Transient and constitutive repression of cytoplasmic translation signaling in cells with mtDNA mutation.

Cytoplasmic translation is under sophisticated control but how cells adapt its rate to constitutive loss of mitochondrial oxidative phosphorylation is unknown. Here we show that translation is repressed in cells with the pathogenic A3243G mtDNA mutation or in mtDNA-less rho(0) cells by at least two distinct pathways, one transiently targeting elongation factor eEF-2 and the other initiation factor eIF-2alpha constitutively. Under conditions of exponential cell growth and mammalian target of rapamycin (mTOR) activation, eEF-2 becomes transiently phosphorylated by an AMP-activated protein kinase (AMPK)-dependent pathway, especially high in mutant cells. Independent of AMPK and mTOR, eIF-2alpha is constitutively phosphorylated in mutant cells, likely a signature of endoplasmic reticulum (ER)-stress response induced by the loss of oxidative phosphorylation. While the AMPK/eEF-2K/eEF-2 pathway appears to function in adaptation to physiological fluctuations in ATP levels in the mutant cells, the ER stress signified by constitutive protein synthesis inhibition through eIF-2alpha-mediated repression of translation initiation may have pathobiochemical consequences.

COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications.

The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.

Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5'-monophosphate (5'-GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic approximately aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2 x 10(-2) M(-1) s(-1)) and the slowest reacting complex (k=0.93 x 10(-2) M(-1) s(-1)) under these conditions. Platination of an oligodeoxynucleotide (30-mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.

Chemical RNA labeling without 3' end bias using fluorescent cis-platin compounds.

Recently, fluorescent, monofunctional cis-platin derivatives have been developed to chemically label nucleic acids for use in fluorescent hybridization assays. Here we show by hybridizations to microarrays containing oligonucleotide probes for the 3' ends, middle parts, and 5' ends of mRNAs, that this labeling methodology bypasses the problem of the 3' end bias that is characteristic of the conventional enzymatic oligo(dT)-primed, reverse transcription labeling of mRNAs.

Insights into the sites of X ray and neutron induced chromosomal aberrations in human lymphocytes using COBRA-MFISH.

A multi-colour fluorescence in situ hybridisation (MFISH) assay has been developed, for simultaneous visualisation of all human chromosomes in 24 different colours. This assay is based on the simultaneous use of combinatorial labelling and ratio labelling, the so called combined binary ratio labelling (COBRA). This technique is used to study the spectra of chromosomal exchanges induced by X ray and neutrons in human lymphocytes. With X rays the dose-effect relationships for both dicentrics and translocations were linear-quadratic, whereas with neutrons these were linear. Among aberrant cells, average estimates of the minimum number of breaks was higher for neutrons than for X rays. Moreover, the induced chromosomal exchange patterns were more complex following neutron irradiation in comparison with X rays. COBRA-MFISH was found to have a greater resolving power over partial labelling for the accurate detection of complex translocations and insertions. With neutrons the frequencies of both were higher than those induced by X rays, and their relative proportions to the total frequencies were independent of dose. These data suggest insertions can be used as the 'signature' of high LET radiation.

A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction.

Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogeneous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology.

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. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

Up-converting phosphor reporters for nucleic acid microarrays.

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.

Overview of fluorescence in situ hybridization techniques for molecular cytogenetics.

This unit presents an overview of the FISH methodology. It covers such topics as direct versus indirect methods, sensitivity, multiplicity, resolution, and applications.

Basic preparative techniques for fluorescence in situ hybridization.

This unit presents protocols for preparing human metaphase chromosome slides from peripheral blood lymphocytes, isolating interphase nuclei from lymphocytes and paraffin-embedded tissues, and preparing DNA fibers. The protocols are designed so that the resulting preparations are amenable to FISH. The methods correspond to a selection of the specimens that can be analyzed with FISH techniques, and the choice of sample preparation method is highly dependent on the molecular cytogenetics question being addressed.

Probe labeling and fluorescence in situ hybridization.

This unit describes in detail basic protocols for probe labeling, denaturing of in situ target DNA, in situ hybridization, and post-hybridization washes. Support protocols for probe labeling cover probe purification and quality control.

Differentially painting human chromosome arms with combined binary ratio-labeling fluorescence in situ hybridization.

Recently we developed a novel strategy for differentially painting all 24 human chromosomes. It is termed COBRA-FISH, short for combined binary ratio labeling-fluorescence in situ hybridization. COBRA-FISH is distinct from the pure combinatorial approach in that only 4 instead of 5 fluorophores are needed to achieve color discrimination of 24 targets. Furthermore, multiplicity can be increased to 48 by introduction of a fifth fluorophore. Here we show that color identification by COBRA-FISH of all of the p and q arms of human chromosomes is feasible, and we apply the technique for detecting and elucidating intra- and interchromosomal rearrangements. Compared with 24-color whole chromosome painting FISH, PQ-COBRA-FISH considerably enhances the ability to determine the composition of rearranged chromosomes as demonstrated by the identification of pericentric inversions and isochromosomes as well as the elucidation of the arm identity of chromosomal material involved in complex translocations that occur in solid tumors.

Individuals with abnormal phenotype and normal G-banding karyotype: improvement and limitations in the diagnosis by the use of 24-colour FISH.

The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.

DNA fiber-FISH staining mechanism.

Fluorescence in situ hybridization to DNA fibers (Fiber-FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber-FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotin-labeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber-FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes. (J Histochem Cytochem 48:743-745, 2000)

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.

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.

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. We also demonstrate the gene mapping possibility of 25-color hybridization by detecting a MYC cosmid on normal metaphase chromosomes. The possibility of mapping single-copy probes in the background of 24-color hybridization expands the tools for cytogenetic mapping of DNA sequences and will contribute to the understanding of the role of viral integration and chromosome rearrangement in virus-mediated carcinogenesis.

A resource of mapped human bacterial artificial chromosome clones.

To date, despite the increasing number of genomic tools, there is no repository of ordered human BAC clones that covers entire chromosomes. This project presents a resource of mapped large DNA fragments that span eight human chromosomes at approximately 1-Mb resolution. These DNA fragments are bacterial artificial chromosome (BAC) clones anchored to sequence tagged site (STS) markers. This clone collection, which currently contains 759 mapped clones, is useful in a wide range of applications from microarray-based gene mapping to identification of chromosomal mutations. In addition to the clones themselves, we describe a database, GenMapDB (http://genomics.med.upenn.edu/genmapdb), that contains information about each clone in our collection.

Universal linkage system: an improved method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) has become a powerful technique for studying gains and losses of DNA sequences in solid tumors. Importantly, DNA derived from archival tumor tissue is also applicable in CGH analysis. However, DNA isolated from routinely processed, formalin-fixed, paraffin-embedded tissue is often degraded, with the bulk of DNA showing fragment sizes of only 400-750 bp. Enzymatic labeling of archival DNA by standard nick translation (NT) decreases DNA size even further, until it becomes too small for CGH (<300 bp). This study presents application in CGH of a commercially available, non-enzymatic labeling method, called Universal Linkage System (ULS), that leaves the DNA fragment size intact. To compare the effect of chemical labeling of archival DNA by ULS vs. enzymatic by NT on the quality of CGH, DNA derived from 16 tumors was labeled by both ULS and NT. In those cases (n = 8), in which the bulk of DNA had a fragment size of 400-1,000 bp, CGH was successful with ULS-labeled probes, but not with NT-labeled probes. In the DNA samples (n = 6) with a fragment size > 1 kb, the intensity of CGH signals was comparable for both ULS- and NT-labeled probes, but CGH with ULS-labeled samples showed a high, speckled, background, which seriously hampered image analysis. In the remaining two cases, which had evenly distributed DNA fragment sizes (range 250-5,000 bp), CGH was successful with both labeling methods. Using DNA fragment size < 1 kb as a selection criterion for ULS labeling, we were able to obtain good quality CGH of a large panel (n = 77) of a variety of archival solid tumors. We conclude that ULS is an excellent labeling method for performing CGH on small-fragment-sized DNA.

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling.

Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH. We introduce the principle of COBRA (COmbined Binary RAtio labelling), which is based on the simultaneous use of combinatorial labelling and ratio labelling. Human chromosome painting in 24 colours is accomplished using four fluorophores only. Three fluorophores are used pair wise for ratio labelling of a set of 12 chromosome painting probes. The second set of 12 probes is labelled identically but is also given a binary label (fourth fluorophore). The COBRA method is demonstrated on normal human chromosomes and on a lymphoma (JVM) cell line, using probes enzymatically labelled with fluorescein, lissamine and cy5 as primary fluorophores, and diethylaminocoumarin (DEAC), a blue dye, as combinatorial fourth label to demonstrate incorporated digoxigenin. In addition, the principle was tested using chemical labelling. The first set of 12 painting probes was therefore labelled by ULS (Universal Linkage System), using DEAC, cy3 and cy5 as primary labels, and the second set was labelled similarly, but also contained a digoxigenin-ULS label, which was indirectly stained with fluorescein. Subsequently, a mathematical analysis is presented and methods are indicated for achieving an MFISH multiplicity of 48, 96 or even higher using existing technology.