Abstract: 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.

Abstract: Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.

Abstract: Mutations in the mitochondrial tRNA(Leu(UUR)) gene are associated with a large variety of human diseases through a largely undisclosed mechanism. The A3243G tRNA(Leu(UUR)) mutation leads to reduction of mitochondrial DNA (mtDNA)-encoded proteins and oxidative phosphorylation activity even when the cells are competent in mitochondrial translation. These two aspects led to the suggestion that a dominant negative factor may underlie the diversity of disease expression. Here we test the hypothesis that A3243G tRNA(Leu(UUR)) generates such a dominant negative gain-of-function defect through misincorporation of amino acids at UUR codons of mtDNA-encoded proteins. Using an anti-complex IV immunocapture technique and mass spectrometry, we show that the mtDNA-encoded cytochrome c oxidase I (COX I) and COX II exist exclusively with the correct amino acid sequences in A3243G cells in a misassembled complex IV. A dominant negative component therefore cannot account for disease phenotype, leaving tissue-specific accumulation by mtDNA segregation as the most likely cause of variable mitochondrial disease expression.

Abstract: PURPOSE: To evaluate the clinical relevance of genomic aberrations in primary cutaneous large B-cell lymphoma (PCLBCL). PATIENTS AND METHODS: Skin biopsy samples of 31 patients with a PCLBCL classified as either primary cutaneous follicle center lymphoma (PCFCL; n = 19) or PCLBCL, leg type (n = 12), according to the WHO-European Organisation for Research and Treatment of Cancer (EORTC) classification, were investigated using array-based comparative genomic hybridization, fluorescence in situ hybridization (FISH), and examination of promoter hypermethylation. RESULTS: The most recurrent alterations in PCFCL were high-level DNA amplifications at 2p16.1 (63%) and deletion of chromosome 14q32.33 (68%). FISH analysis confirmed c-REL amplification in patients with gains at 2p16.1. In PCLBCL, leg type, most prominent aberrations were a high-level DNA amplification of 18q21.31-q21.33 (67%), including the BCL-2 and MALT1 genes as confirmed by FISH, and deletions of a small region within 9p21.3 containing the CDKN2A, CDKN2B, and NSG-x genes. Homozygous deletion of 9p21.3 was detected in five of 12 patients with PCLBCL, leg type, but in zero of 19 patients with PCFCL. Complete methylation of the promoter region of the CDKN2A gene was demonstrated in one PCLBCL, leg type, patient with hemizygous deletion, in one patient without deletion, but in zero of 19 patients with PCFCL. Seven of seven PCLBCL, leg type, patients with deletion of 9p21.3 and/or complete methylation of CDKN2A died as a result of their lymphoma. CONCLUSION: Our results demonstrate prominent differences in chromosomal alterations between PCFCL and PCLBCL, leg type, that support their classification as separate entities within the WHO-EORTC scheme. Inactivation of CDKN2A by either deletion or methylation of its promoter could be an important prognostic parameter for the group of PCLBCL, leg type.

Abstract: The 3243A>G mutation in mitochondrial DNA (mtDNA) is a genetic variant that is associated with a high risk of developing diabetes during life. Enhanced aging of pancreatic beta-cells, a reduced capacity of these cells to synthesize large amounts of insulin,and a resetting of the ATP/ADP-regulated K-channel seem to be the pathogenic factors involved.

Abstract: 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.

Abstract: Chromosomal translocations involving the immunoglobulin (Ig) receptor loci usually disrupt and silence these loci. On the basis of observations in follicular lymphoma (FL) with downstream Ig heavy chain (IGH) class switch recombination (CSR), we hypothesized that downstream CSR-mediated chromosomal translocations would leave the V(D)J-Cmu transcription unit intact, thereby still allowing IgM expression from the IGH allele involved in the translocation. To test this hypothesis, we analyzed biallelic IGH translocations in the IgM-expressing cell line Z-138 by interphase FISH, DNA fiber-FISH, long-distance vectorette PCR, and DNA sequencing. One IGH allele was involved in a t(11;14), showing a break in the JH region that juxtaposed the Emu enhancer and the 3' Calpha enhancers to the cyclin D1 gene. The other IGH allele contained a t(8;14) breakpoint involving the 3' end of a Sgamma region, whereas the reciprocal breakpoint at 8q24 was approximately 40 kb centromeric of MYC. Molecular analysis showed that this IGH allele harbored a normal V(D)J-Cmu complex, which is responsible for IgM expression. These data show that chromosomal breakpoints such as the t(8;14) can occur in downstream IGH constant regions and do not necessarily interfere with Ig expression.

Abstract: Knowledge of how molecules interact in space and time is crucial for understanding cellular processes. A host of novel techniques have been developed for the visualisation of single target molecules in living cells, many based on fluorescence in situ hybridisation (FISH) or immunocytochemistry (IC). To extend the applicability of FISH to living cells, special backbone-modified probes and specific conformations (molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding and the use of quantum dots, which have proven potential for cellular labelling.

Abstract: We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver-operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6-BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.

Abstract: The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.

Abstract: The hSNF5 subunit of human SWI/SNF ATP-dependent chromatin remodeling complexes is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). Here, we report that loss of hSNF5 function in MRT-derived cells leads to polyploidization and chromosomal instability. Re-expression of hSNF5 restored the coupling between cell cycle progression and ploidy checkpoints. In contrast, cancer-associated hSNF5 mutants harboring specific single amino acid substitutions exacerbated poly- and aneuploidization, due to abrogated chromosome segregation. We found that hSNF5 activates the mitotic checkpoint through the p16INK4a-cyclinD/CDK4-pRb-E2F pathway. These results establish that poly- and aneuploidy of tumor cells can result from mutations in a chromatin remodeler.

Abstract: Cytogenetic studies in a patient with acute myelocytic leukemia (AML) revealed as the sole karyotypic alteration a half-cryptic rearrangement, identified with 48-color combined binary ratio-labeled fluorescence in situ hybridization (pq-COBRA-FISH) as a reciprocal t(6;14)(q?;q?). The breakpoints were later assigned on the basis of G-banding to t(6;14)(q25-q26;q32). FISH experiments using genomic probes showed that the breakpoint on 14q32.2 was within bacterial artificial chromosome RP11-782I5 and revealed BCL11B as the only candidate gene in the region. BCL11B is a homolog to BCL11A (2p13), a highly conserved gene implicated in mouse and human leukemias. To our knowledge, this is the first report implicating BCL11B in hematological malignancies. Because of lack of material, the translocation partner remains unknown.

Abstract: Fluorescent cis-platinum compounds that react with the N7 atom of guanine are useful for labeling nucleic acids influorescence hybridization applications. Here we report that cyanin (CyN) cis-platinum labeling of DNA samples for array comparative genomic hybridizations (arrayCGH) can be achieved reproducibly and reliably. We demonstrate that degrees of labeling of approximately 1% of all nucleotides in test and reference DNA samples with CyN3- and CyN5-cis-platinum produces arrayCGH signal-to-background ratios ranging from 30 to 40. The arrayCGH results achieved during analyses of mouse and human tumor samples were comparable to those achieved using enzymatic labeling. Thus, we conclude that Cy-cis-platinum labeling is an alternative to enzymatic labeling for arrayCGH.

Abstract: Mutations in mitochondrial DNA (mtDNA) associate with various disease states. A few mtDNA mutations strongly associate with diabetes, with the most common mutation being the A3243G mutation in the mitochondrial DNA-encoded tRNA(Leu,UUR) gene. This article describes clinical characteristics of mitochondrial diabetes and its molecular diagnosis. Furthermore, it outlines recent developments in the pathophysiological and molecular mechanisms leading to a diabetic state. A gradual development of pancreatic beta-cell dysfunction upon aging, rather than insulin resistance, is the main mechanism in developing glucose intolerance. Carriers of the A3243G mutation show during a hyperglycemic clamp at 10 mmol/l glucose a marked reduction in first- and second-phase insulin secretion compared with noncarriers. The molecular mechanism by which the A3243G mutation affects insulin secretion may involve an attenuation of cytosolic ADP/ATP levels leading to a resetting of the glucose sensor in the pancreatic beta-cell, such as in maturity-onset diabetes of the young (MODY)-2 patients with mutations in glucokinase. Unlike in MODY2, which is a nonprogressive form of diabetes, mitochondrial diabetes does show a pronounced age-dependent deterioration of pancreatic function indicating involvement of additional processes. Furthermore, one would expect that all mtDNA mutations that affect ATP synthesis lead to diabetes. This is in contrast to clinical observations. The origin of the age-dependent deterioration of pancreatic function in carriers of the A3243G mutation and the contribution of ATP and other mitochondrion-derived factors such as reactive oxygen species to the development of diabetes is discussed.

Abstract: Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.

Abstract: A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.

Abstract: Molecular cytogenetic analysis frequently shows human papillomavirus (HPV) integration near translocation breakpoints in cervical cancer cells. We have recently described a cluster of HPV18 integrations in the distal end of the common fragile site FRA8C at 8q24 in primary cervical carcinoma samples. Chromosome band 8q24 contains the MYC gene (alias c-MYC), FRA8C, and FRA8D. The MYC gene is frequently deregulated--usually by translocation or amplification--in various tumor types. In the present study, we performed a molecular cytogenetic analysis of HPV18 integration patterns and the 8q24 translocation in a primary cervical carcinoma and in HeLa cells using combined binary ratio-fluorescence in situ hybridization. Our aim was to determine how the chromosomal breaks involved in these events relate physically to the MYC gene; whether they map to the FRA8C site, the FRA8D site, or both; and how they correlate with the occurrence of DNA flexibility domains. The 8q24 translocation breakpoints mapped between stretches of integrated HPV18 sequences in the distal end of FRA8C. This region contained DNA helix flexibility clusters, several of which mapped in the vicinity of HPV integration sites and translocation breakpoints in cervical carcinomas. DNA helix flexibility clusters were also found near known MYC translocation breakpoints in Burkitt lymphomas (BL), but most BL breakpoints mapped clearly outside FRA8C. Our data revealed that FRA8C is involved in HPV integration and chromosomal translocations in cervical carcinoma; however, this fragile site is not involved in classical MYC translocations in most BLs. In the context of the familial nature of cervical cancer, FRA8C may be considered a candidate susceptibility region for cervical carcinoma.

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Abstract: 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.

Abstract: 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.

Abstract: Fluorescent and haptenized, monofunctionally binding platinum compounds are increasingly used for chemically labeling nucleic acids for hybridization detection purposes. Here we analyze in detail the effect of labeling density of the cyanin-3 and -5 platinum DNA adducts on fluorescence and thermal stability. We also analyzed the kinetics of the reaction of the cyanin platinum compounds with DNA. The data provided are important for the design of optimal platinum DNA labeling and hybridization conditions for fluorescence hybridization applications.

Abstract: Chemical induction of premature chromosome condensation (PCC) was investigated and optimized to be able to analyze the chromosomal constitution of cancer cells independent of mitosis and with minimal culture artifacts. A potent protein phosphatase inhibitor, calyculin A, was used to induce PCC in normal diploid cells, in several established human tumor cell lines, and in cells isolated from freshly dissected adenomatous polyps of a patient with hereditary colorectal cancer. In parallel, mitotic arrest was pursued by use of Colcemid. In cell lines, a difference of up to 10-fold was found between frequency of cells with PCC induced by calyculin A (PCC index) and the mitotic index after treatment with Colcemid. In the fresh tumor specimens, Colcemid failed to result in metaphase formation, whereas a regimen of 80 nM calyculin A for 75 min, after only 2 days of culturing, resulted in a PCC index of 2-5%. pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization) was used for a detailed analysis of four cell lines treated with calyculin A, which proved that PCC spreads are amenable to molecular karyotyping, and a comparison between PCC spreads and metaphases from mitotic arrest revealed no discrepancies in karyotypes. pq-COBRA-FISH on PCC spreads from fresh colon tumor samples revealed only numerical and no structural abnormalities. Calyculin A-induced PCC combined with multicolor FISH gives a new opportunity for analysis of the chromosomal constitution of G(1) and G(2) cancer cells and may find application in the study of the role of chromosome instability in cancer development.

Abstract: Cryptic subtelomeric chromosome rearrangements play an important role in the aetiology of mental retardation, congenital anomalies, miscarriages and neoplasia. To facilitate a comprehensive molecular-cytogenetic analysis of these extremely gene-rich and mutation-prone chromosome regions, novel multicolour fluorescence in situ hybridisation (FISH) techniques are being developed. As yet, subtelomeric FISH methods have either had limited multiplicities, making it necessary to perform many hybridisations per patient, or a limited scope of analysable chromosome mutation types, thus not detecting some aberration types such as pericentric inversions or very small aberrations. COBRA (COmbined Binary RAtio) labelling is a generic multicolour FISH technique that combines ratio and combinatorial labelling to attain especially high multiplicities with few fluorochromes. The Subtelomere COBRA FISH method ("S-COBRA FISH") described here detects efficiently all 41 BAC and PAC FISH probes necessary for a complete subtelomere screening in only two hybridisations. It was applied to the analysis of 10 cases with known and partially known aberrations and successfully detected balanced and unbalanced translocations, deletions and an unbalanced pericentric inversion in a mosaic situation. The ability of S-COBRA FISH to efficiently detect all types of balanced and unbalanced subtelomeric chromosome aberrations makes it the most comprehensive diagnostic procedure for human subtelomeric chromosome regions described to date.

Abstract: Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.

Abstract: Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.

Abstract: 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.

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Abstract: We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.

Abstract: Growth hormone (GH) has direct effects on the growth plate to stimulate longitudinal growth, but it is not clear which chondrocyte populations GH acts on. The dual effector theory suggests that GH would act primarily on the "stem cells." However, staining with a GH receptor (GHR) antibody is found in all layers of the growth plate in rabbits and humans. We now have investigated the localization and regulation of GHR and the related GH binding protein (GHBP) in the rat growth plate using a sensitive immunohistochemical method involving tyramide signal amplification (TSA) and antibodies specific for GHR or GHBP. Both GHR and GHBP were shown in the germinal and proliferative chondrocytes, but most clearly in early maturing chondrocytes at the interface between proliferative and hypertrophic cells. Staining for GHR and GHBP was located in both the cytoplasm and the nucleus. Expression of GHR mRNA and GHBP mRNA in the growth plate was confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining for GHR and GHBP decreased with age; in 12-week-old normal rats, only the early maturing chondrocytes were stained. In GH-deficient dwarf rats, staining seemed less than in normal rats, and in hypophysectomized (Hx) rats, staining for GHBP was clearly reduced. Treatment of Hx rats with thyroid hormones (T3 + T4), via subcutaneously (sc) implanted osmotic minipumps, induced little growth and induced a small layer of GHR-positive and GHBP-positive early maturing chondrocytes. Treatment with GH and thyroid hormones (TH) resulted in greater growth and a broader layer of GHR-positive and GHBP-positive cells, indistinguishable from normal rats. In contrast, dexamethasone treatment of normal rats inhibited their growth and reduced GHR and GHBP staining in the growth plate. These results show that GHR and GHBP in the growth plate are under hormonal control. The localization of GHR/GHBP suggests that in addition to actions on germinal and proliferative cells in young rats, GH also has effects on early maturing chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte.

Abstract: 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.

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

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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)

Abstract: Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene has limited the possibilities for a FISH assay to an approach based on colocalization of probes for BCL2 and the immunoglobulin heavy chain (IGH) locus. Intrinsically high rates of false positive nuclei and high interobserver variability make such assays unsuitable for use on lymphoma tissue samples, where tumor cells often form only a minority of the cell population. Using YAC end cloning techniques and screening of a PAC library, we have isolated PAC clones flanking the BCL2 gene. Using these PACs, and several cosmid clones in the second BCL2 intron, we developed a segregation-based interphase FISH assay with two probe combinations enabling separate detection of 5' and 3' (mbr/mcr) breakpoints. The assay was applied to a series of 40 follicular lymphomas. To evaluate the results, the same lymphomas were analyzed by DNA fiber FISH with a 600-kb set of BCL2 DNA clones labeled in alternating colors in combination with a color barcode covering the IGH locus. This approach allowed precise mapping of BCL2 breakpoints, and simultaneously showed juxtaposition of IGH genes to BCL2. Comparison of the results of interphase and fiber FISH showed complete correlation. Five cases were negative with both FISH techniques as well as with Southern blotting. Interestingly, all of these 5 cases lacked BCL2 overexpression as determined by immunohistochemistry, against 3 of 35 rearrangement-positive follicular lymphomas. Furthermore, absence of t(14;18) seemed to be correlated with a higher histologic grade (grades 2 and 3 according to Berard). These data indicate that the segregation-based interphase FISH assay detects 100% of BCL2 rearrangements. Because interpretation of the results is straightforward and requires no extensive experience, this assay may be the best available diagnostic test for BCL2 rearrangement. Genes Chromosomes Cancer 27:85-94, 2000.

Abstract: 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.

Abstract: 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.

Abstract: The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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. This was confirmed by double staining of cells with anti-digoxin and anti-alpha-tubulin antibodies. We concluded that anti-digoxin-specific antibody shows crossreactivity with a component present in the midbody.

Abstract: Telomeres are G/C-rich repetitive DNA sequences at the end of all eukaryotic chromosomes. The loss of telomeric repeat sequences during cell divisions has been proposed as a possible mechanism for cell senescence. The standard procedure for measurement of telomere length is Southern blot (SB) hybridization with a telomere-specific probe. However, in using this technique no information can be obtained on variation in telomeric fragments due to interchromosomal, intrachromosomal, and intercellular differences. Lansdorp et al. (Hum Mol Genet 5:685-691, 1996) developed a method to measure individual telomeres, using in situ hybridization on metaphase chromosomes, employing peptide nucleic acid (PNA) probes and digital fluorescence microscopy. In this paper we describe a method that can be used to assess telomeric length in interphase cells. An algorithm was developed to measure the total intranuclear fluorescence in situ hybridization (FISH) signal, which features accurate correction for the local autofluorescence. Application of this methodology to samples of fetal liver, umbilical cord blood, and adult bone marrow cells showed a gradual decrease of average telomeric length. Southern blot analysis and PNA FISH measurements on chromosomes in the same samples showed similar results. Advantages of interphase measurements include the possibility of studying nonproliferating cells, thus avoiding selection and cell culturing.

Abstract: 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. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

Abstract: No data on the chromosomal constitution of spermatocytic seminomas are available thus far because of their rarity. Ploidy analysis performed on paraffin-embedded cases showed varying results from (near-) diploid to aneuploid. We applied comparative genomic hybridization on four snap-frozen primary spermatocytic seminomas of three different patients. Conventional cytogenetic analysis was successful in one, and "interphase cytogenetics" with centromeric region-specific probes was applied to another. The results from comparative genomic hybridization showed almost exclusively numerical chromosomal aberrations, in agreement with the data from karyotyping. Despite the limited number of cases studied, a nonrandom pattern of chromosome imbalances was detected: chromosome 9 was gained in all spermatocytic seminomas. This suggests that that this aberration plays a role in the development of this cancer. Interphase cytogenetics shows that the copy number of most chromosomes ranges from two to four, with an average of near trisomic. This constitutes the first report on the chromosomal constitution of spermatocytic seminomas.

Abstract: To further define the structure of the pericentromeric region of human chromosome 7, we have identified and characterized a YAC clone (YAC 311.H5) containing the D7S1480 locus, which maps to the short arm near the centromere of this chromosome, by linkage in CEPH families and radiation hybrid analysis. This YAC contains two new blocks of alphoid DNA (named Z5 and Z6). Both Z5 and Z6 show monomeric structures and a lack of higher-order repeats, and, therefore, belong to suprachromosomal family type 4 (M1). The orientation of the two blocks and the physical distances over the region were defined by pulsed-field gel electrophoresis (PFGE) and fluorescence in situ hybridization on chromatin fibers (FiberFISH). A YAC contig spanning the centromeric region has been developed by STS content.

Abstract: 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.

Abstract: 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.

Abstract: Biotin deoxyuridine 5'-triphosphate (dUTP) and digoxigenin dUTP are the labels most commonly used in comparative genomic hybridization (CGH). The relative infrequent use of direct fluorochrome-labeled nucleotides in CGH is related to the lower sensitivity they provide. Here we report the evaluation of two fluorochrome-conjugated nucleotides that have not been previously used in CGH (lissamine-5-dUTP and fluorescein-N6dATP) and show that this direct label combination performs at least as well as the indirect biotin/digoxigenin pair.

Abstract: Immunoglobulin class switching usually involves deletion of part of the immunoglobulin CH region. By DNA fiber fluorescence in situ hybridization (FISH) with a barcode of probes covering the DH, JH, and CH genes, the configuration of the entire CH region can be visualized on single DNA molecules. Using this technique, we have studied class switching in three types of B-cell neoplasia, mantle-cell lymphoma (MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL), representing B cells in, respectively, pregerminal center, germinal center, and postgerminal center stages of development. In MCL and FL, simultaneous detection of the t(11;14) and t(14;18) breakpoint with probes for the BCL-1 and BCL-2 loci, respectively, allowed differentiation between productive and nonproductive alleles. In none of 10 MCL cases was class switching detected. In 21 HCL, all nonimmunoglobulin M (IgM) cases had class-switch deletion consistent with the expressed isotype on at least one allele. In FL, however, a peculiar pattern of CH rearrangement was observed. In IgM expressing FL, the translocated alleles had switched in 11 of 13 cases, and the nontranslocated allele showed complex rearrangements downstream from the Cmu-Cdelta genes in 9 of 13 cases. These downstream rearrangements may reflect tumor-specific deregulation of the class-switch machinery. All seven immunoglobulin G (IgG) expressing FL showed class switching on both alleles. Fiber FISH analysis also showed several polymorphisms. The most frequent one, present on 38% of all analyzed alleles, consisted of an extra Cgamma gene or pseudogene in the 3' cluster.

Abstract: The techniques of in situ hybridization (ISH) are widely applied for analyzing the genetic make-up and RNA expression patterns of individual cells. This review focusses on a number of advances made over the last 5 years in the fluorescence ISH (FISH) field, i.e., Fiber-FISH, Multi-colour chromosome painting, Comparative Genomic Hybridization, Tyramide Signal Amplification and FISH with Polypeptide Nucleic Acid and Padlock probes.

Abstract: 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.

Abstract: Hypotriploidy/hyperdiploidy ("intermediate ploidy") often occurs in testicular germ cell tumors of adolescents and adults. Disomic and trisomic chromosomes represent significant parts of the tumor genome and a few chromosomes fall outside the two- to three-copy number range. We performed comparative genomic hybridization (CGH) with DNA isolated from a cell line from a case of testicular germ cell tumor of adolescents and adults and found most of the ratio values to be dislocated from the baseline 1.0 and placed adjacent of the diagnostic thresholds of 0.8 and 1.2. We attributed that to the fact that, in current software packages for analysis of CGH, the fluorescence ratio baseline is assumed to correspond to the copy number of most loci of the genome. We then evaluated, instead of the commonly used fluorescent ratio value from the whole metaphase, the use of the fluorescence ratios of single chromosomes. The results permitted a clear distinction between the chromosomes with two and three copies and, in particular, of the regions deleted or amplified outside the two- to three-copy range. We concluded that the evaluation of unbalances of DNA copy number in intermediate ploidy cases is best carried out using multiple normalization.

Abstract: The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

Abstract:

Abstract: Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin's lymphoma, which has been recently recognized and defined as a disease entity. We investigated 25 cases (18 at primary diagnosis and seven during the course of disease) of MZBCL by comparative genomic hybridization (CGH) and compared these results with cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data. Twenty of the 25 cases (80%) showed gains (total 49) or losses (total 15) of genetic material. In extranodal, nodal, and splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). High-level amplifications involving the regions 18q21-23 and 18q21-22, respectively, were detected in two cases. Gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p occurred more frequently in relapsed or progressive lymphomas. For all of the frequently affected chromosomes, CGH allowed narrowing of the relevant subregions including 3q21-23, 3q25-29 and 18q21-23. By Southern blot analysis, the BCL2, BCL6, and CMYC proto-oncogenes were found to be a part of the over-represented regions in two cases, one case, and two cases, respectively, with gains involving 18q, 3q or 8q. In 13 cases, CGH revealed chromosomal imbalances which were not detected by cytogenetic analysis but could be confirmed by FISH or Southern blot analysis in all cases investigated. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low proportion of tumor cells bearing these abnormalities, as shown by interphase FISH. The characteristic pattern of chromosomal gains and losses detected in this study confirms the distinct nature of MZBCL and may point to chromosomal regions involved in the pathogenesis of these neoplasms.

Abstract: 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.

Abstract: The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

Abstract: DNA fibers can be released from cell suspensions and frozen tissue and can be used as a template for hybridization with multiple differently labeled probes. Simultaneous hybridization with 5-10 adjacent or partly overlapping probes generates a highly specific "color barcode" for individual DNA segments. Rearrangements in this barcode can be easily detected and mapped. The resolution of DNA fiber FISH is between 2 and 500kb. In mixing experiments of cell lines with different structural abnormalities, we found a sensitivity of approximately 10%. We applied DNA fiber FISH for detection of t(11;14) in mantle cell lymphoma (MCL) and immunoglobulin (Ig) class switching in hairy cell leukemia (HCL). Using a barcode for the Ig and BCL-1 loci at 14q32 and 11q13, we detected and mapped a t(11;14) breakpoint in 35 of 36 MCL. In 5 cases complex mono-allelic rearrangements at both sides of the cyclin D1 gene were identified. In 13 HCL with phenotypic evidence of Ig class switching, including 2 cases with solely IgD, fiber FISH revealed concordant Ig class switch deletions. In most cases both alleles were affected. These results indicate that DNA fiber FISH is a very powerful method to detect and map structural DNA alterations.

Abstract: 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. Combined detection of HCMV-IE mRNA/DNA and splicing factors in rat 9G cells that can be induced for IE gene expression shows that IE genes are not associated with speckled regions enriched for splicing factors when transcriptionally inactive, but 'attract' splicing factors when transcriptionally activated. This process proved reversible after transcription inhibition. IE transcripts appeared to be retained near the transcription site in track-like domains by splicing factors associated with them until splicing has been completed. Double-hybridization experiments revealed that a substantial part of the accumulated transcripts contain a poly(A) tail suggesting that most, if not all, IE transcripts are polyadenylated at the site of transcription. These results indicate that RNA processing may occur independent of the position of the gene in the cell nucleus relative to speckle domains.

Abstract: The relevant microscopical conditions for the optimal visualization of ratio-color FISH stained cells were investigated. Special attention was given to the influence of the type of illumination, (semi)-critical vs. Köhler type illumination, in combination with the use of multi-band excitation and emission filters, on the registration of the colors of ratio labelled probes. Due to chromatic errors, many collecting lenses were found to cause a wavelength dependent excitation pattern with critical illumination. This resulted in a change of the observed color of microscopic objects when stained with a mixture of two dyes and excited with a dual band pass filter. A quantitative study of this effect for semi-critical illumination of FISH ratio-labelled chromosomes revealed a difference of 20% between highest and lowest ratio values depending on the position of the object in the microscopic field vs. only 2.5% for Köhler type of illumination. The impact of these errors on the identification of ratio-labelled probes and on the sensitivity of comparative genomic hybridization (CGH) to detect gene amplifications or losses is discussed. Standard preparations consisting of solutions of defined mixtures of fluorescent dyes or objects stained with defined ratios of fluorophores, are proposed to correct for the errors observed.

Abstract: We have prepared avidin-labelled antibodies ('shuttles') with the aim of increasing the sensitivity of detecting mouse IgG and human complement factors in ELISA tests and of detecting monoclonal antibodies and digoxigenin haptens (DIG) in hybridization and immunoblot procedures. Avidin-D was conjugated to goat IgG anti-mouse IgG or to anti-digoxigenin antibodies by thiol/maleimide chemistry. Conjugates of different molecular weight were obtained by Superdex 200 gel filtration. The avidin-D-labelled antibodies were then incubated with biotinylated horseradish peroxidase or with biotinylated alkaline phosphatase. Such preformed enzyme-labelled complexes were subsequently used in the various assays. A 5-8-fold increase in sensitivity was found when the preformed enzyme-labelled antibody-avidin-D complexes were compared to directly enzyme-labelled antibodies or antibody fragments. Furthermore it was shown that ELISA procedures employing digoxigenin-labelled polyclonal antibodies detected by shuttle conjugates were approximately five times more sensitive than biotinylated antibodies detected by avidin-biotin complexes (ABC method). The greatest sensitivity was obtained using antibody-avidin complexes which consisted of two IgG molecules and 4-6 avidin-D molecules.

Abstract: We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

Abstract: Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparations techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference. AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (-15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).

Abstract: Rearrangements within the chromosome 11q13 region are frequent in hematologic malignancies. 50% of 75% of mantle cell lymphomas (MCLs) carry a translocation t(11;14) (q13;q32). Using Southern blot analysis, a BCL1 breakpoint can be detected in approximately 50% of MCLs. It is not known whether other MCLs harbor also breakpoints at 11q13. Breakpoints in this region not involved in t(11;14), are detected in chronic lymphocytic leukemia and acute myeloid leukemia. To detect and localize breakpoints at 11q13 more accurately, we have developed fluorescence in situ hybridization using two probe sets of differently labeled cosmids, symmetrically localized at either side of the major translocation cluster of BCL1. These probes span a region of 450 to 750 kb. We applied this assay to a series of hematologic malignancies with 11q13 abnormalities identified by classical cytogenetics. All four samples with a t(11;14) (q13;q32) showed dissociation of the differently colored signals in metaphase and interphase cells, thereby indicating a chromosomal break in the region defined by the probe sets. The frequency of abnormal metaphase and interphase cells was comparable with that observed in any of the 13 malignancies with other chromosomal 11q13 abnormalities, indicating that these chromosomal breaks occurred outside the 450- to 750-kb region covered by the probes. One patient showed triplication and one patient showed monoallelic loss of this region. The current data show that double-color fluorescence in situ hybridization is a simple and reliable method for detection of the t(11;14)(q13;q32) in interphase cell nuclei and that is can be used to distinguish this translocation from other 11q13 rearrangements in hematologic malignancies.

Abstract: The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibroblasts (rat 9G). Experimental variables to balance penetration of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was obtained using mild cross-linking fixation followed by treatment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.

Abstract: Fluorescence in situ hybridization (FISH) applied to metaphase chromosomes provides a mapping resolution of 1 to 3 Mb. FISH applied to interphase nuclei has a resolution of 50 kb and ranges 1-2 Mb. This better resolution is attributed to the higher degree of chromatin decondensation. Here, we describe FISH applied to naked DNA fibers (fiber FISH) and show that with such fully decondensed chromatin a resolution range of at least 1-400 kb can be obtained. Furthermore, we show that DNA fiber FISH provides a mapping tool that is highly supplementary to restriction mapping, because it permits very accurate gap and overlap sizing. Also, DNA fiber FISH provides the means to generate "color bar codes" for disease regions, which can be used to inspect patient DNAs for suspected gene rearrangements.

Abstract: In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5-10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction.

Abstract: With the advent of in situ hybridization techniques for the analysis of chromosome copy number or structure in interphase cells, the diagnostic and prognostic potential of cytogenetics has been augmented considerably. In theory, the strategies for detection of cytogenetically aberrant cells by in situ hybridization are simple and straightforward. In practice, however, they are fallible, because false classification of hybridization spot number or patterns occurs. When a decision has to be made on molecular cytogenetic normalcy or abnormalcy of a cell sample, the problem of false classification becomes particularly prominent if the fraction of aberrant cells is relatively small. In such mosaic situations, often > 200 cells have to be evaluated to reach a statistical sound figure. The manual enumeration of in situ hybridization spots in many cells in many patient samples is tedious. Assistance in the evaluation process by automation of microscope functions and image analysis techniques is, therefore, strongly indicated. Next to research and development of microscope hardware, camera technology, and image analysis, the optimization of the specimen for the (semi)automated microscopic analysis is essential, since factors such as cell density, thickness, and overlap have dramatic influences on the speed and complexity of the analysis process. Here we describe experiments that have led to a protocol for blood cell specimen that results in microscope preparations that are well suited for automated molecular cytogenetic analysis.

Abstract: 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.

Abstract: The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients. In all cases, the DNA index assessed by a propidium iodide/CD38 double-staining technique with flow cytometry was simultaneously investigated for correlation, with fluorescence in situ hybridization results. Additional aims of this study were 1) to analyze whether the abnormalities detected were common to all plasma cells or were present in only a subpopulation of tumor cells, 2) to explore changes caused by disease progression, and 3) to establish possible associations among the altered chromosomes. Although the overall incidence of numerical abnormalities was 67%, this frequency increased to 80% in the 41 cases in which 7 or more chromosomes were analyzed. Trisomies were significantly more common than monosomies (84% versus 16%). Chromosomes 9 and 15 were the most frequently altered (52% and 48% of cases, respectively), with all of their abnormalities corresponding to trisomies. The most frequent losses involved chromosomes 13 (26%) and X in females (32%). Other common numerical changes corresponded to chromosomes 1 (39%), 11 (37%), 6 (32%), 3 (31%), 18 (29%), 7 (28%), and 17 (22%). By contrast, chromosomes 8(13%), 10(8%), and 12(3%) were rarely altered. DNA aneuploidy by flow cytometry was detected in 67% of patients, and a high degree of correlation was observed between the DNA index obtained by flow cytometry and the chromosome index derived from fluorescence in situ hybridization studies, calculated according to two mathematical formulas (coefficient of correlation of 0.82 and 0.91 when at least 7 or 12 chromosomes were considered, respectively). The frequency of numeric chromosome aberrations was higher in those patients with progressive disease and, interestingly, trisomy of chromosome 8 was exclusively detected in this latter group of patients. Our study shows that, with the exception of chromosome 8, a possible marker of clonal evolution, the numeric chromosome changes are present in nearly all malignant plasma cells (r > 0.84). Finally, frequent associations between chromosomal aberrations were observed (ie, chromosomes 6, 7, 9, and 17; 7 and 15; and 11 and 17). By excluding them, it was found that two triple combinations of chromosome-specific probes, chromosomes 1 and 9 together with either chromosome 13 or 15, could be a useful marker for detection of residual disease, as it permits the identification of most MM patients displaying numerical changes.

Abstract: Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.

Abstract: In this study we systematically assessed the sensitivity limits of fiber-FISH in model experiments. Exonic fragments and cDNAs with exon sizes of >/=200 bp could be mapped on their cognate cosmid. This positional fiber-FISH mapping was validated by long-range PCR. It is expected that these two independent mapping approaches will help to refine current available gene maps and show their applicability in fine mapping of sequence-tagged sites or expressed sequence tags. Also, they will be useful in resolving gene structures by mapping exon and intron locations.

Abstract: We investigated the feasibility of automated counting of in situ hybridization signals (ISH) in interphase cells isolated from paraffin embedded prostate tissue. In total, 34 specimens from 7 patients with prostate cancer were stained with probes specific for the centromeric regions of chromosomes Y, 1, 7, 8, 10, and 15, using an immunoperoxidase based technique suitable for bright-field microscopy. Enumeration of the number of ISH spots of 500 nuclei per specimen was performed (1) using an automatic system developed without any human intervention and (2) using the same system, but including verification of the counts based on visual inspection of the stored images. As reference from each specimen, 200 cell nuclei were evaluated manually, using conventional microscopy. A typical analysis procedure (including user verification) took 35 min. The difference (root mean error) between the automated counting and the counting after visual interaction was relatively small (15%). The percentage of cells with incorrect counts by automated analysis was 20.2%, a number that could easily be improved by user interaction. Detection of cells with aneusomy proved to be more sensitive compared to the routine manual counting, in cases where aberrant frequencies were low. Automated counting of samples with low frequencies (< 10%) resulted in a higher frequency of aberrant cells in 9 of 11 cases, probably due to the fact that an unbiased cell selection is guaranteed. Automated assessment of ISH signals is considered useful for the evaluation of chromosomal aberrations in prostate tumor cells, provided that the counts are visually confirmed.

Abstract: Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.

Abstract: Recently, a peroxidase-mediated amplification system has been described for immunofluorescence and fluorescence in situ hybridization studies. It is based on the deposition of hapten- or fluorochrome-labeled tyramide molecules. Although providing a significantly increased detection sensitivity compared to conventional procedures, its localization properties are inferior because of free diffusion of intermediate reaction products before they are immobilized. In enzyme cytochemistry, it is well established that improved localization of enzyme activity can be achieved through the addition of viscosity-increasing polymers to the incubation media. In this study we analyzed the effect of different polymers on the localization sharpness and sensitivity of the tyramide-peroxidase reaction in FISH applications. Significantly improved localization of the fluorescent endproduct was observed using dextran sulfate or polyvinylalcohol (PVA) with, respectively, no or little loss of sensitivity.

Abstract: Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.

Abstract: Vertebrate chromosomes terminate in variable numbers of T2AG3 nucleotide repeats. In order to study telomere repeats at individual chromosomes, we developed novel, quantitative fluorescence in situ hybridization procedures using labeled (C3TA2)3 peptide nucleic acid and digital imaging microscopy. Telomere fluorescence intensity values from metaphase chromosomes of cultured human hematopoietic cells decreased with the replication history of the cells, varied up to six-fold within a metaphase, and were similar between sister chromatid telomeres. Surprisingly, telomere fluorescence intensity values within normal adult bone marrow metaphases did not show a normal distribution, suggesting that a minimum number of repeats at each telomere is required and/or maintained during normal hematopoiesis.

Abstract: Twenty-seven samples (cell cultures prepared for routine cytogenetics) of leukemia patients with known cytogenetic abnormalities were stained by in situ hybridization for interphase cytogenetics with centromere specific probes for chromosome Nos 4, 6, 7, 8, 9, 12, 17, 18, X and Y. The number of hybridization domains per nucleus was quantified using a semi-automated system developed in our laboratory. Results of this automated counting procedure (with and without verification of the counting results by the operator) were compared with conventional cytogenetic data and with visual scoring of the number of hybridization dots. The findings show that the system is capable of analysing 1000 cell nuclei in less than 30 min, including the necessary verification of the results by the operator. Automated counting and visual scoring were in good agreement. Conventional cytogenetics and interphase cytogenetics agreed in only 50% of the cases, confirming other studies showing that conventional cytogenetic results are not always representative for the majority of the cell population.

Abstract: The cellular origin of the sarcomatous component of gliosarcomas is controversial. It is not clear if the sarcoma arises in transition from the glial cells that comprise the gliomatous component or independently arises from non-neoplastic mesenchymal cells of the tumor stroma. Using comparative genomic hybridization (CGH) along with cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis, and polymerase chain reaction (PCR) analysis of microsatellite allelic imbalance, we have evaluated the genetic alterations in the gliomatous and sarcomatous components of five gliosarcomas. The glial element was grade 4 fibrillary astrocytoma (glioblastoma multiforme) in all five tumors. The sarcoma elements were fibroblastic without osseous, chondroid, or angiosarcomatous differentiation. Gain of chromosome 7, loss of chromosome 10, deletions of the chromosome 9 p-arm, and alterations of chromosome 3 were frequently observed, demonstrating that gliosarcomas can be genetically classified as belonging to the spectrum of glioblastomas. Furthermore, the sarcomatous and gliomatous portions of each gliosarcoma investigated were similar with respect to both the presence and absence of specific genetic alterations. This observation supports the hypothesis that the sarcomatous component of a gliosarcoma either arises from the same common precursor cell as the gliomatous portion, or it arises from the gliomatous portion itself.

Abstract: Fluorescent in situ hybridization (FISH) is a powerful, direct and sensitive technique with a wide resolution range that enables the simultaneous study of multiple targets, labelled in different colours. Spreading techniques, denoted here as 'Fiber-FISH', increase FISH-resolution to the DNA fiber, using decondensed nuclear DNA as hybridization target. FISH could be a powerful analytical tool for thorough physical examination of yeast artificial chromosomes (YACs) which are often chimaeric or contain internal deletions. However, with one exception restricted to meiotic yeast chromosomes, FISH has not been used successfully on yeast/YAC DNA. We have developed a fast and simple method that can be applied routinely for compositional and structural analysis of cosmid and YAC DNA in yeast. It enables precise localization and ordering of clones, resolves overlaps and distances and gives a detailed picture of the integrity and colinearity of both probe and target. The combination of high resolution, signal abundance and short yeast cell cycle allows direct visualization of replicating DNA fibers. In a 400 kb region of the human dystrophin gene, we identified two replication origins, demonstrating that human DNA cloned in yeast is capable of initiating its own replication.

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Abstract: Molecular biology techniques allow the unraveling of the genetic alterations that cause or accompany malignant disease. Since tumors are often heterogeneous, biochemical analysis of tissue homogenates is of limited diagnostic value. This paper gives examples of methods that are presently operational to analyze the genetic composition of individual cells. They are based on fluorescence in situ hybridization (FISH) and digital imaging microscopy. First, the current status of indirect and direct FISH staining methods with respect to probe labeling, detection sensitivity, multiplicity, and DNA resolution is summarized. Microscope hardware as well as charge-coupled device (CCD) cameras required for FISH analysis are then described. Applications potentially important for the analysis of urological malignancies, such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells) and high-resolution DNA mapping on highly extended chromatin, are described in detail. Finally, the limitations of the present methodology and its future prospects are discussed.

Abstract: We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.

Abstract: We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.

Abstract: Fluorescence in situ hybridization (FISH) has profoundly altered the aspect of genome research and molecular diagnostics. Deletions of only a few kilobases can be detected by hybridizing probes to naked DNA fibers. Loss or gain of chromosomal material in tumor cells can be visualized using comparative genome hybridization. Further diversification of FISH application will result from new ultrasensitive detection techniques.

Abstract: Genes for Epstein-Barr virus, human cytomegalovirus immediate early antigen and luciferase are abundantly transcribed in Namalwa, rat 9G and X1 cells, respectively. The EBV transcripts and HCMV-IE transcripts are extensively spliced, while in the luciferase transcript only a small intron sequence has to be spliced out. EBV transcripts are hardly localized in the cytoplasm while the luciferase and HCMV-IE transcripts are present in the cytoplasm and translated into proteins. We have correlated these characteristics with nuclear RNA distribution patterns as seen by fluorescence in situ hybridization. Transcripts of the HCMV-IE transcription unit were shown to be present in a main nuclear signal in the form of a track or elongated dot and as small nuclear RNA signals that radiate from this site towards the cytoplasm. A similar distribution pattern of small RNA signals was observed for transcripts of the luciferase gene, whereas the main nuclear signal was always observed as a dot and never as a track or elongated dot. In Namalwa cells, EBV transcripts were only present as track-like signals. The results suggest that when the extent for splicing is high, unspliced or partially spliced mRNAs begin to occupy elongated dot or track-like domains in the vicinity of the gene. When the extent of splicing is low, splicing is completed co-transcriptionally, leading to a bright dot-like signal. The presence of small nuclear spots in addition to the main signal correlates with cytoplasmic mRNA expression. The small spots most likely represent, therefore, mRNAs in transport to the cytoplasm.

Abstract: We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.

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Abstract: We have applied two-colour fluorescence in situ hybridization (FISH) to DNA fibers and combined it with digital imaging microscopy for the mapping of large cosmid contigs. The technique was validated using a set of unique plasmids and a cosmid contig both originating from the thyroglobulin (Tg) gene and previously mapped by restriction analysis. The resolution proved to be close to the theoretical lower limit of approximately 1 kb, ranging > or = 400 kb. Subsequently a 400 kb cosmid contig derived from a DMD-YAC was directly mapped by Fiber-FISH. The resulting map is in full agreement with the restriction map. Two-colour Fiber-FISH mapping thus showed to be capable for accurately sizing gaps and overlaps, and to identify chimeric or repeat sequence containing cosmids across a 400 kb region at once. The generated 400 kb 'colour bar-code' was subsequently used to map two DMD deletion breakpoints in patient DNA with an accuracy of 1-2 kb. The results underscore the value of this method for the delineation of chromosomal rearrangements for positional cloning and single patient clinical studies.

Abstract: 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.

Abstract: This paper reviews methods and applications of CCD microscopy for analysing cells and chromosomes subjected to fluorescence in situ hybridization (FISH). The current status of indirect and direct FISH staining methods with respect to probe labelling, detection sensitivity, multiplicity and DNA resolution is summarized. Microscope hardware, including special multi-band pass filters and CCD cameras required for FISH analysis, is described. Then follows a detailed discussion of current and emerging applications such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells), comparative genomic hybridization, automated evaluation of radiation-induced chromosomal translocations, and high-resolution DNA mapping on highly extended chromatin. Finally, the limitations of the present methodology and future prospects are discussed.

Abstract: The utility of p-phenylenediamine, 1,4-di-azobicyclo-(2.2.2.)-octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half-life times of 96, 330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH 8.5), these half-life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes.

Abstract: We have used a fluorescent in situ hybridization procedure to detect human satellite 1 DNA, the simple sequence family that constitutes the non-male-specific fraction of classical satellite 1 DNA. Satellite 1 appears to be located on pericentromeric regions of chromosomes 3, 4 and 13, and on satellites of each acrocentric chromosome. These results suggest a possible relationship between quinacrine fluorescence of heterochromatin and DNA composition. Furthermore, by means of multicolour in situ hybridization, we have spatially resolved satellite 1 sequences and centromeric alpha-satellite within heterochromatic blocks.

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Abstract: Human cytomegalovirus (HCMV) infections cause considerable morbidity and mortality in immunocompromised patients. During HCMV infection, leukocytes appear in the circulation in low frequencies that express the HCMV pp65 protein antigen. Since there is evidence that changes in the frequency of antigen-positive cells in the early phase of the infection have prognostic value, we applied automated image cytometry to quantify these antigen-positive cells. For this purpose weekly peripheral blood leukocyte samples of 80 kidney transplant recipients were visually examined for the presence of antigen-positive cells using an immunocytochemical detection method. Seventeen patients, who reacted positive with this assay, were identified. Next, automated image cytometry was applied to quantitate the frequency of antigen-positive cells in sequential blood samples from the 17 patients. Patients who developed a period of HCMV viremia had a significantly longer antigenemic period and a significantly higher frequency of antigen-positive cells than patients with a HCMV infection who remained nonviremic. Therefore, automated image immunocytometry based screening can be used to distinguish patients at risk for the development of a HCMV viremia. Moreover, automated quantitation reveals prognostic information about the HCMV infection at a more sensitive level than other HCMV detection techniques.

Abstract: A cell detection method based upon automated screening is described for recognition of low frequencies (1 in 100,000) of immuno-enzymatically labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide-field, fully automated microscope (Autoplan) and a modular image analysis computer (MIAC), both from Leica, Wetzlar, Germany. The MIAC contains image boards for optimum use of mathematical morphology algorithms. Communication with the MIAC is via a personal computer. Programs for automated cell analysis have been written in C language. Main features of the system are fast analysis of large microscope fields including a count of all cells, selection of objects of interest (alarms), and display of digitally stored images of these alarms. We tested this system for the detection of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno-enzymatic (peroxidase) staining was performed with DAB and counterstaining with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. The lowest frequency detected was 1 antigen-positive cell/3 x 10(5) antigen-negative cells. Screening time was about 60 min for one million cells.

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Abstract: The human interleukin-6 receptor alpha chain (IL6R alpha) gene was regionally mapped to chromosome 1 band q21 by fluorescence in situ hybridization. As hybridization probes, partially overlapping lambda clones encompassing 28 kb of the genomic region of the gene were used. These clones were isolated using a polymerase chain reaction (PCR)-generated fragment of the 3' non-coding region of the gene. This localization confirms and extends the provisional assignment of the IL6R alpha gene to chromosome 1, for which a panel of somatic cell hybrids was used.

Abstract: The human genome is made up of long DNA segments, the isochores, which are compositionally homogeneous and can be subdivided into a small number of families characterized by different G+C levels. Chromosome in situ suppression hybridization (in which excess unlabeled human DNA is added to suppress hybridization of repeated sequences present in the probe, enabling enhanced observation of single-copy sequences) of DNA fractions characterized by an increasing G+C level was carried out to determine the distribution of "single-copy" sequences corresponding to isochore families L1+L2, H1, H2, and H3 on metaphase chromosomes. This produced a banding pattern progressing from a relatively diffuse staining to an R-banding, to a T-banding. More specifically, our results showed that (i) T-bands are formed by the G+C-richest isochores of the H3 family and by part of the G+C-rich isochores of the H1 and H2 families (with a predominance of the latter); (ii) R'-bands (namely, R-bands exclusive of T-bands) are formed to almost equal extents by G+C-rich isochores of the H1 families (with a minor contribution of the H2 and H3 families) and by G+C-poor isochores of the L1+L2 families; (iii) G-bands essentially consist of G+C-poor isochores from the L1+L2 families, with a minor contribution of isochores from the H1 family. These results not only clarify the correlations between DNA base composition and chromosomal bands but also provide information on the distribution of genes in chromosomes, gene concentration increasing with the G+C levels of isochores.

Abstract: A large number of human hematopoietic growth factor and growth factor receptor genes are localized at the long arm of chromosome 5. In this study we have determined the order of the human interleukin-3 (IL3), IL4, IL5, IL9, granulocyte macrophage-colony stimulating factor (GMCSF), and the MCSF receptor (MCSFR) genes by fluorescence in situ hybridization. Genomic lambda-clones were isolated using polymerase chain reaction (PCR)-generated probes and labeled with biotin and/or digoxigenin. These clones were first individually mapped: IL3, IL4, IL5, IL9, and GMCSF to 5q31 and MCSFR to 5q33. For ordering purposes multiple probe combinations were hybridized to metaphase chromosomes and interphase nuclei. The interphase hybridizations were evaluated by image analysis, which also allowed the measurement of the physical distance between the hybridization spots. These mapping results suggest the gene order 5cen-IL3/GMCSF-IL5-IL4-IL9-MCSFR+ ++-qter. The known genomic distance between the IL4 and IL5 genes allowed the estimation of the physical distances between the 5q31-specific genes, demonstrating that they are all within about 1.5 Mb of DNA.

Abstract: We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

Abstract: Atomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1-79 results in clear marking of the telomere region 1p36. The diameter of the probe p1-79 linked reaction product was 75-100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material.

Abstract: We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifluorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and, to the human eye, the green signals appeared less bright. With a blue fluorescent coumarin-labeled dUTP, only highly repetitive target sequences could be visualized directly. Imaging with a cooled CCD (charged coupled device) camera for prolonged integration times permitted localization of small targets using probes labeled with red and green fluorochromes. Visualization of a 1.8-kb single copy sequence with a rhodamine-labeled cDNA proved feasible. The strategy of identifying hybridization sites with multiple colors was successfully applied for multiple hybridizations to repetitive and unique targets, using the red and green labels directly and the biotin label indirectly with one layer of avidin-coumarin.

Abstract: Malignant cells from 24 cases of hairy cell leukemia were studied by in situ hybridization for evidence of selective aneuploidy using alphoid and satellite probes specific for 16 human chromosomes. Based on these data, hairy cell leukemia appears to be diploid for the chromosomes studied and is a malignancy which displays the phenomenon of pairing of the centromere and p arm of chromosome 15 during interphase.

Abstract: The technique of in situ hybridization has been used to evaluate the expression of an ovulation hormone mRNA (caudodorsal cell hormone; CDCH) in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. Hybridization with radioactive as well as with nonradioactive labeled oligonucleotide and plasmid probes revealed a specific labeling on cell bodies of caudodorsal cells (CDCs), which are known to produce CDCH, on the light microscopical level. In addition, specific labeling was observed outside the cell bodies, as far as the cerebral commissure, where CDCH is released in the haemolymph. To investigate whether these signals represent an axonal localization of the CDCH mRNA, we performed in situ hybridization at the electron microscopical (EM) level. The results showed an intraaxonal localization of CDCH mRNA with digoxigenin labeled oligonucleotide and plasmid probes. Gold labeling was observed in secretion granules, and double labeling experiments showed that these granules also contain CDCH. This specific intragranular localization suggest that CDCH mRNA is transported through the axon and released by exocytosis in the haemolymph.

Abstract: Normal human bone marrow and peripheral blood leukocytes as well as malignant cells from a variety of leukemias and lymphomas, demonstrate somatic pairing of centromeres and p arms of chromosome 15 during interphase. This phenomenon, effected by sequences on the p arm and requiring the intranuclear transport of spatial domains for at least one of the homologs, was not seen in amniotic fluid cells, uterine cervical tissue or in tissue fibroblasts. These studies contribute to the recent evidence of somatic pairing of homologous chromosomes in man and provide support for mobile chromosomal domains in interphase. It appears that sequences on the p arm of chromosome 15, possibly the nucleolar organizing genes, are uniquely important in the maturation of benign and malignant cells of hemato-lymphopoietic origin.

Abstract: The pericentric inversion of chromosome 16 and the t(16;16) are two recurrent aberrations in bone marrow of patients with acute nonlymphocytic leukemia subtype M4 Eo, characterized by abnormal eosinophilic granulation. We describe here the precise localization of the breakpoints using fluorescence in situ hybridization (FISH) with cosmids spread over the short arm of chromosome 16 and the detection, isolation and characterization of a 14Kb EcoRI fragment containing a cluster of breakpoints. First, cosmids were mapped to intervals defined by constitutional 16p rearrangements, second, the inv(16) and t(16;16) breakpoints were mapped to one of the intervals using FISH with the mapped cosmids and third, cosmids within this interval were ordered using two color interphase FISH. An STS of the cosmid closest to the breakpoints was then used to isolate five YACs, which did span all of the 16 inv(16) breakpoints and one t(16;16) breakpoint analysed. In the DNA of one inv(16) patient we detected an additional submicroscopic deletion immediately proximal to the 16p breakpoint. Since this patient has the same phenotype, the 16p sequences proximal to the breakpoint seem non-essential to M4 Eo. This implies that the pathologic event is the juxtaposition of sequences distal to the 16p breakpoint with sequences proximal to the 16q breakpoint. While four of the five YACs showed instability of the region around the inv(16) breakpoint, DNA halo analysis allowed us to identify one YAC which was co-linear with normal genomic DNA and has yielded the actual breakpoint sequences which could be subcloned into cosmids and fosmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The proto-oncogene bmi-1 is frequently activated by Moloney murine leukemia proviral insertions in E mu-myc transgenic mice1,2. Using a mouse bmi-1 cDNA probe a transcript of 3.3 kb was detected on Northern blots of human Burkitt's lymphoma cell lines. We have isolated and sequenced cDNA clones from a human erythroleukemia cell line (K562) derived cDNA library, using different mouse bmi-1 cDNA fragments as a probe. Analysis of genomic BMI-1 sequences reveals a gene structure which is very similar to that of the mouse, consisting of at least 10 exons. The human cDNA is 3203 bp in length and shows 86% identity to the mouse nucleotide sequence. The open reading frame encodes a protein of 326 amino acids which shares 98% identity to the amino acid sequence of mouse bmi-1 protein. In vitro translation experiments show that human cDNA derived RNA translates into a protein with a mobility of 44-46 kD on SDS polyacrylamide gels. Fluorescence in situ hybridization (FISH) on metaphase chromosome spreads located the human BMI-1 gene to the short arm of chromosome 10 (10p13), a region known to be involved in translocations in various leukemias.

Abstract: In situ hybridization (ISH) techniques on interphase cells, or interphase cytogenetics, have powerful potential clinical and biological applications, such as detection of minimal residual disease, early relapse, and the study of clonal evolution and expansion in neoplasia. Much attention has been paid to issues related to ISH data acquisition, i.e., the numbers, colors, intensities, and spatial relationships of hybridization signals. The methodology concerning data analysis, which is of prime importance for clinical applications, however, is less well investigated. We have studied the latter for the detection of small monosomic and trisomic cell populations using various mixtures of human female and male cells. With a chromosome X specific probe, the male cells stimulated monosomic subpopulations of 0, 1, 5, 10, 50, 90, 95, 99, and 100%. Analogously, when a (7 + Y) specific probe combination was used, containing a mixture of chromosome No. 7 and Y-specific DNA, the male cells simulated trisomic cell populations. Probes specific for chromosomes Nos. 1, 7, 8, and 9 were used for estimation of ISH artifacts. Three statistical tests, the Kolmogorov-Smirnov test, the multiple-proportion test, and the z'-max test, were applied to the empirical data using the control data as a reference for ISH artifacts. The Kolmogorov-Smirnov test was found to be inferior for discrimination of small monosomic or trisomic cell populations. The other two tests showed that when 400 cells were evaluated, and using selected control probes, monosomy X could be detected at a frequency of 5% aberrant cells, and trisomy 7 + Y at a frequency of 1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Interphase cytogenetics is the application of nonradioactive in situ hybridization with chromosome-specific DNA probes to interphase nuclei. The possibilities and limitations of this new technique for the study of chromosomal aberrations in gliomas are discussed.

Abstract: The development of the inflammatory response within the brain, meninges and cerebrospinal fluid (CSF) compartment has been studied for the first time simultaneously in experimental herpes simplex virus (HSV) encephalitis after inoculation via the cornea. Two major viral pathways were found from the eye to the brain: one through the trigeminal nerve to the brain stem and one through the nasolacrimal duct to the olfactory system. Viral antigen was found to be present in the CNS before there were clinical signs or cellular infiltration of brain tissue. Subsequently, the virus spread to all parts of the trigeminal brain stem complex. This phenomenon was accompanied by severe inflammation of the meninges covering the trigeminal root near its entry into the brain stem. The meninges near the entry of the olfactory fila also contained antigen. However, HSV-1 did not spread along meningeal rami of the trigeminal nerve and, consequently, is--at least in this experimental model--not a route to reach the inferior frontal and temporal lobes. The development of CSF changes followed the histopathological development of meningitis and encephalitis closely. HSV-DNA could be detected in the CSF from day 4 post inoculation (p.i.) and HSV-1-specific immunofluorescence in CSF cells was convincingly present on day 5 p.i.; on the same days (4 and 5 p.i.) inflammatory cells were found in apposition to infected cells in the brain. We postulate that HSV is carried to the CSF by infected leukocytes rather than a direct spread to the CSF by simple extension of the encephalitic process to the meningeal surface.(ABSTRACT TRUNCATED AT 250 WORDS)

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Abstract: In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.

Abstract: Interphase cytogenetics is the application of nonradioactive in situ hybridization with chromosome-specific DNA probes to interphase nuclei. In this study, interphase cytogenetics was used to investigate 66 primary brain tumors (33 gliomas, 30 meningiomas, and three medulloblastomas) for numerical chromosomal aberrations of chromosomes 1, 6, 7, 10, 11, 17, 18, X, and Y. Of the 33 gliomas (17 astrocytomas grades II, III, and IV, five oligoastrocytomas, seven oligodendrogliomas, and four ependymal tumors), 22 were near diploid, while the remaining 11 showed a significant triploid or tetraploid component. The predominant specific aberrations in gliomas were an over-representation of chromosome 7 (13 cases) and an under-representation of chromosome 10 (16 cases). These changes were observed in grade III and grade IV astrocytomas, as well as in oligodendrogliomas. Other frequent numerical changes were a gain of chromosome 17 (six cases) and a loss of chromosome 18 (seven cases). This loss of chromosome 18 seemed relatively specific for gliomas with an oligodendroglial component (six cases). Only two of 33 gliomas displayed no genetic abnormality with the probes used. Seven patients with astrocytomas died of their brain tumor during the clinical follow-up period. Their astrocytomas did not show a different chromosomal constitution compared to the other gliomas. For the meningiomas, the probe panel was extended with a probe specific for chromosome 22. Loss of chromosome 22 was obvious in 21 of the 30 meningiomas, and was the sole abnormality in 11 meningiomas; in the other 10, this loss was associated with other chromosomal changes. Five of these tumors with additional aberrations were recurrent or atypical meningiomas. It is suggested that interphase cytogenetics can contribute to a better understanding of the biological behavior of these tumors and possibly result in better insights into prognosis and strategies for therapy.

Abstract: To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 microns, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5-10 cells only. The resolution range of the technique is at least 10-200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.

Abstract: Fluorescence in situ hybridization (FISH) is now widely used for the localization of genomic DNA fragments, and the identification of chromosomes by painting. We now show that half of the chromosomal complement can be painted in twelve different colors by using human chromosome specific libraries carrying three distinct labels mixed in multiple ratios. The photographs are in 'real' color rather than 'colorized'. The painting technique described here can be used for the identification of small or complex chromosomal rearrangements and marker chromosomes in humans or in any other species for which well defined chromosome specific libraries exist in a laboratory equipped with a conventional fluorescence microscope. The versatility of this novel cytogenetic technology may well constitute an advancement comparable to the introduction of chromosome banding and high resolution analysis of chromosomes in prometaphase.

Abstract: The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

Abstract: The development of a sensitive, non-isotopic filter hybridization method based on the peroxidase catalyzed luminol reaction is described. High sensitivity was achieved by optimizing the conditions of the hybridization procedure, the immunochemical detection and the peroxidase/luminol reaction. This resulted in the reproducible detection of 10-30 femtogram of target DNA on blots within minutes when a cooled charge coupled device (CCD) camera was used to record the luminescence signal. After optimalization, the system was successfully applied for the qualitative and quantitative analysis of small amounts of DNA in dot-blots as well as in Southern blot analysis.

Abstract: Bone marrow cells of four patients with t(1;7) and myelodysplasia or acute myeloid leukemia were analyzed using nonradioactive in situ hydridisation. As probes, centromeric alphoid DNA sequences of chromosomes 1 and 7, a satellite DNA probe for 1q12, and chromosome-specific libraries of chromosomes 1 and 7 were used. The breakpoints of the t(1;7)(p11;p11) as determined by banding analysis could be studied more accurately, and the recently proposed designation t(1;7)(cen;cen) was confirmed in all four cases. Colocalization of alphoid DNA sequences of chromosomes 1 and 7 by double target in situ hybridisation was demonstrated in metaphase cells and also in interphase nuclei. The in situ hybridisation method described is applicable for the screening of peripheral blood cells or archival material.

Abstract: Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.

Abstract: This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.

Abstract: In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.

Abstract: An alphoid repetitive DNA (D8Z2) probe specific for the pericentromeric region of chromosome 8 was used to detect extra copies of chromosome 8 in bone marrow cells obtained from 10 patients with hematological disorders and five controls. Numerical aberrations of chromosome 8 were established by conventional banding techniques. Trisomy 8 was found in four patients with myelodysplastic syndrome (MDS) and three with acute myeloid leukemia (AML). Three additional patients with MDS exhibited an extra chromosome 8 in only one metaphase. In five of the seven trisomy cases, the presence of the trisomy 8 clone was confirmed by in situ hybridization (ISH). In one case of AML with trisomy 8, detected by GTG-banding, no significant numbers of cells containing three spots were found using the alphoid repetitive probe; however, hybridization with a chromosome 8-specific library revealed that the alleged extra chromosome 8 was a translocation chromosome containing only the long arm of chromosome 8. Due to a lack of material, it was not possible to achieve optimal ISH results on the trisomy 8 bone marrow cells of patient 7. In the three MDS patients with a single trisomy 8 metaphase, a slight, albeit significant, increase of trisomy 8 interphase cells was found with ISH. We conclude that this probe is useful for cytogenetic studies. Moreover, ISH, in general, is a powerful tool for precise classification of chromosomal aberrations and can also contribute significantly to the clinical evaluation of patients with hematological disorders.

Abstract: Normal lymphocyte prometaphase chromosome spreads were hybridized in situ using single- and double-color fluorescence techniques. The results obtained with either the 1.8-kb protective protein cDNA or a 12-kb genomic fragment of the human protective protein gene as probe demonstrate that the PPGB gene is localized on the long arm of chromosome 20. This assignment was confirmed by hybridization with whole chromosome DNA libraries.

Abstract: Chromosome aberrations of a hypodiploid ovarian carcinoma cell line (modal chromosome number 38) having a complex karyotype were analyzed using biotinylated DNA library probes that specifically hybridize to chromosomes 3, 6, 7, 8, 11, 13, and 16 from telomere (pter) to telomere (qter). A series of cosmid probes localized to the short arm of chromosome 16 were used to further investigate one of the two aberrant chromosomes 16 present in this cell line. The competitive in situ suppression (CISS) hybridization of DNA-libraries was mostly performed subsequent to GTG-banding of the same metaphase cell in order to interpret the hybridization signals optimally. This combined approach made it possible to detect the origin of chromosomal material that could not be identified using GTG-banding. Furthermore, the in situ hybridization techniques appeared to be helpful in the characterization of complex translocations and for accurate breakpoint determination.

Abstract: We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.

Abstract: We have used fluorescein-, digoxigenin- and biotin-(di)deoxyXTPs and terminal deoxynucleotidyl transferase for small scale labeling of synthetic oligonucleotide probes and here we show the applicability of such probes for the in situ detection of multiple RNA sequences. The enzymatic 3'-end-labeling methods proved to be good alternatives for the chemical fluorochrome and hapten labeling of 5'-end alkylamino-derivatized oligonucleotides. By combining 3'-end fluorescein-, biotin-, and digoxigenin-labeled oligonucleotides, double and triple hybridizations are feasible. For example, we demonstrated simultaneously mRNAs coding for caudodorsal cell hormone, a molluscan insulin-related peptide, and 28 S ribosomal RNA in cryostat sections of the cerebral ganglia of the pond snail Lymnaea stagnalis.

Abstract: Forty-five Swiss albino mice were inoculated with HSV-1 (strain McKrae) by the corneal route. The spread of HSV to the brain stem and the ventricular ependyma was investigated. The polymerase chain reaction (PCR) was used to detect HSV-DNA in the CSF during the course of infection. The ependyma of the third and fourth ventricle and the central canal contained viral antigen at a late stage of infection in up to 60% of mice. At this stage we found that many animals gave a positive PCR in the CSF although no antigen could be detected in the ependymal cells. The presence or absence of antigen containing cells could not be related to detection of HSV-DNA in the CSF. The results show that infection of the ventricular wall is not important for the spread of HSV to the CSF.

Abstract: The feasibility was studied of in situ hybridisation using chromosome specific DNA probes on paraffin wax embedded normal and malignant tissues from different organs. Both isolated nuclei and 5 microns sections were used in in situ hybridisation experiments with biotinylated repetitive DNA probes specific for the centromeric regions of chromosomes 1 and 17. The hybridisation results were visualised with peroxidase-diaminobenzidine. The optimal pretreatments with sodium thiocyanate and pepsin were experimentally defined for the different tissues. Although interphase cytogenetics on paraffin wax embedded tissue is possible, the results indicate that it has its limitations, compared with investigations on fresh tumour tissue.

Abstract: The technique of nonradioactive in situ hybridization has been used to visualize the DNA and mRNA expression of human cytomegalovirus (HCMV) immediate early antigen (IEA) in a transfected rat fibroblast cell line. Expression of the transfected HCMV immediate early DNA can be induced by a cycloheximide treatment and is S-phase-dependent. In addition to cytoplasmic mRNA localization, a nuclear RNA hybridization signal was found. In a substantial part of the cells the nuclear signal was in the form of a "track," possibly showing transport of IEA mRNA from the site of transcription to the cytoplasm. The use of PCR-generated intron- and exon-specific probes in a double hybridization revealed that intron and exon mRNA sequences coexist in the nuclear RNA signal. This shows the applicability of multiple-color fluorescence hybridization to obtain information about the site of pre-mRNA splicing in the nucleus. In addition, by combining the technique of in situ hybridization with an immunocytochemical procedure we illustrate the possibility of visualizing transcribed mRNAs simultaneously with their translation products.

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Abstract: Nuclei isolated from normal human brain tissue, collected from six autopsies, were hybridized with a panel of nine satellite DNA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, and the X and Y chromosomes. The results did not confirm the recently reported trisomy 7 and loss of sex chromosomes observed in metaphases obtained from normal brain tissue after short-term cultures; however, cells of all six brains displayed somatic pairing of the chromosome 17 centromeres in approximately 50% of the nuclei.

Abstract: The development and application of a procedure for interphase cytogenetics on brain tumor material is described. Nuclei isolated from freshly removed brain tumor tissue were investigated for chromosomal aberrations by nonradioactive in situ hybridization with a panel of chromosome-specific probes. The panel consisted of nine satellite DNA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, X, and Y. For each probe, the number of hybridization signals per cell was determined in 200 nuclei. It was inferred from the hybridization results that in 11 gliomas (seven astrocytomas grade II-IV, three oligodendrogliomas, and one ependymoma) the numerical aberrations were gains of chromosomes 1 (once), 7 (twice), 10 (once), 11 (twice), and X (twice); losses of chromosomes 1 (once), 10 (twice), 17 (twice), and Y (once); and complete tetraploidy (once). Among the 18 investigated meningiomas monosomy 18 and trisomy 17 were observed once and twice, respectively. An additional hybridization with a cosmid probe for the BCR gene on 22q11 indicated monosomy 22q in 11 meningiomas. These results show the value of interphase cytogenetics for the analysis of solid tumors for which it is relatively difficult to obtain sufficient metaphases of good quality for conventional cytogenetics.

Abstract: A series of experiments was carried out using a mouse HSV-1 encephalitis model for detecting HSV-DNA in CSF by the polymerase chain reaction (PCR). The results were correlated with the time period after corneal inoculation, clinical signs and symptoms, and progression of infection in brain tissue (antigen detection and histopathology), and were compared with virus isolation by spin-amplified culture (SAC/IF). The PCR proved to be superior to SAC/IF, both with respect to early detection and the percentage of positive samples. The results of PCR did not correlate with signs of disease, nor with the interval post-inoculation. A correlation with the spread of HSV within the brain tissue was found. The possible pathways for virus spread from brain parenchyma to the CSF as well as a possible explanation for the low amounts of virus and viral-DNA within the CSF are discussed.

Abstract: Interstitial pneumonia after allogeneic bone marrow transplantation is frequently associated with human cytomegalovirus (HCMV) infection. However, in a considerable proportion of the cases, no infectious agent can be determined and the interstitial pneumonia is then classified as idiopathic. Hypothetically, idiopathic interstitial pneumonia could be caused by HCMV present in such small amounts or such conformation that the virus cannot be detected by routine histopathologic analysis or viral culture techniques. To check this hypothesis, three sensitive methods for HCMV detection (in situ hybridization, the polymerase chain reaction for HCMV-DNA detection, and immunohistochemistry for the detection of HCMV immediate early antigens) have been applied on lung tissue sections of bone marrow transplant patients who died with interstitial pneumonia. Three categories were distinguished: (1) patients with HCMV-related interstitial pneumonia (n = 5); (2) patients with idiopathic interstitial pneumonia (n = 10); and (3) patients with HCMV interstitial pneumonia who had been treated with antiviral therapy (n = 2). In the first category, all three techniques yielded clearly positive results, whereas these techniques indicated that one of the patients of the second category had HCMV-related pneumonia. In the third category no positive signals could be obtained. The presented data indicate that a direct involvement of HCMV in idiopathic interstitial pneumonia is unlikely. However, a PCR performed for Epstein-Barr virus (EBV) was positive in two patients with idiopathic interstitial pneumonia. These data indicate that the introduction of new sensitive techniques such as in situ hybridization, immunohistochemistry, and the polymerase chain reaction revives the interest for HCMV and other causative infectious agents in idiopathic interstitial pneumonia.

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Abstract: In two female patients with a 45,X/46,X, +mar karyotype the marker chromosomes were identified as normal length nonfluorescent Y chromosomes (nlYnf) using non-isotopic in situ hybridization (NISH) complementary to routine cytogenetic analysis and Southern hybridization. The recognition of the nlYnf as isodicentric in both patients illustrates and confirms the usefulness and importance of NISH in the identification and characterization of this and many other types of complex chromosome rearrangements.

Abstract: Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.

Abstract: Double fluorescence in situ hybridization was used to detect Philadelphia (Ph) chromosomes in interphase nuclei and metaphases of patients with chronic myeloid leukemia. Application of cosmid probes for 3' ABL and 5' BCR sequences gave better results than libraries for chromosomes 9 and 22. The present approach may provide an alternative method for monitoring minimal residual disease in Ph+ CML patients.

Abstract: DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescence microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.

Abstract: Nonradioactive in situ hybridization (ISH) using biotinylated centromere probes for chromosomes 1, 6, 7, 10, 16, 17, 18, and the X, respectively, was combined with GTG-banding to study cytogenetic changes in two different ovarian cancer cell lines. ISH was performed after GTG-banding on the same metaphase. The use of a low trypsin concentration (0.01%) in the banding procedure was essential for subsequent ISH. This combined approach allows the detection of subtle chromosomal rearrangements and appears to aid the identification of marker chromosomes.

Abstract: A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

Abstract: A method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC. Three chromosome-specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined. Three-color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.

Abstract: Attempts were made to detect human parvovirus B19-DNA by in situ hybridisation and the polymerase chain reaction in placental and fetal tissues from a case of intrauterine fetal death. In the in situ hybridisation experiments radioactive and non-radioactive (labelled with 2-acetyl-aminofluorene, AAF) DNA probes were used. B19-DNA was detectable in paraffin wax embedded fetal tissue from the liver, heart, lung, brain and thymus. The resolution with the AAF-labelled probes was higher than with the radiolabelled DNA. Parvovirus B19 DNA sequences were also detected in these tissues by an enzymatic in vitro amplification technique--the polymerase chain reaction. Amplification of a B19-DNA sequence before detection increases the rapidity and sensitivity of detection. The rapid, specific, and sensitive analysis of parvovirus B19 in normal and diseased tissues using these techniques may contribute considerably to determining the role of this virus as a risk factor in the outcome of pregnancy.

Abstract: In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.

Abstract: Peripheral blood leukocyte samples (n = 458) of 24 bone marrow transplant and 52 kidney transplant patients were examined weekly for the presence of human cytomegalovirus (HCMV) using an improved culture technique (DEAFF; detection of early antigen fluorescent foci). In total 5 (21%) bone marrow transplant and 11 (21%) kidney transplant patients developed a viremia. Patients' samples were investigated for the presence of HCMV DNA using an in vitro DNA amplification technique, the polymerase chain reaction (PCR). From the statistically evaluable viremic patients (n = 13), 110 blood samples were analyzed. In 5 of these patients, the DEAFF and PCR led to identical results. In 8 patients however the PCR was more sensitive, i.e. HCMV DNA was detected for a longer period of time. Applying statistical analysis using the McNemar test, this result was significant (P less than 0.05). The PCR applied on leukocyte samples did not detect HCMV DNA in viruric patients without viremia. Moreover, the current PCR never led to positive results with peripheral blood leukocyte samples of healthy seropositive or seronegative controls. Since the PCR can be performed in 6 hr, this technique will contribute to rapid detection of HCMV DNA in peripheral blood leukocytes and therefore to optimal clinical management of HCMV-infected transplant recipients.

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Abstract: A recently described immunoperoxidase method for the detection of nuclear human cytomegalovirus (HCMV) immediate early antigen (IEA) directly on peripheral blood leucocytes suffers from the drawback that the antigen is vulnerable to endogenous peroxidase inactivation procedures. To solve this problem a procedure is developed in which endogenous peroxidase is inactivated after binding and immobilization of the primary antibody with 4% formaldehyde. In combination with this procedure, three types of inactivation were investigated: glucose/glucose oxidase, hydrochloric acid and methanol/H2O2. Of these three, the first gives optimal results, especially in combination with methanol/acetic acid (20/1 v/v) as the primary fixative. This procedure results in preparations which allow for a more objective evaluation and enable automated examination using bright field microscopy. As a second improvement we developed a simple adherence method in order to diminish the risk of infection for the laboratory staff during processing of unknown blood samples. The protocol described shows great clinical potential for the diagnosis of HCMV infections.

Abstract: To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

Abstract: The results of a comparison of three DNA-detection methods for human parvovirus B19 DNA are described. The sensitivity of detection of virus from hybridization assays using 32P-radiolabeled DNA and RNA probes was compared with a method for enzymatically amplifying specific target DNA sequences (polymerase chain reaction). B19 virus DNA was detected using a radiolabeled DNA probe at serum dilutions of 10(-3), equivalent to approximately 3 pg of viral DNA. Using radiolabeled RNA probes even 0.3 pg of viral DNA was detectable. The polymerase chain reaction was more sensitive than the hybridization assays: 100 fg of viral DNA was easily detectable by electrophoresis on agarose and after subsequent hybridization with a radiolabeled probe approximately 10 fg of B19 DNA was detected. The sensitivity of the PCR, combined with the simplicity and reduced time scale, demonstrates the potential of this technique as an additional method for routine diagnosis of B19 infections.

Abstract: In situ hybridisation with acetyl-aminofluorene (AAF) and 35S-labelled DNA probes for polyomaviruses, was used to detect JC virus DNA in brain necropsy material in a patient with progressive multifocal leucoencephalopathy (PML). In a second patient PML was diagnosed from a brain necropsy specimen using the same technique. The main infected cell type were oligodendrocytes; dot hybridisation was used to estimate the number of viral copies in each infected cell. Southern blot hybridisation for further analysis of the viral genome was also carried out. In situ hybridisation with non-radioactive labelled polyomavirus DNA provides a simple and specific means for studying viral DNA in formaldehyde fixed tissue sections from patients with suspected PML. Even in small biopsy samples hybridisation results can be correlated with standard histopathological, immunocytochemical, and electron microscopic findings.

Abstract: Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.

Abstract: Interphase nuclei isolated from paraffin-embedded tissue of four normal brains were hybridized with biotinated repetitive DNA probes specific for the (peri)centromeric regions of chromosomes 1 and 7. Hybridization results were visualized with a peroxidase-DAB system after which the number of specific signals per nucleus was counted using bright field microscopy. Using the probe specific for chromosome 7 (p7t1), both the cerebral and the cerebellar samples showed 2 spots in 82% and 83%, respectively, of the nuclei. In situ hybridization with the chromosome 1 probe (pUC1.77) showed two spots in 69% of the cerebral nuclei. In cerebellar samples, hybridization with pUC1.77 resulted in only one large spot per nucleus in 82% of the cells. The average spot size in nuclei with one signal was about 1.6 times as large as that in nuclei with two signals. These observations suggest that the single large spot in the cerebellar cells is not the result of monosomy of chromosome 1 but that it reflects somatic pairing of the two chromosome 1 centromeres. Based on the size and the fraction of nuclei with one large spot, the small granular neuron is the most likely candidate. The difference between cerebral and cerebellar samples indicates that this somatic pairing of chromosome 1 is a cell-type-dependent phenomenon.

Abstract: A technique is described for the detection of HSV-DNA in very small volumes (5-10 microliters) of cerebrospinal fluid (CSF). The method was evaluated in CSF samples of 4-6-week-old mice inoculated with HSV-1 via the corneal route. The sensitivity of the PCR assay was compared with results of spin-amplified viral culture with immunofluorescent visualization (SAC/IF), routine viral culture (RVC) and radioactive dot-blot hybridization (DBA) in CSF samples obtained from other mice. The results show the PCR to be superior over the other techniques: infectious virus or viral DNA in CSF was demonstrated by PCR, SAC/IF, RVC and DBA in 68, 55, 20 and 2.5%, respectively. These results show the feasibility of the PCR for a rapid, non-invasive diagnosis of human HSV-encephalitis.

Abstract: Immunohistochemical techniques with monoclonal antibodies against cytomegalovirus (CMV) immediate early (IEA) and early antigens (EA), and in situ hybridisation, were used to detect CMV infection in routinely obtained, formaldehyde fixed and paraffin wax embedded tissues taken from bone marrow transplant patients, who had died form interstitial pneumonia. To improve the rates of detection of CMV-IEA and EA the wax embedded material was pretreated with 0.4% pepsin/HCl at 37 degrees C for 30 minutes. This pretreatment was also advantageous for in situ hybridisation. In the patients with histological evidence of CMV infection or positive viral culture from the lung tissue, or both, viral proteins and nucleic acids were detected in lung, as well as in other organs. Immunohistochemical techniques proved superior in heavily infected but necrotic tissues. In control patients (patients who had died from interstitial pneumonia without any evidence of CMV, or with no interstitial pneumonia at all) in situ hybridisation showed no positive signal, while immunohistochemical techniques showed only a few positive cells in lung tissue of one of nine patients. In addition to CMV-DNA analysis, formaldehyde-fixed, paraffin wax embedded tissue is amenable to immunohistochemical analysis with CMV monoclonal antibodies.

Abstract: The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material. A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as "ectopic" CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral ganglion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Experimentally infected mice were used to assess the value of immunofluorescence (IF) procedures in cerebrospinal fluid (CSF) cells in comparison with routine viral culture of CSF for diagnosis of herpes simplex type-1 (HSV-1) encephalitis. Virus specific antigen was detected by immunofluorescence in the majority of CSF cells in 75% of infected animals. In contrast, only 20% of infected mice had positive viral cultures, which sometimes took as long as a week to show a cytopathological effect (CPE). It is concluded that antigen detection by immunofluorescence is a rapid, specific and sensitive technique for demonstrating HSV-1 antigen in CSF cells of experimentally infected mice. Our results put forward a plea for further studies using these techniques on CSF samples from patients with suspected HSV-1 encephalitis. The prerequisites for reliable interpretation of results have been defined in this study.

Abstract: In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.

Abstract: Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.

Abstract: Acetylaminofluorene (AAF) modified cytomegalovirus (CMV) DNA probes have been applied for the rapid detection of CMV genomes by non-radioactive in situ hybridization in routinely obtained pathological material. To establish proper protocols, AAF modified mouse satellite DNA and mouse liver were used to investigate the procedural variables. Among these were type and time of fixation, glass slide coating for improved tissue adherence, protease permeabilization of sections, type and time of denaturation and hybridization, probe concentration, post-hybridization washing conditions and immunocytochemical detection. This research has led to a user-friendly procedure which, in addition to cells displaying a cytopathological effect typical for CMV infection, detects with high sensitivity CMV carrying cells that show no histo-pathological alterations. It can be readily applied in routine clinical-diagnostic laboratories.

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Abstract: Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell plays an important role in its susceptibility to ozone. The intracellular levels of reduced and oxidized glutathione were affected as well. The ATP content, however, proved to be insensitive to ozone exposure.