Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization.

African American dietary patterns at the beginning of the 20th century.

Early field studies in human nutrition documented the eating habits of African Americans living in a variety of circumstances. We compare the results of these investigations. Our analysis shows systematic differences along a continuum reaching from remote, rural communities in the South toward increasingly metropolitan locations.

Methods for visualizing RNA processing and transport pathways in living cells.

Recent advances in fluorescence microscopy, imaging, and probe technology provided possibilities to study the spatial and temporal distribution of RNA species in living cells. While some methods have been developed to localize all nascent or poly (A) containing transcripts others have been developed to study the in vivo distribution of specific RNA species. Irrespective of the method that has been used, the results of these studies provided important information concerning the localization and the cellular transport pathways of RNAs.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR.

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes.

Mutational analysis of fibrillarin and its mobility in living human cells.

Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs.

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.

Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH.

Combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) allows 24-color FISH karyotyping of human metaphase chromosomes utilizing only four fluorochromes, instead of the five required for combinatorial labeling procedures. Here we show that by introduction of a fifth fluorochrome, COBRA-FISH permits molecular cytogenetic mapping of viral integration sites in complex karyotypes in the context of a 24-color hybridization. We were able to detect a single copy of the human papillomavirus 16 in the SiHa cell line and to confirm the site of integration at 13q21-31.

Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus.

Fluorescence in situ hybridization and immunocytochemical techniques have contributed significantly to our current understanding of how transcription, RNA processing, and RNA transport are spatially and temporally organized in the cell nucleus. New technologies enabling the visualization of nuclear components in living cells specifically advanced our knowledge of the dynamic aspects of these nuclear processes. The picture that emerges from the work reviewed here shows that the positioning of genes within the three-dimensional nuclear space is of crucial importance, not only for its expression, but also for the efficient processing of its transcripts.

Dynamics of RNA polymerase II localization during the cell cycle.

Mitosis is characterized by condensation of chromatin, cessation of RNA transcription, and redistribution of nuclear proteins. We investigated the distribution of the hypo- and hyperphosphorylated forms of RNA polymerase II in mitotic cells from different cell lines by immunofluorescence. In interphase cells, the hyperphosphorylated RNA polymerase II (Pol IIO) is present in speckles and diffusely throughout the nucleoplasm.

RNA polymerase II localizes at sites of human cytomegalovirus immediate-early RNA synthesis and processing.

Pre-mRNA synthesis in eukaryotic cells is preceded by the formation of a transcription initiation complex and binding of unphosphorylated RNA polymerase II (Pol II) at the promoter region of a gene. Transcription initiation and elongation are accompanied by the hyperphosphorylation of the carboxy-terminal domain (CTD) of Pol II large subunit. Recent biochemical studies provided evidence that RNA processing factors, including those required for splicing, associate with hyperphosphorylated CTDs forming "transcription factories.

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.

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.

Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei.

In Trypanosoma brucei, transcription by RNA polymerase II and 5' capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I.

Experiment station dietary studies prior to World War II: a bibliography for the study of changing American food habits and diet over time.

Dietaries reported by previous generations of researchers, originally collected to advance the science of human nutrition, now have considerable value as primary historical documents. USDA experiment station reports and bulletins represent the single largest collection of such documents. Our annotated bibliography of experiment station studies is meant to facilitate their use in historical and other diachronic studies of American food habits and diet.

Staining of the midbody by an anti-digoxin-specific antibody.

Using RNA in situ hybridization to reveal cytoplasmic localization patterns of mRNAs in cultured cells, we noted unexpected staining of a cytoplasmic component in telophase cells. Control experiments revealed that the anti-digoxin-specific antibody was responsible for this staining. Because the staining was observed only at a position where both daughter cells are still connected, we identified the stained component as the midbody.

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.

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.

Species-specific regulation and switching of transcription between stage-specific ribosomal RNA genes in Plasmodium berghei.

Malaria parasites (Plasmodium spp.) differentially express structurally distinct sets of rRNA genes in a stage-specific manner. The four rRNA genes of the rodent malaria parasite, P.

Splicing factors associate with nuclear HCMV-IE transcripts after transcriptional activation of the gene, but dissociate upon transcription inhibition: evidence for a dynamic organization of splicing factors.

Before being transported to the cytoplasm, intron-containing pre-mRNAs have to be spliced somewhere in the cell nucleus. Efficient splicing requires an ordered assembly of splicing factors onto the pre-mRNAs. To accomplish this, intron containing genes may be preferentially localized at nuclear sites enriched for splicing factors or alternatively, splicing factors may circulate throughout the nucleus and have the ability to associate with randomly positioned nascent transcripts.

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.

Identification of regulatory sequences in the promoter of the PDGF B-chain gene in malignant mesothelioma cell lines.

Platelet-derived growth factor (PDGF) B-chain mRNA is readily detectable in malignant mesothelioma (MM) cell lines, but not in normal mesothelial (NM) cell lines. The high affinity receptor for PDGF B-chain dimers, the PDGF beta-receptor, is expressed in MM cell lines. NM cell lines predominantly express the PDGF alpha-receptor.

A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation.

Petiole protoplasts from in vitro-grown carrot plants are a very good alternative to traditionally obtained protoplasts from suspension cultures. High plating and regeneration efficiencies were obtained in most of the breeding lines that were tested. The embedding of the protoplasts in alginate was crucial for initiating cell division and further development.