibility of cells colonizing implanted cardiac assist devices; and (2) the functionality of cells seeded onto biomaterials.

A novel class of nuclear RNA (nRNA) with a long life span as a gene repressor candidate.

Different systemic organs of fetal mice were continuously labelled with 5-3H-uridine during the organogenesis periods, and chased for various lengths of time after birth. In the autoradiographs made on paraffin-embedded sections of the organs taken after the chase for longer periods than 1 week, including a 12-months chase, specific labels were present exclusively in all the nuclei. The specific nuclear labels were resistant to RNase A or H digestion and to acid hydrolysis with 1 N HCl at 60 degrees C for 5 min, but were completely abolished by DNase digestion or prolonged acid hydrolysis for 10 min, the optimum condition for the Feulgen reaction to stain DNA. Electrophoretic analysis of the total nucleic acids extracted from the different organs chased for 3 or 12 months showed all the tritium radioactivity to be present in the DNA fraction before digestion with DNase or RNase A, and to be completely absent from the DNA fraction and shifted to the RNA fraction, or to be largely destroyed by degradation, after each digestion, respectively. By HPLC analysis of the total nucleic acid extract after further successive digestions with nuclease P1 and alkaline phosphatase into the constituent nucleotides, it was shown that all tritium activity was incorporated in uridine, without any detectable label in other nucleotides. By the simultaneous labeling of human peripheral lymphocytes at the late S-phase with 5-3H-uridine and BrdU, it was demonstrated that the autoradiographic labels, which, this time, were labile to RNase A digestion, were present in the G-bands of the spread chromosomes as identified by BrdU immunohistochemistry. The findings strongly indicate the presence of a novel class of nuclear RNA (nRNA). This type of RNA (a) may be localized in the nucleus in close association or hybridization with nuclear DNA, (b) have a life span as long as that of the cell, and (c) be duplicated in the late phase of DNA replication. The nRNA may play a fundamental role as gene repressor existing in the G-bands of metaphase chromosomes in the process of ontogeny and cytodifferentiation.