In situ detection of precursor and mature microRNAs in paraffin embedded, formalin fixed tissues and cell preparations

The in situ detection of microRNAs (miRs) expression offers several challenges. It would be advantageous to have a method which can be used in paraffin embedded, formalin fixed tissue to be able to access the large data bank of archival material. Further, it would be helpful if one could differentiate between precursor and mature, active forms of the miR. In this review, two different methods for the in situ detection of miR in paraffin embedded, formalin fixed tissues are described. Detection of the inactive precursor miR can be accomplished by RT in situ PCR. This will allow the detection of one copy of a given pre-miR per cell. Detection of the mature form of a given miR can be accomplished with in situ hybridization with a labeled probe in which some of the nucleotides have been modified; this is referred to as a locked nucleic acid (LNA) probe. An intense signal after in situ detection with the LNA probe documents marked up-regulation of the, typically, mature miR. Further, one can easily determine the specific subcellular compartmentalization of the precursor and mature forms which may provide insight into the modulation of these important regulatory molecules and their targets.     

RNA Synthesis in isolated living and fixed nuclei ofAcetabularia

Summary Isolated, gently fixed nuclei ofAcetabularia mediterranea are capable of incorporating nucleotides into RNA. This has been demonstrated by (a) sensitivity of the synthesized products to RNase, (b) inhibition of RNA synthesis by actinomycin D and (c) incubation of nuclei without CTP and GTP. Experiments with -amanitin show that at least two RNA polymerases are active in isolated fixed nuclei. Also isolated, living nuclei incorporate nucleotides into RNA. RNA synthesis was located autoradiographically and quantified by a TV-analysator.     

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells

In the presence of 1–5 mM n-butyrate, murine leukemic L1210 cells cease proliferation and become arrested in the G1A compartment of the G1 phase. Cells in this compartment, in comparison with the remaining cells of the G1 phase (G1B), are characterized by low RNA content and more condensed chromatin. During unperturbed growth the cell residence times in G1A are of indeterminate duration (exponentially distributed); the half-time of L1210 cell residence in G1A is about 1.4 h. The effect of n-butyrate in arresting cells in G1A was concentration-dependent. However, the sensitivity of L1210 cells to this drug was markedly enhanced when cells were treated for longer than one generation (12 h). Cells arrested in G1A remained viable and when n-butyrate was removed, after a lag period, they resumed progression through the cycle.The effect of n-butyrate on cell progression through various parts of the cycle was studied in a stathmokinetic experiment. The rate of cell entrance into mitosis was decreased by 30, 60 and 110%, in the presence of 1, 2.5 and 5 mM n-butyrate respectively, thus indicating a slowdown in cell progression through G2 and S. The duration of G2 was prolonged by 20, 70 and 140% at 1, 2.5 and 5 mM n-butyrate respectively. The half-time of cell residence in G1A was increased by as much as 1.5-, 6.3- and 15.6-fold by 1, 2.5 and 5 mM n-butyrate. Progression through late G1 (G1B) was not affected at 1 mM, and could not be estimated at higher drug concentrations. The effects on cell cycle progression were evident 1 h after addition of n-butyrate.DNA in situ in nuclei of n-butyrate-treated cells had lowered (by 2–8 °C) stability to thermal denaturation and increased (by 15%) accessibility to DNase I. The decrease in DNA stability to heat was more pronounced when permealized cells were heated in the presence of 1 mM MgCl₂ rather than EDTA. DNA in situ in the nuclei of n-butyrate-treated cells also showed decreased sensitivity to acid-induced denaturation. Changes in chromatin were seen in all cells, regardless of cell cycle phase, within the first hours after addition of n-butyrate. Mitotic cells, however, reacted to n-butyrate more rapidly than interphase cells. The observed changes in L1210 cells are most likely a consequence of histone modifications (acetylation of inner histones, dephosphorylation of histone H1) induced by n-butyrate.     

Age-related changes in chromatin of liver cell nuclei of different ploidity.

A comparison of the Feulgen hydrolysis curves and the chromatin compactness of the liver cell nuclei of young and old rats was made. It was found that the rate of DNA depurination and chromatin compactness are higher in the liver cell nuclei of old rats, both in di-and tetraploidal cells. The effect of fixation upon the course of the hydrolysis curves is discussed.     

Activation of nuclear polypeptide synthesis in rat liver cells after inhibition of template protein synthesis. The role of nuclear polypeptide synthesis in the changes of the chromatin structure

The correlation between the rates of nuclear polypeptide synthesis (NPS) and matrix protein synthesis in rat liver cells was investigated. It was shown that NPS is activated under conditions of protein synthesis inhibition in the cytoplasm. Model experiments revealed that the NPS and ADP ribosylation systems compete for chromatin structure: ADP ribosylation induces condensation, while NPS--decondensation of chromatin.     

Histone gene expression and chromatin structure in mammalian cell hybrids

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.     

Age-related changes in chromatin of liver cell nuclei of different ploidity

Summary A comparsion of the Feulgen hydrolysis curves and the chromatin compactness of the liver cell nuclei of young and old rats was made. It was found that the rate of DNA depurination and chromatin compactness are higher in the liver cell nuclei of old rats, both in di-and tetraploidal cells. The effect of fixation upon the course of the hydrolysis curves is discussed.This investigation was supported by grant 474/VI Committee of Cell Pathology, Polish Academy of Sciences     

Reinitiation of synthesis of small cytoplasmic RNA species K and L in isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.