Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.

Detection of mitochondrial DNA deletions in human skin fibroblasts of patients with Pearson's syndrome by two-color fluorescence in situ hybridization.

Pearson's marrow/pancreas syndrome is a disease associated with a large mitochondrial DNA (mtDNA) deletion. The various tissues of a patient contain heteroplasmic populations of wild-type (WT) and deleted mtDNA molecules. The clinical phenotype of Pearson's syndrome is variable and is not correlated with the size and position of the deletion. The histo- and cytological distribution of WT and deleted mtDNA molecules may be factors that correlate with the phenotypical expression of the disease. Here we introduce a new application of two-color FISH to visualize WT and deleted mtDNA simultaneously in a cell population of in vitro cultured skin fibroblasts of two patients with Pearson's syndrome. At the third passage of culturing, fibroblasts showed a remarkable heterogeneity of WT and deleted mtDNA: about 90% of the cells contained almost 100% WT mtDNA, and 10% of the cells contained predominantly deleted mtDNA. At the tenth passage of culturing, fibroblasts showed a reduction of intercellular heteroplasmy from 10% to 1%, while intracellular heteroplasmy was maintained. This new approach enables detailed analysis of distribution patterns of WT and deleted mtDNA molecules at the inter- and intracellular levels in clinical samples, and may contribute to a better understanding of genotype-phenotype relationships in patients with mitochondrial diseases.

Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.

Nanoparticle-induced fluorescence lifetime modification as nanoscopic ruler: demonstration at the single molecule level.

We combine interferometric detection of single gold nanoparticles, single molecule microscopy, and fluorescence lifetime measurement to study the modification of the fluorescence decay rate of an emitter close to a nanoparticle. In our experiment, gold particles with a diameter of 15 nm were attached to single dye molecules via double-stranded DNA of different lengths. Nanoparticle-induced lifetime modification (NPILM) has promise in serving as a nanoscopic ruler for the distance range well beyond 10 nm, which is the upper limit of fluorescence resonant energy transfer (FRET). Furthermore, the simultaneous detection of single nanoparticles and fluorescent molecules presented in this work provides new opportunities for single molecule biophysical studies.

Fluorescence in situ hybridization analysis of transcript dynamics in cells.

Since the first description of in situ hybridization in 1969 the technique has advanced to allow sensitive detection of DNA and mRNA molecules at the cellular and subcellular levels. In particular fluorescence in situ hybridization (FISH) has become a frequently used tool in basic and applied biomedical research since detection is sensitive and allows discrimination of multiple targets in the same sample. By using RNA-FISH we have been able to detect primary transcripts of the human embryonic, fetal, and adult globins in erythroid cells to study the competitive transcription mechanism or variegated expression patterns of the human beta-globin locus. We have correlated such expression patterns with other parameters such as cell type, cell cycle, replication, and stage of differentiation by simultaneous detection of, e.g., incorporated BrdUTPs, proteins (e.g., cyclins A and E, PCNA, histones), and globin (primary) transcripts and/or locus integration sites. Thus a combination of FISH and immunofluorescence methods allow the visualization of different processes taking place in the nucleus relative to each other in terms of three-dimensional space and structure and time (development, cell cycle).

In situ hybridization in living cells: detection of RNA molecules.

Fluorescence hybridization is a widely used technique in cell biology and pathology for detecting specific nucleic acid (DNA and RNA) sequences in fixed cells. This technique does not, however, provide dynamic information on the intracellular behavior of the targeted molecules. The aim of this work was to investigate possibilities of labeled DNA probes for RNA detection in cells that are maintained alive. Such techniques will provide useful tools for studying dynamic cellular processes such as RNA distribution and transport from transcription sites to translation sites by means of fluorescence microscopy. First a reversible, nonperturbing cell permeabilization procedure was developed using streptolysin O. This procedure was used to introduce oligodeoxynucleotides and fluorochrome-labeled DNA probes specific for 28S ribosomal RNA (2.1 kb) into living cells, which were then analyzed by fluorescence microscopy. The results showed that: (i) no increased cell death or growth perturbation was observed after permeabilization, (ii) introduction of a 28S RNA-specific probe (plasmid and oligonucleotides) into living cells led to bright nucleoli and a low cytoplasmic signal, and (iii) negative control probes did not lead to any fluorescent staining. These results indicate that specific hybridization of labeled nucleic acid probes takes place while cells are maintained under normal physiological conditions.

Oestradiol, a new hapten for detecting nucleic acid sequences by FISH.

Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin, biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications, oestradiol is a welcome addition to the collection of haptens employed in FISH.

Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.

We describe a detection principle for indirect fluorescence in situ hybridization (FISH) methods that with only one or two antibody layers dramatically improves FISH signal intensities. The method uses as a first layer an anti-hapten immunoglobulin [or (strept)avidin] conjugated to peroxidase. The quintessence of the method is the use of fluorochrome- or biotin labelled tyramides as peroxidase substrates to generate and deposit many fluorochrome or biotin molecules close to the in situ bound peroxidase. These may either be directly evaluated under the fluorescence microscope or after another incubation with fluorochrome-labelled (strept)avidin.

Imaging of RNA in situ hybridization by atomic force microscopy.

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase-labelled antibody and visualized with 3.3'-diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate-buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock-hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA--in situ hybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before dehydration.

Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization.

A method is presented to conjugate horseradish peroxidase (HRP) to oligodeoxynucleotides for fluorescence in situ hybridization assays employing tyramide signal amplification (TSA). HRP is covalently bound to the oligonucleotide by thiol ether linkage and purified by high-performance liquid chromatography. With TSA detection, a single HRP-labeled oligonucleotide probe is sufficient for in situ detection of clustered DNA repeat sequences with a degree of repetition between 20 and 50.

Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection.

We have used horseradish peroxidase-labeled 40 mer oligodeoxynucleotides (HRP-ODNs) specific for the human cytomegalovirus immediate early gene (HCMV-IE) and a novel dinitrophenol-tyramide signal amplification reagent (DNP-TSA plus) to evaluate their utility in fluorescence in situ hybridization (FISH). For DNA FISH, single or cocktails of HRP-ODNs were hybridized to metaphase chromosomes of rat 9G cells which, as determined by DNA fiber FISH, carry an integrated tandem repeat of 50-60 copies of the HCMV-IE gene. With one layer of DNP-TSA plus deposition and subsequent detection with a fluorochrome-conjugated antibody, four HRP-ODNs were needed to detect the HCMV-IE integration site. When employing two TSA amplification rounds, one HRP-ODN was sufficient for obtaining a strong signal of the integrated gene cluster, indicating that 50-60 HRP molecules can be detected with ease. In addition to DNA FISH, we report here the first use of HRP-ODN probes for mRNA detection by FISH. A single HRP-ODN and one DNP-TSA plus step resulted in clear visualization of the HCMV-IE gene transcripts in rat 9G cells induced for HCMV-IE expression by cycloheximide. Two TSA detection steps enhanced signal intensities even further. Parallel experiments with hapten-labeled ODN and cDNA probes and conventional detection methods illustrated the superiority of the HRP-ODN/TSA approach in DNA and RNA FISH.

Activation of the beta globin locus by transcription factors and chromatin modifiers.

Locus control regions (LCRs) alleviate chromatin-mediated transcriptional repression. Incomplete LCRs partially lose this property when integrated in transcriptionally restrictive genomic regions such as centromeres. This frequently results in position effect variegation (PEV), i.e. the suppression of expression in a proportion of the cells. Here we show that this PEV is influenced by the heterochromatic protein SUV39H1 and by the Polycomb group proteins M33 and BMI-1. A concentration variation of these proteins modulates the proportion of cells expressing human globins in a locus-dependent manner. Similarly, the transcription factors Sp1 or erythroid Krüppel-like factor (EKLF) also influence PEV, characterized by a change in the number of expressing cells and the chromatin structure of the locus. However, in contrast to results obtained in a euchromatic locus, EKLF influences the expression of the gamma- more than the beta-globin genes, suggesting that the relief of silencing is caused by the binding of EKLF to the LCR and that genes at an LCR proximal position are more likely to be in an open chromatin state than genes at a distal position.