Sample records for fluorescence in situ hybridization

  1. Routine fluorescence in situ hybridization in soil

    Microsoft Academic Search

    J. Bertaux; U. Gloger; M. Schmid; A. Hartmann; S. Scheu

    2007-01-01

    The use of fluorescence in situ hybridization (FISH) to identify and enumerate soil bacteria has long been hampered by the autofluorescence of soil particles masking the bacterial signals and because the need of counting hundreds of bacteria in order to achieve statistically reliable data is time consuming. Recently, it was demonstrated that Nycodenz facilitates FISH in soil by concentrating bacteria

  2. Molecular cytogenetics using fluorescence in situ hybridization

    SciTech Connect

    Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

    1990-12-07

    Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

  3. Classification of fluorescence in situ hybridization images using belief networks

    E-print Network

    Lerner, Boaz

    of genetic abnormalities. FISH offers numerous advantages compared with con- ventional cytogenetic techniques hybridization (FISH); Image classification; Naive Bayesian classifier; K2 algorithm 1. Introduction Automatic fluorescence in situ hybridization (FISH) signal classification is essential for efficient clinical diagnosis

  4. Fluorescence in situ hybridization on membrane filters

    SciTech Connect

    Mahr, A.; Bowe, A.; Chin, K. [Integrated Genetics, Framingham, MA (United States)] [and others

    1994-09-01

    Processing large numbers of clinical specimens for fluorescence in situ hybridization (FISH) using currently available slide preparation methods is both time consuming and laborious. In contrast, FISH on membrane filters would allow easy, efficient sample preparation, and analogous to other molecular hybridization techniques, would permit batch hybridization and washing. We therefore investigated a variety of filter membranes, cell types, and application methods in order to develop a protocol more amenable to clinical applications. Many membrane types were shown to be acceptable for FISH, including those made of mixed esters of cellulose, nitrocellulose, polycarbonate, polysulfonate, and polyvinylidene fluoride. Glass fiber filters and polytetrafluoroethylene membranes were unsuitable due to innate characteristics such as swelling and autofluorescence, respectively. Amniocytes, whole blood, as well as mononuclear cell preparations of adult peripheral blood, cord blood, fetal blood, fetal liver cell suspensions, and HEL cell lines were applied to filters and successfully hybridized. Successful application methods included: applying a drop of cell suspension onto the filter and air drying; using a settling method developed for glass slides; vacuum filtering cells onto membranes; and culturing cells directly onto membranes. Cell retention for unfixed fetal liver cells on filters was 93% compared to 47-64% for settling on glass slides and 85% for drying onto glass slides. Hybridization efficiency for the last method was poor. Optimal signal intensity for membrane filters was achieved with adaptations of standard FISH protocols. Hybridization efficiencies as high as 100% have been observed using autosomal cosmid contig probes covering 100 to 140 kb of target DNA. Advantages of filter-based over slide-based FISH include adaptability to mass processing and increased cell retention while retaining high signal quality.

  5. Supernumerary ring chromosome 17 identified by fluorescent in situ hybridization

    SciTech Connect

    Fagan, K. [Hunter Area Pathology Service, New South Wales (Australia)] [Hunter Area Pathology Service, New South Wales (Australia); Edwards, M. [Western Suburbs Hospital, New South Wales (Australia)] [Western Suburbs Hospital, New South Wales (Australia)

    1997-04-14

    We present a patient with multiple anomalies and severe developmental delay. A small supernumerary ring chromosome was found in 40% of her lymphocyte cells at birth. The origin of the marker chromosome could not be determined by GTG banding, but fluorescent in situ hybridization (FISH) later identified the marker as deriving from chromosome 17. 20 refs., 2 figs., 1 tab.

  6. Minimizing off-target signals in RNA fluorescent in situ hybridization

    E-print Network

    Tomkins, Andrew

    is often accomplished with fluorescence in situ hybridization (FISH) using antisense probes. Analysis of high specificity has been to produce chemically- synthesized oligonucleotides that are directly labeled

  7. Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

    PubMed Central

    Iampietro, Carole; Bergalet, Julie; Lefebvre, Fabio Alexis; Moquin-Beaudry, Gaël; Zhang, Olivia; Wang, Xiaofeng; Lécuyer, Eric

    2013-01-01

    Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples. PMID:23407302

  8. Fluorescence in situ hybridization techniques for cytogenetic and genomic analyses.

    PubMed

    Walling, Jason G; Zhang, Wenli; Jiang, Jiming

    2013-01-01

    Fluorescent in situ hybridization (FISH) is a powerful method to visualize DNA sequences in the context of the whole chromosome. Yet despite the value of FISH analysis for cytogenetic studies, there are surprisingly few labs that are able to adapt the technique for their experiments in chromosomal and genome biology. Here we present a comprehensive FISH protocol acquired from over 20 years of collective experience using different plant species. Our description uses rice as a model for performing a complete FISH procedure, but the protocol can be readily adapted for other plant species. We have provided more specialized instruction beyond routine FISH, which includes the preparation of meiotic and mitotic samples suitable for FISH analysis, procedures for direct and indirect labeling of DNA probes, and techniques for increasing signal strength using layers of antibodies. PMID:23135841

  9. Differential destabilization of repetitive sequence hybrids in fluorescence in situ hybridization.

    PubMed

    Hozier, J C; Scalzi, J M; Clase, A C; Davis, L M; Liechty, M C

    1998-01-01

    A method for painting a chromosome or chromosome region by fluorescence in situ hybridization (FISH) without blocking DNA is described. Both unique sequence and repetitive sequence components of a fluorescently labeled probe are hybridized under low-stringency conditions, but the chromosomes are washed in such a manner that repetitive sequences are differentially removed, while region-specific unique sequence fragments remain bound to the target chromosomes. We refer to this differential retention and removal of probe components as differential stability FISH. PMID:9925929

  10. Mapping Small DNA Sequences by Fluorescence in situ Hybridization Directly on Banded Metaphase Chromosomes

    Microsoft Academic Search

    Yao-Shan Fan; Lisa M. Davis; Thomas B. Shows

    1990-01-01

    A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photo-graphic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as

  11. Fluorescence in-situ hybridization analysis for melanoma diagnosis.

    PubMed

    Senetta, Rebecca; Paglierani, Milena; Massi, Daniela

    2012-04-01

    Melanocytic proliferation constitutes a heterogeneous group of lesions with remarkable differences in their biology and clinical outcome. Thus, accurate histological diagnosis of these cases is mandatory to establish the most appropriate surgical treatment and follow-up. Although histological examination alone is usually sufficient to identify melanomas among the greater number of nevi, the definition of the benign or malignant nature of a subset of melanocytic tumours, exhibiting atypical features, is a challenging task. Novel techniques that may assist in the histopathological diagnosis in difficult cases have been extensively researched over recent years. Fluorescence in-situ hybridization (FISH), performed with a panel of four probes, including three locus-specific identifier (RREB1, MYB, and CCND1) genes, seems to represent a sensitive and specific molecular tool for the diagnosis of non-ambiguous melanocytic lesions. Some studies have agreed that FISH may be an ancillary diagnostic instrument, but cannot replace light microscopy, to distinguish benign nevi from malignant melanomas in daily practice. However, in the context of ambiguous melanocytic tumours, results are still controversial, and additional and substantial work is needed to develop reliable probes that may identify, with high sensitivity, specific subsets of ambiguous melanocytic lesions, including spitzoid proliferation. PMID:22007736

  12. MONITORING INTRAVESICAL THERAPY FOR SUPERFICIAL BLADDER CANCER USING FLUORESCENCE IN SITU HYBRIDIZATION

    Microsoft Academic Search

    BENJAMIN R. KIPP; R. JEFFREY KARNES; SHANNON M. BRANKLEY; AARON R. HARWOOD; V. SHANE PANKRATZ; THOMAS J. SEBO; MICHAEL M. BLUTE; MICHAEL M. LIEBER; HORST ZINCKE; KEVIN C. HALLING

    2005-01-01

    Purpose:We evaluated fluorescence in situ hybridization (FISH) for assessing the response to therapy in patients with superficial bladder cancer receiving bacillus Calmette-Guerin or other intravesical therapies.

  13. Mapping neurofibromatosis 1 homologous loci by fluorescence in situ hybridization

    SciTech Connect

    Viskochil, D.; Breidenbach, H.H.; Cawthon, R. [Univ. of Utah, Salt Lake City (United States)] [and others

    1994-09-01

    Neurofibromatosis 1 maps to chromosome band 17q11.2 and the NF1 gene is comprised of 59 exons that span approximately 335 kb of genomic DNA. In order to further analyze the structure of NF1 from exons 2 through 27b, we isolated a number of cosmid and bacteriophage P-1 genomic clones using NF1-exon probes under high-stringency hybridization conditions. Using tagged, intron-based primers and DNA from various clones as a template, we PCR-amplified and sequenced individual NF1 exons. The exon sequences in PCR products from several genomic clones differed from the exon sequence derived from cloned NF1 cDNAs. Clones with variant sequences were mapped by fluorescence in situ hybridization under high-stringency conditions. Three clones mapped to chromosome band 15q11.2, one mapped to 14q11.2, one mapped to both 2q14.1-14.3 and 14q11.2, one mapped to 2q33-34, and one mapped to both 18q11.2 and 21q21. Even though some PCR-product sequences retained proper splice junctions and open reading frames, we have yet to identify cDNAs that correspond to the variant exon sequences. We are now sequencing clones that map to NF1-homologous loci in order to develop discriminating primer pairs for the exclusive amplification of NF1-specific sequences in our efforts to develop a comprehensive NF1 mutation screen using genomic DNA as template. The role of NF1-homologous sequences may play in neurofibromatosis 1 is not clear.

  14. Detection of dengue group viruses by fluorescence in situ hybridization

    PubMed Central

    2012-01-01

    Background Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays. PMID:23110979

  15. The application of fluorescence in situ hybridization in different ploidy levels cross-breeding of lily.

    PubMed

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid 'Freya' had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

  16. The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

    PubMed Central

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

  17. Fluorescent in situ hybridization diagnosis of extramedullary nodal blast crisis.

    PubMed

    Jin, Guan-Nan; Zou, Ping; Chen, Wan-Xin; Ding, Ze-Yang; Zhou, Hao

    2013-03-01

    The t(9;22)(q34;q11) translocation between bcr and abl genes plays a pivotal role in the pathogenesis and diagnosis of chronic myelogenous leukemia (CML). Fluorescence in situ hybridization (FISH) using specific DNA probes provides a useful and accurate way for the detection of bcr/abl fusion gene in single cell. Here, we report an unusual case of a patient with no prior hematologic disease who initially manifested lymphadenopathy. The lymph node findings were suspicious for T-lineage lymphoblastic lymphoma, however, his blood and bone marrow at that time were in chronic phase of CML. This presented difficulty for accurate discrimination between CML blast crisis (BC) and non-Hodgkin's lymphomas (NHLs). To discern where the extramedullary nodal malignancy originated from, we cytologically analyzed lymph node biopsies and bone marrow with FISH to detect bcr/abl fusion signals. Together with the morphology, immunohistochemistry, cytogenetics as well as molecular analysis, the patient was diagnosed as extramedullary T-lymphoid BC of Ph+ CML. In conclusion, this case is unusual at three levels: first, extramedullary nodal BC as a presenting manifestation of CML is rare and the blasts are of precursor T lymphoblastic lineage, rather than the more common B-cell lineage; second, this case suggests that extramedullary lymphoid nodal BC of CML can exist independently without the bone marrow developing into BC; and third, FISH analysis on the single neoplastic cell is an accurate way to confirm that the neoplasm is either extramedullary localized blasts of CML or genetically distinct neoplasm. PMID:23417967

  18. Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

    Microsoft Academic Search

    Janet M. Holmes; Renee H. Martin

    1993-01-01

    Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was

  19. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    Microsoft Academic Search

    Galina G Hovhannisyan

    2010-01-01

    Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and\\/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH) techniques. FISH

  20. Multiplex fluorescence in situ hybridization (M-FISH).

    PubMed

    Anderson, Rhona

    2010-01-01

    Multiplex in situ hybridization (M-FISH) is a 24-color karyotyping technique and is the method of choice for studying complex interchromosomal rearrangements. The process involves three major steps. Firstly, the multiplex labeling of all chromosomes in the genome with finite numbers of spectrally distinct fluorophores in a combinatorial fashion, such that each homologous pair of chromosomes is uniquely labeled. Secondly, the microscopic visualization and digital acquisition of each fluorophore using specific single band-pass filter sets and dedicated M-FISH software. These acquired images are then superimposed enabling individual chromosomes to be classified based on the fluor composition in accordance with the combinatorial labeling scheme of the M-FISH probe cocktail used. The third step involves the detailed analysis of these digitally acquired and processed images to resolve structural and numerical abnormalities. PMID:20809305

  1. Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures

    Microsoft Academic Search

    VOLKHARD A. J. KEMPF; KARLHEINZ TREBESIUS; INGO B. AUTENRIETH

    2000-01-01

    Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide

  2. Evolution of Chromosome 6 of Solanum Species Revealed by Comparative Fluorescence in Situ Hybridization Mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome mapping is an important tool in evolutionary research. Here we demonstrate a comparative fluorescent in situ hybridization (FISH) mapping strategy. A set of 13 bacterial artificial chromosome (BAC) clones derived from potato chromosome 6 was used for FISH mapping in seven differen...

  3. Resolution of sex chromosome constitution by genomic in situ hybridization and fluorescence in situ hybridization with (TTAGG) n telomeric probe in some species of Lepidoptera

    Microsoft Academic Search

    Atsuo Yoshido; František Marec; Ken Sahara

    2005-01-01

    We have developed a simple method to resolve the sex chromosome constitution in females of Lepidoptera by using a combination\\u000a of genomic in situ hybridization (GISH) and fluorescence in situ hybridization with (TTAGG)\\u000a n\\u000a telomeric probe (telomere-FISH). In pachytene configurations of sex chromosomes, GISH differentiated W heterochromatin and\\u000a telomere-FISH detected the chromosome ends. With this method we showed that Antheraea

  4. Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm

    Microsoft Academic Search

    F. Z. Bischoff; D. D. Nguyen; K. J. Burt; L. G. Shaffer

    1994-01-01

    Single color fluorescence in situ hybridization (FISH) has been utilized on sperm to estimate nondisjunction rates for chromosomes 1, 12, 15, 16, X and Y. Using single-color FISH, one cannot distinguish nonhybridization from nullisomy nor disomy from diploidy. In order to provide an internal control, a multicolor FISH strategy was employed. Satellite probes specific for 13 human chromosomes were used

  5. Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

    Microsoft Academic Search

    Annelie Pernthaler; Jakob Pernthaler; Rudolf Amann

    2002-01-01

    Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for

  6. Isolation and fluorescence in situ hybridization mapping of 60 cosmid clones on human chromosome 18

    SciTech Connect

    Nakashima, Hitoshi; Sakai, Masako; Inaba, Rie; Imamura, Takashi (National Institute of Genetics, Yata (Japan))

    1994-02-01

    The authors have mapped 60 new cosmids on the short and long arms of chromosome 18 either by R- or by DAPI-banding and simultaneous fluorescence in situ hybridization. These markers were isolated from hybrid MS126-21 made from a human-rodent hybrid cell line that retained human chromosome 18. Twenty-two of the cosmid probes map on the short arm, and 31 probes cluster in the distal half of the long arm between bands 18q21.1 and 18q23, while 7 other probes are mapped more proximal to the centromere around bands 18q1.1-q12.3. The technique of fluorescence in situ hybridization has proven to be a very efficient methodology for gene mapping. These 60 probes will be useful in the elucidation of genetic alterations associated with diseases such as tetrasomy 18p syndrome, 18q- syndromes, and colorectal cancer. 19 refs., 2 figs.

  7. Comparison of Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization for Low and High Throughput HER2 Genetic Testing

    PubMed Central

    Poulsen, Tim S.; Espersen, Maiken L. M.; Kofoed, Vibeke; Dabetic, Tanja; Høgdall, Estrid; Balslev, Eva

    2013-01-01

    The purpose was to evaluate and compare 5 different HER2 genetic assays with different characteristics that could affect the performance to analyze the human epidermal growth factor 2 (HER2) gene copy number under low and high throughput conditions. The study included 108 tissue samples from breast cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region of interest was identified from a serial H&E stained slide following tissue cores were transferred to a tissue microarrays (TMA). When using TMA in a routine flow, all patients will be tested for HER2 status with IHC followed by CISH or FISH, thereby providing individual HER2 results. In conclusion, our results show that the differences between the HER2 genetic assays do not have an effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing. PMID:24383005

  8. Highly sensitive analysis for N- myc amplification in neuroblastoma based on fluorescence in situ hybridization

    Microsoft Academic Search

    Tatsurou Tajiri; Kumiko Shono; Yoshimitsu Fujii; Shinichi Noguchi; Yoshiaki Kinoshita; Masazumi Tsuneyoshi; Sachiyo Suita

    1999-01-01

    Background\\/Purpose: The N-myc amplification status in neuroblastoma has been evaluated previously for the whole tumor by the Southern blot method. The aim of this study is to evaluate the effectiveness of the fluorescence in situ hybridization (FISH) method to analyze N-myc amplification in neuroblastoma and compare the findings with those using the Southern blot method.Methods: In 26 neuroblastoma primary tumors

  9. Novel chromosome findings in bladder cancer cell lines detected with multiplex fluorescence in situ hybridization

    Microsoft Academic Search

    Jon C. Strefford; Debra M. Lillington; Martin Steggall; A. M. E. Nouri; Bryan D. Young; R. T. D. Oliver

    2002-01-01

    Bladder cancer is a common neoplasm worldwide, consisting mainly of transitional cell carcinomas, while squamous, adenocarcinoma, and sarcomatoid bladder cancers account for the remaining cases. In the present study, multiplex fluorescence in situ hybridization (M-FISH) has been used to characterize chromosome rearrangements in eight transitional and one squamous cell carcinoma cell line, RT112, of UMUC-3, 5637, CAT(wil), FGEN, EJ28, J82,

  10. Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol

    Microsoft Academic Search

    Cleber C. Ouverney; Jed A. Fuhrman

    1997-01-01

    Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA

  11. HER2\\/neu Testing in Breast Carcinoma: A Combined Immunohistochemical and Fluorescence In Situ Hybridization Approach

    Microsoft Academic Search

    Ren L. Ridolfi; Mehdi R. Jamehdor; Janet M. Arber

    2000-01-01

    We evaluated 750 consecutive invasive breast carcinomas for HER-2\\/neu utilizing a combination of immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) methodologies. IHC reactions of 3+ were considered HER-2\\/neu positive and 0 and 1+ IHC reactions were considered HER-2\\/neu negative. IHC reactions of 2+ were considered inconclusive and reflexed to FISH analysis. In addition, a 10% sampling and validation FISH

  12. Automated analysis of fluorescence in situ hybridization on fixed, paraffin-embedded whole tissue sections in B-cell lymphoma.

    PubMed

    Reichard, Kaaren K; Hall, Bryan K; Corn, Ayumi; Foucar, M Kathryn; Hozier, John

    2006-08-01

    Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information. PMID:16680153

  13. Classification of Multicolor Fluorescence In Situ Hybridization (M-FISH) Images With Sparse Representation

    Microsoft Academic Search

    Hongbao Cao; Hong-Wen Deng; Marilyn Li; Yu-Ping Wang

    2012-01-01

    There has been a considerable interest in sparse representation and compressive sensing in applied mathematics and signal processing in recent years but with limited success to medical image processing. In this paper we developed a sparse representation-based classification (SRC) algorithm based on L1-norm minimization for classifying chromosomes from multicolor fluorescence in situ hybridization (M-FISH) images. The algorithm has been tested

  14. Fluorescent in situ hybridization of mitochondrial DNA and RNA.

    PubMed

    Alán, Lukáš; Zelenka, Jaroslav; Ježek, Jan; Dlasková, Andrea; Ježek, Petr

    2010-01-01

    To reveal nucleic acid localization in mitochondria, we designed molecular beacon fluorescent probes against: i) the light strand complementary to ND5 mitochondrial DNA (mtDNA) gene (annealing also to corresponding mRNA); ii) displacement (D) loop 7S DNA (annealing also to parallel heavy strand mtDNA and corresponding light strand transcript); iii) the proximal D-loop heavy strand displaced by the light strand promoter minor RNA. Confocal microscopy demonstrated ND5 probe spreading (less for other probes) in mitochondrial reticulum tubules but upon RNase A treatment all probes contoured mtDNA nucleoid localization. DNase I spread the signal over mitochondrial tubules. Future applications are discussed. PMID:21125028

  15. Fluorescent In Situ Hybridization to Detect Transgene Integration into Plant Genomes

    NASA Astrophysics Data System (ADS)

    Schwarzacher, Trude

    Fluorescent chromosome analysis technologies have advanced our understanding of genome organization during the last 30 years and have enabled the investigation of DNA organization and structure as well as the evolution of chromosomes. Fluorescent chromosome staining allows even small chromosomes to be visualized, characterized by their composition and morphology, and counted. Aneuploidies and polyploidies can be established for species, breeding lines, and individuals, including changes occurring during hybridization or tissue culture and transformation protocols. Fluorescent in situ hybridization correlates molecular information of a DNA sequence with its physical location on chromosomes and genomes. It thus allows determination of the physical position of sequences and often is the only means to determine the abundance and distribution of DNA sequences that are difficult to map with any other molecular method or would require segregation analysis, in particular multicopy or repetitive DNA. Equally, it is often the best way to establish the incorporation of transgenes, their numbers, and physical organization along chromosomes. This chapter presents protocols for probe and chromosome preparation, fluorescent in situ hybridization, chromosome staining, and the analysis of results.

  16. Polymerase Chain reaction generated probes for fluorescence in situ hybridization.

    PubMed

    Dupont, J M; Lebbar, A; Dupuy, O; Frydman, N; Letessier, D; Auvinet, P; Rabineau, D

    1998-01-01

    The extended use of Fish with centromeric probes in many cytogenetic laboratories is often impaired by the cost of this technique. Polymerase Chain Reaction (PCR) constitutes a simple way to generate and label such centromeric probes at low cost. Two types of human DNA source can be used: 1--Somatic hybrid cell lines containing a unique human chromosome. The specific amplification of the human subset of alphoid DNA is realised with a primer pair specific for the consensus region of human alpha satellite sequence. 2--Total Human DNA. This time, a primer pair specific for the alpha satellite DNA of the chromosome of interest must be designed. These probes, labelled during the PCR reaction by direct incorporation of modified dUTP, are actually widely used in our laboratory, alone or mixed with other probes (chromosome painting or locus specific probes). PMID:11928124

  17. Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes

    SciTech Connect

    Fan, Y.S.; Davis, L.M.; Shows, T.B. (New York State Department of Health, Buffalo (USA))

    1990-08-01

    A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4{prime},6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders.

  18. Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes.

    PubMed Central

    Fan, Y S; Davis, L M; Shows, T B

    1990-01-01

    A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4',6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders. Images PMID:2201023

  19. Single-Step Multicolor Fluorescence In Situ Hybridization Using Semiconductor Quantum Dot–DNA Conjugates

    PubMed Central

    Bentolila, Laurent A.; Weiss, Shimon

    2011-01-01

    We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD–FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD–FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD’s broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD–FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD–FISH probes detection down to the single molecule level. PMID:16679564

  20. Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

    Microsoft Academic Search

    Tatsuhiko Hoshino; L. Safak Yilmaz; Daniel R. Noguera; Holger Daims; Michael Wagner

    2008-01-01

    Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is

  1. Physical mapping of chromosome 17 cosmids by fluorescence in situ hybridization and digital image analysis

    SciTech Connect

    Kallioniemi, O.P.; Kallioniemi, A.; Sudar, D.; Pinkel, D.; Gray, J. (Univ. of California, San Francisco, CA (United States)); Mascio, L. (Lawrence Livermore National Lab., CA (United States)); Deaven, L. (Los Alamos National Lab., NM (United States))

    1994-03-01

    The authors used fluorescence in situ hybridization and digital image analysis to localize cosmids along human chromosome 17. Seventy-one cosmids were selected at random from a chromosome 17 library constructed from a partial Sau3AI digest of flow-sorted chromosomes from a mouse-human hybrid cell line. Sixty-three of these (89%) gave a signal only on chromosome 17. The 40 cosmids producing the most distinct hybridization signals in metaphase and interphase cells were precisely mapped using digital image analysis. An additional 20 cosmids, previously mapped by linkage analysis, were also mapped. The order of these probes determined by metaphase mapping was consistent with the order determined by linkage analysis. 13 refs., 1 fig., 1 tab.

  2. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  3. Genomic analysis of sorghum by fluorescence in situ hybridization 

    E-print Network

    Kim, Jeong-Soon

    2004-11-15

    The reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to ...

  4. Genomic analysis of sorghum by fluorescence in situ hybridization

    E-print Network

    Kim, Jeong-Soon

    2004-11-15

    of genetics and cell biology, e.g., aneuploid identification (Ji et al. 1997, 1999a), polyploid genome evolution (Hanson et al. 1998), meiotic recombination (Ji et al. 1999b), recombination analysis (Reyes-Valdes et al. 1996), karyotyping (Chen et al. 2000... of identification. The utility of BACs as molecular cytogenetic probes in plants became evident through early studies using marker-selected BACs for FISH (Hanson et al. 1995; Jiang et al. 1995). Utilizing marker-selected BACs from euchromatin in the distal...

  5. In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH).

    PubMed

    Bourdon, Matthieu; Coriton, Olivier; Pirrello, Julien; Cheniclet, Catherine; Brown, Spencer C; Poujol, Christel; Chevalier, Christian; Renaudin, Jean-Pierre; Frangne, Nathalie

    2011-06-01

    Endopolyploidy, i.e. amplification of the genome in the absence of mitosis, occurs in many plant species and happens along with organ and cell differentiation. Deciphering the functional roles of endopolyploidy is hampered by the fact that polyploid tissues generally comprise cells with various ploidy levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels present at the end of development range from 2C to 256C in the same tissue. To investigate the temporal and spatial distribution of endopolyploidy it is necessary to address the DNA content of individual nuclei in situ. Conventional methods such as fluorometry or densitometry can be used for some tissues displaying favorable characteristics, e.g. small cells, small nuclei, organization in a monolayer, but high levels of varying polyploidy are usually associated with large sizes of nuclei and cells, in a complex three dimensional (3-D) organization of the tissues. The conventional methods are inadequate for such tissue, becoming semi-quantitative and imprecise. We report here the development of a new method based on fluorescent in situ bacterial artificial chromosome hybridizations that allows the in situ determination of the DNA ploidy level of individual nuclei. This method relies on the counting of hybridization signals and not on intensity measurements and is expected to provide an alternative method for mapping endopolyploidy patterns in mature, 3-D organized plant tissues as illustrated by the analysis of ploidy level and cell size in pericarp from mature green tomato fruit. PMID:21418357

  6. Assignment of the human prosaposin gene (PSAP) to 10q22.1 by fluorescence in situ hybridization

    Microsoft Academic Search

    I. Bar-Am; L. Avivi; M. Horowitz

    1996-01-01

    The human prosaposin gene (PSAP) was previously localized to 10q21?q22 by isotopic in situ hybridization using a human prosaposin cDNA as a probe. The present study, using fluorescence in situ hybridization with a mouse genomic prosaposin fragment as probe, confirms the localization of PSAP and precisely maps it to band 10q22.1.Copyright © 1996 S. Karger AG, Basel

  7. Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH).

    PubMed

    Zhong, X B; Hans de Jong, J; Zabel, P

    1996-01-01

    Fluorescence in situ hybridization (FISH) is an increasingly powerful tool with a variety of applications in both basic and applied research. With excellent genetic, cytogenetic and molecular maps available, the tomato genome provides a good model to benefit from the full potential of FISH. Tomato chromosomes at mitotic metaphase are small and not particularly suitable for high-resolution FISH. In contrast, chromosomes at meiotic pachytene are about 15 times longer, and easier to identify by their differences in chromosome arm lengths and chromomere pattern. We have developed a technique for preparing chromosomal spreads of young pollen mother cells at mid-prophase I which is suitable for FISH. In a first series of experiments, the hybridization patterns of three classes of repetitive DNA sequences were studied in single and multicolour FISH. PMID:8653264

  8. Exploring the origin of the D genome of oat by fluorescence in situ hybridization.

    PubMed

    Luo, Xiaomei; Zhang, Haiqin; Kang, Houyang; Fan, Xing; Wang, Yi; Sha, Lina; Zhou, Yonghong

    2014-09-01

    Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat. PMID:25478818

  9. Detection of a complex translocation using fluorescent in situ hybridization (FISH)

    SciTech Connect

    Rosen, B.A. [Brandeis Univ., Waltham, MA (United States); Abuelo, D.N. [Rhode Island Hospital, Providence, RI (United States); Mark, H.F. [Brown Univ. School of Medicine, Providence, RI (United States)

    1994-09-01

    The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

  10. In situ hybridization.

    PubMed

    Tsai, Chung-Jui; Harding, Scott A

    2013-01-01

    A non-radioactive in situ hybridization protocol is presented for localization of mRNA transcripts in thin tissue sections. In situ hybridization provides spatial resolution of transcript distribution at the cellular level that is not attained by Northern hybridization or PCR-based methods using organ- or tissue-derived RNA. With appropriately designed gene-specific probes, in situ hybridization can discriminate closely related gene family members. Although the procedure for tissue section preparation is tedious and lengthy, once prepared, the probe labeling, hybridization and signal detection steps can be easily scaled to handle multiple genes for comparative analysis. PMID:23317910

  11. Mapping Chromosome Rearrangement Breakpoints to the Physical Map of Caenorhabditis Elegans by Fluorescent in Situ Hybridization

    PubMed Central

    Albertson, D. G.

    1993-01-01

    A scheme for rapidly mapping chromosome rearrangements relative to the physical map of Caenorhabditis elegans is described that is based on hybridization patterns of cloned DNA on meiotic nuclei, as visualized by fluorescent in situ hybridization. From the nearly complete physical map, DNA clones, in yeast artificial chromosomes (YACs), spanning the rearrangement breakpoint were selected. The purified YAC DNAs were first amplified by degenerate oligonucleotide-primed polymerase chain reaction, then reamplified to incorporate fluorescein dUTP or rhodamine dUTP. The site of hybridization was visualized directly (without the use of antibodies) on meiotic bivalents. This allows chromosome rearrangements to be mapped readily if the duplicated, deficient or translocated regions do not pair with a normal homologous region, because the site or sites of hybridization of the probe on meiotic prophase nuclei will be spatially distinct. The pattern, or number, of hybridization signals from probes from within, or adjacent to, the rearranged region of the genome can be predicted from the genetic constitution of the strain. Characterization of the physical extent of the genetically mapped rearrangements places genetic landmarks on the physical map, and so provides linkage between the two types of map. PMID:8514130

  12. Fluorescent in situ hybridization analyses of human oocytes in trisomy 18 and 21

    SciTech Connect

    Cheng, E.Y.; Chen, Y.J.; Gartler, S.M. [Univ. of Washington School of Medicine, Seattle, WA (United States)

    1994-09-01

    The commonly accepted view of synapsis is that only 2 homologues can synapse at any one site and that this restriction applies to polyploids as well. However, triple synapsis has been observed is some triploid plants and in triploid chicken. In humans, triple synapsis of the long arm of chromosome 21 was detected in sperm of a trisomic 21 individual. More recently, studies of oocytes from trisomic 21 and 18 fetuses also indicated extensive triple synapsis along the entire length of the chromosomes. To further investigate this question, we undertook an evaluation of trivalent synapsis in fetal oocytes from 2 trisomic 21 and 2 trisomic 18 fetuses using fluorescent in situ hybridization (FISH) with whole chromosome probes. Oocytes were hybridized with whole chromosome probes obtained from ONCOR, Inc. after fixation with methanol and acetic acid. Slides were scored for the distribution of prophase stages, hybridization efficiency, and hybridization characteristics of chromosomes 18 and 21 in the trisomic 18 and 21 fetuses respectively. Fifty-eight per cent (379/650) of pachytenes analyzed for chromosome 18 contained a conspicous trivalent and 319 (48%) of these nuclei contained a single, thick, continuous fluorescent signal consistent with complete triple synapsis along the entire length of all 3 chromosomes. Sixteen per cent (104/650) of pachytene contained 2 signals consistent with a bivalent and a univalent, and 9 cells contained 3 thin signals consistent with asynapsis of all 3 chromosomes. The remaining 158 pachytenes had unusual pairing configurations that we could not classify, but they most likely represent trivalents with partial pairing between different homologues. In the 2 trisomic 21 fetuses, the majority (143/232) of pachytenes also contained one signal while only 52 cells contained a bivalent and univalent. Five cells contained 3 separate signals. These results confirm the existence of triple synapsis in human meiosis.

  13. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage

    Microsoft Academic Search

    Duongruitai Nicomrat; Warren A. Dick; Olli H. Tuovinen

    2006-01-01

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised

  14. Chromosome orientation fluorescence in situ hybridization (CO-FISH) to study sister chromatid segregation in vivo

    PubMed Central

    Falconer, Ester; Chavez, Elizabeth; Henderson, Alexander; Lansdorp, Peter M.

    2013-01-01

    Previously, assays for sister chromatid segregation patterns relied on incorporation of BrdU and indirect methods to infer segregation patterns after two cell divisions. Here we describe a method to differentially label sister chromatids of murine cells and directly assay sister chromatid segregation patterns following one cell division in vitro and in vivo by adaptation of the well-established CO-FISH (chromosome orientation fluorescent in situ hybridization) technique. 5-bromo-2?-deoxyuridine (BrdU) is incorporated into newly-formed DNA strands, followed by photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially-labeled sister chromatids in post-mitotic cells are visualized using fluorescence microscopy and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires four days for in vivo mouse tissues, and two days for in vitro cultured cells. PMID:20595964

  15. Rapid Identification and Enumeration of Antibiotic Resistant Bacteria in Urban Canals by Microcolony-Fluorescence in Situ Hybridization

    Microsoft Academic Search

    Takehiko Kenzaka; Nobuyasu Yamaguchi; Fuangfa Utrarachkij; Orasa Suthienkul; Masao Nasu

    2006-01-01

    The abundance and phylogenetic composition of antibiotic resistant bacteria in canals of metropolitan Bangkok, Thailand, were investigated using a microcolony method and fluorescence in situ hybridization (FISH). Cells were directly trapped from aquatic samples onto polycarbonate membranes and incubated for 24 hr on selective agar containing antibiotics. Individual antibiotic resistant bacterial microcolonies samples were classified on the filter using FISH

  16. Frequency of Chromosome 17 Aneuploidy in Primary and Recurrent Pterygium by Interphase-Fluorescence in situ Hybridization

    Microsoft Academic Search

    Umit Kamis; Hurkan Kerimoglu; Ahmet Ozkagnici; Hasan Acar

    2006-01-01

    Aim:To investigate chromosome 17 numerical aberrations by using fluorescence in situ hybridization (FISH) in pterygia and to find out whether there is any association between chromosome 17 aneuploidy and recurrent pterygia. Methods: Pterygium tissue samples were taken from 21 patients by surgical excision. Eighteen of them had primaryand 3 had recurrent pterygium. Peripheral whole blood interphase cells obtained from 11

  17. Assignment of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO) to Human Chromosome 4q33 by Fluorescence in Situ Hybridization and Somatic Cell Hybridization

    Microsoft Academic Search

    Elaine B. Spector; William K. Seltzer; Stephen I. Goodman

    1999-01-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization.

  18. Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH).

    PubMed

    Deloge-Abarkan, Magali; Ha, Thi-Lan; Robine, Enric; Zmirou-Navier, Denis; Mathieu, Laurence

    2007-01-01

    Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria. PMID:17213948

  19. Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization

    SciTech Connect

    Chen, H.; Tuck-Muller, C.M.; Wertelecki, W. [Univ. of South Alabama, Mobile, AL (United States)] [and others

    1995-03-27

    We report on a 15-year-old black boy with severe mental retardation, multiple congenital anomalies, and a supernumerary ring chromosome mosaicism. Fluorescence in situ hybridization with a chromosome 1 painting probe (pBS1) identified the ring as derived from chromosome 1. The karyotype was 46,XY/47,XY,+r(1)(p13q23). A review showed 8 reports of ring chromosome 1. In 5 cases, the patients had a non-supernumerary ring chromosome 1 resulting in partial monosomies of the short and/or long arm of chromosome 1. In 3 cases, the presence of a supernumerary ring resulted in partial trisomy of different segments of chromosome 1. In one of these cases of the supernumerary ring was composed primarily of the centromere and the heterochromatic region of chromosome 1, resulting in normal phenotype. Our patient represents the third report of a supernumerary ring chromosome 1 resulting in abnormal phenotype. 28 refs., 5 figs., 1 tab.

  20. Chromosome translocations measured by fluorescence in-situ hybridization: A promising biomarker

    SciTech Connect

    Lucas, J.N.; Straume, T.

    1995-10-01

    A biomarker for exposure and risk assessment would be most useful if it employs an endpoint that is highly quantitative, is stable with time, and is relevant to human risk. Recent advances in chromosome staining using fluorescence in situ hybridization (FISH) facilitate fast and reliable measurement of reciprocal translocations, a kind of DNA damage linked to both prior exposure and risk. In contrast to other biomarkers available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time post exposure, has a rather small inter-individual variability, and can be measured accurately at the low levels. Here, the authors discuss results from their studies demonstrating that chromosome painting can be used to reconstruct radiation dose for workers exposed within the dose limits, for individuals exposed a long time ago, and even for those who have been diagnosed with leukemia but not yet undergone therapy.

  1. Single copies of HIV proviral DNA detected by fluorescent in situ hybridization.

    PubMed

    Spadoro, J P; Payne, H; Lee, Y; Rosenstraus, M J

    1990-08-01

    A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency of detection of individual loci of proviral DNA was 57% to 78%. Only 1% of uninfected control cells exhibited a false-positive signal. HIV proviral DNA could be accurately identified in mixed populations comprised of only 5% infected cells. Thus, this assay could be used to identify cells that harbor HIV proviral DNA and to monitor the status of proviral DNA throughout the course of HIV infection. PMID:2400600

  2. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    NASA Astrophysics Data System (ADS)

    Yi, Dingrong; Xie, Shaochuan

    2013-12-01

    Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

  3. Mosaic dup (9p) diagnosed by fluorescence in situ hybridization (FISH).

    PubMed

    Petty, E M; Gibson, L H; Breg, W R; Burns, J P; Yang-Feng, T L

    1993-03-15

    We describe a girl with some manifestations of the dup (9p) syndrome. High-resolution Giemsa-banded karyotype of her lymphocytes documented that she was mosaic with 80% of cells being 46,XX, and 20% 46,XX,-20, + der(20;?) (p13;?). The additional material on 20p could not be defined clearly by high-resolution Giemsa banding, as the banding pattern appeared consistent with either distal 9p or distal 13q. In order to make a definitive cytogenetic diagnosis, we used fluorescence in situ hybridization (FISH) with a chromosome 9 specific DNA library to establish that the origin of the additional chromosomal material on chromosome 20 was from 9p. FISH used in this situation enabled us to counsel the family specifically regarding the prognosis and manifestations of distal 9p duplication. PMID:8456860

  4. Nasopharyngeal hyalinizing clear cell carcinoma with EWSR1 rearrangements diagnosed by fluorescence in situ hybridization.

    PubMed

    Fukuda, Atsushi; Tagami, Yohei; Takasawa, Akira; Sugita, Shintaro; Kuramoto, Rinnosuke; Imai, Suguru; Hasegawa, Tadashi; Iizuka, Keiji

    2015-10-01

    Hyalinizing clear cell carcinoma (HCCC) is a rare, low-grade salivary gland neoplasm with a predilection for the palate and tongue. A 63-year-old woman presented a 14×14×17-mm mass at the roof of the nasopharynx. Endoscopic resection was performed via a transnasal approach. Histopathological findings of the salivary gland tumor indicated hyalinization of the stroma and neoplastic cells with clear cytoplasm without mucin. Fluorescence in situ hybridization analysis revealed that the tumor cells were positive for EWSR1 rearrangement. We finally diagnosed this case as HCCC of the nasopharynx. EWSR1 rearrangements are non-existent in other salivary gland tumors with clear cell change; thus, the identification of this rearrangement was very useful in accurately diagnosing HCCC. PMID:25805066

  5. Partial trisomy 13q identified by sequential fluorescence in situ hybridization

    SciTech Connect

    Gopal Rao, V.V.N.; Carpenter, N.J.; Gucsavas, M. [Institute of Medical Genetics, Tulsa, OK (United States)] [and others

    1995-07-31

    We report on a 19-month-old boy with partial trisomy 13q resulting from a probable balanced translocation involving chromosomes 1 and 13. The infant presented with omphalocele, malrotation, microcephaly with overriding skull bones, micrognathia, apparently low-set ears, rocker-bottom feet, and congenital heart disease, findings suggestive of trisomy 13. Karyotypic studies from peripheral blood lymphocytes documented an unbalanced karyotype 46,XY,-1,+der. The mother`s chromosomes were normal, and the father was not available. Conventional cytogenetic techniques were unable to identify the extra material on the terminal 1q. Using fluorescence in situ hybridization (FISH) on the GTL-banded metaphases, the extra material on 1q was identified as the terminal long arm of 13, thus resulting in partial trisomy 13 (q32-qter). 8 refs., 2 figs., 1 tab.

  6. A 30-Mb metric fluorescence in situ hybridization map of human chromosome 19q

    SciTech Connect

    Gordon, L.A.; Bergmann, A.; Christensen, M.; Danganan, L. [Lawrence Livermore National Lab., CA (United States)] [and others] [Lawrence Livermore National Lab., CA (United States); and others

    1995-11-20

    A high-resolution metric physical map of chromosome 19q has been constructed by fluorescence in situ hybridization. The map locates 136 cosmid reference points that span 30 Mb. The reference points are sequentially ordered from centromere to telomere, and the distance between neighboring cosmids is known from 240 partially overlapping, redundant estimates of genomic distances in kilobases separating pairs of cosmids. The average spacing between cosmid reference points is 220 kb, with over 75% of intervals less than 300 kb. Eighty-four genes and polymorphic markers have been assigned to mapped cosmids. The information on order and genomic distances separating pairs of cosmids, both key elements for building physical maps, has furthered the construction and integration of the genetic and physical maps of chromosome 19. 24 refs., 3 figs.

  7. RAPID IDENTIFICATION OF CANDIDA ALBICANS DIRECTLY FROM YEAST POSITIVE BLOOD CULTURE BOTTLES BY FLUORESCENCE IN SITU HYBRIDIZATION USING PNA PROBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for identification of Candida albicans directly from yeast-positive blood culture bottles is described. The test (C. albicans PNA FISH) is based on a fluorescein-labeled PNA probe targeting C. albicans 26...

  8. Detection of Dehalococcoides spp. by peptide nucleic acid fluorescent in situ hybridization.

    PubMed

    Danko, Anthony S; Fontenete, Silvia J; de Aquino Leite, Daniel; Leitão, Patrícia O; Almeida, Carina; Schaefer, Charles E; Vainberg, Simon; Steffan, Robert J; Azevedo, Nuno F

    2014-01-01

    Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread contamination. Anaerobic treatment technologies that utilize Dehalococcoides spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of Dehalococcoides spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the Dehalococcoides spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct Dehalococcoides strains, and they can have strong implications in the monitoring and differentiation of Dehalococcoides populations in laboratory cultures and at contaminated sites. PMID:24970105

  9. Chromosomal loci of 50 human keratinocyte cDNAs assigned by fluorescence in situ hybridization

    SciTech Connect

    Morishima, Yohich; Ariyama, Takeshi; Yamanishi, Kiyofumi [Uyoto Prefectural Univ. of Medicine (Japan)] [and others] [Uyoto Prefectural Univ. of Medicine (Japan); and others

    1995-07-20

    The chromosomal loci of expressed genes provide useful information for a candidate gene approach to the genes responsible for genetic diseases. A large set of randomly isolated cDNAs catalogued by partial sequencing can serve as a resource for accessing and isolating these disease genes. Using fluorescence in situ hybridization, we examined the chromosomal loci of 217 human keratinocyte-derived cDNAs, with independent novel sequence tags at the 3{prime} end region. Among them, we determined the loci of 50 cDNAs. Single-pass sequencing of these from the 5{prime} ends indicated that 39 cDNAs still can be produced for new genes. These cDNAs with identified chromosomal loci are powerful tools that can be used to help elucidate the genes responsible for hereditary skin disorders. 42 refs., 3 figs., 2 tabs.

  10. Three dimensional dual labelled DNA fluorescent in situ hybridization analysis in fixed tissue sections

    PubMed Central

    Kernohan, Kristin D.; Bérubé, Nathalie G.

    2014-01-01

    Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:•developed a FISH technique using fixed, frozen cryosections;•provide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;•include optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections.

  11. Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

    Microsoft Academic Search

    HENRIK STENDER; CLETUS KURTZMAN; JENS J. HYLDIG-NIELSEN; D. Sorensen; ADAM BROOMER; KENNETH OLIVEIRA; HEATHER PERRY-O' KEEFE; ANDREW SAGE; BARBARA YOUNG; JAMES COULL

    2001-01-01

    A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different

  12. X chromosome aneuploidy in infertile women: Analysis by interphase fluorescent in situ hybridization

    SciTech Connect

    Morris, M.A.; Moix, I.; Mermillod, B. [Univ. and Cantonal Hospital, Geneva (Switzerland)] [and others

    1994-09-01

    Up to 1 in 3 couples have a problem of infertility at some time in their lives. Sex chromosome anomalies are found in 5-10% of couples, with mosaic aneuploidy being a common finding in primary infertility. Recurrent spontaneous abortion (RSA), in contrast, is frequently associated with autosomal structural anomalies. We hypothesized that low-level mosaic X chromosome aneuploidy was associated with primary infertility but not with RSA. Three groups were studied: women from couples with primary infertillity (n=26); women with three or more spontaneous abortions (n=22); and age-matched normally fertile women (at least two pregnancies; n=28). Interphase fluorescent in situ hybridization (FISH) was used to determine X chromosome ploidy in 100 nuclei per patient, using a contig of three cosmids from MAO locus (kindly donated by W. Berger, Nijmegen). A control probe (chr. 15 centromere) was simultaneously hybridized, and only nuclei containing two control signals were scored for the X chromosome. The mean numbers of nuclei with two X chromosome signals were the same in all groups (Welch equality of means test: p>0.97). However, there is a significant difference between the variances of the primary infertile and RSA groups (Levene`s test: p=0.025 after Bonferrone correction for multiple testing). This provides preliminary support for the hypothesis of an association between primary infertility and low-level mosaic X chromosome aneuploidy.

  13. In situ hybridization

    SciTech Connect

    Valentino, K.L.; Eberwine, J.H.; Barchas, J.D.

    1987-01-01

    These proceedings collect papers on genetic aspects of neurobiology. Topics include: gene expression in mammalian cells, in situ hybridization with RNA probes, mRNA in brain cells, neuropeptide gene expression, genetic mapping, and localization of peptide hormone gene expression.

  14. Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense

    PubMed Central

    Chen, Guofu; Zhang, Chunyu; Zhang, Baoyu; Wang, Guangce; Lu, Douding; Xu, Zhong; Yan, Peishen

    2011-01-01

    Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1–D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future. PMID:22022408

  15. Combined Morphological and Interphase Fluorescence in Situ Hybridization Study in Multiple Myeloma of Chinese Patients

    PubMed Central

    Ng, Margaret Heung-Ling; Kan, Amanda; Chung, Yuk-Fei; Wong, Ivy Hing-Nam; Lo, Kwok-Wai; Wickham, Nicolas William Rupert; Lei, Kenny Ieng-Kit; Lee, Joseph Chuen-Kwan

    1999-01-01

    To gain insight into the real incidence of the numeric chromosomal aberrations and the cell lineage involvement of the neoplastic process in multiple myeloma (MM), we examined 18 Chinese MM patients by May-Grunwald-Giemsa (MGG) staining and fluorescence in situ hybridization using three DNA centromeric probes specific for chromosomes 3, 7, and 9. In this investigation, cytogenetic abnormalities were detected in plasma cells (PCs), myeloid cells (MCs), and lymphoid cells (LCs) in all of the MM patients studied. This is the first demonstration of the cytogenetic aberration involved in the myeloid series. Furthermore, the MCs and PCs of 16 MM patients had the same aneuploidies in one or more of the chromosomes analyzed. These data suggest that the neoplastic transformation of MM may occur early in the hematopoietic development. Chromosomal aberrations involving mainly subclones and considerable cellular heterogeneity with gain of a variety of copy numbers of the same chromosome were demonstrated within PCs, which may possibly be the result of an underlying defect of PCs in the control of their number of chromosomes. Whereas PCs showed evidence suggestive of increased polyploidization, MCs and LCs, which exhibited similar chromosomal patterns as the former, rarely did. Thus, the clonal evolution from LC to PC, if that happens in MM, is characterized by chromosomal instability favoring growth of tumor cells with polysomies and polyploidies. PMID:9916913

  16. Locus/chromosome aberrations in intraductal papillary mucinous neoplasms analyzed by fluorescence in situ hybridization.

    PubMed

    Miyabe, Katsuyuki; Hori, Yasuki; Nakazawa, Takahiro; Hayashi, Kazuki; Naitoh, Itaru; Shimizu, Shuya; Kondo, Hiromu; Nishi, Yuji; Yoshida, Michihiro; Umemura, Shuichiro; Kato, Akihisa; Ohara, Hirotaka; Joh, Takashi; Inagaki, Hiroshi

    2015-04-01

    Locus and chromosome abnormalities have not been well clarified in intraductal papillary mucinous neoplasms (IPMNs). The aim of this study was to retrospectively examine these abnormalities using fluorescence in situ hybridization. IPMNs (n=28) were histopathologically classified into noninvasive IPMN (n=17) and IPMN with an associated invasive carcinoma (invasive IPMN, n=11) groups. Noninvasive IPMNs possessed non-neoplastic and noninvasive spots in their tissues, and invasive IPMN cases possessed non-neoplastic, noninvasive, and invasive spots. Non-neoplastic (n=28), noninvasive (n=28), and invasive (n=11) spots were then analyzed for aneuploidy of chromosomes 3, 6, 7, 8, 17, and 18 and deletions of p16 and p53 loci. Polysomy 6 and p16 deletion were significantly more frequent in noninvasive than in non-neoplastic spots. Polysomy 7, polysomy 18, p16 deletion, and p53 deletion were significantly more frequent in invasive than in noninvasive spots. Detection of polysomy 7 and p53 deletion gave a high diagnostic accuracy for invasive IPMN (sensitivity, 90.9%; specificity, 94.1%; and accuracy, 92.5%). Our study suggests that: (1) polysomy 6 and p16 deletion may contribute to adenomatous change of IPMN; (2) polysomy 7, polysomy 18, p16 deletion, and p53 deletion play roles in malignant transformation of noninvasive IPMN; and (3) polysomy 7 and p53 deletion may be excellent diagnostic markers for invasive IPMN. PMID:25517961

  17. Ecophysiological Analysis of Microorganisms in Complex Microbial Systems by Combination of Fluorescence In Situ Hybridization with Extracellular Staining Techniques

    NASA Astrophysics Data System (ADS)

    Nielsen, Jeppe Lund; Kragelund, Caroline; Nielsen, Per Halkjær

    Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems.

  18. Comparative genome mapping: mouse and rat homologies revealed by fluorescence in situ hybridization.

    PubMed

    Scalzi, J M; Hozier, J C

    1998-01-01

    Mouse and rat genome studies are vital to the use of rodents as models of biology and human genetic disease. In this study, comparative cytogenetic maps of individual homologous mouse (Mus musculus) and rat (Rattus norvegicus) chromosomal regions are presented as defined by cross-species fluorescence in situ hybridization. Such "Zoo-FISH" methods permit direct visual observation of the location of DNA segments from one species on mitotic chromosomes of evolutionarily diverged species. Mouse whole chromosome paint (WCP) probes generated from microdissection and degenerate oliogonucleotide primed (DOP) PCR were hybridized on slides containing a mixture of both mouse (the reference species) and rat (the diverged/ comparative species) metaphase chromosomes. Using six different mouse WCPs, eight regions on seven rat chromosomes were shown to be evolutionarily conserved between these species. The specific chromosomal sites of homology delineated in this study between mouse (MMU) and rat (RNO) genomes include the following: MMU 1 to RNO 9q21-q36 and to RNO 13 from bands q11 to the telomere, MMU 4 to all of RNO 5, MMU 11 to all of RNO 10 and the distal region of RNO 14 (14q21-q22), MMU 7 and MMU 19 both to RNO 1, from bands 1q21 to 41 (MMU 7) and 1q42 to the telomere (MMU 19), and MMU X to all of RNO X. Additionally, several new mouse and rat map assignments have been predicted based on the observed cross-species hybridization patterns in conjunction with known mapping data for mouse or rat genes. PMID:9465294

  19. Localization of seed protein genes on metaphase chromosomes of Vicia faba via fluorescence in situ hybridization

    Microsoft Academic Search

    J. Fuchs; I. Schubert

    1995-01-01

    Using fluorescencein situ hybridization (FISH), four different seed protein genes were physically mapped on metaphase chromosomes ofVicia faba L. dropped on slides. FISH with a 2.8 kb genomic probe of a legumin B4 gene resulted in reproducible signals on the long arm of chromosome III near the centromere. The same clone cross-hybridized at a lower frequency to the short arm

  20. The importance of using fluorescence in situ hybridization for the diagnosis of Smith-Magenis syndrome

    SciTech Connect

    Juyal, R.C.; Greenberg, F.; Lupski, J.R. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Smith-Magenis syndrome (SMS) is a clinically recognizable multiple congenital anomaly/mental retardation syndrome associated with deletion of chromosome 17p11.2. Quality metaphase preparations are required for unambiguous detection of the deletion. We and others have reported cases of SMS due to mosaicism for del(17)(p11.2). Examination of peripheral blood lymphocyte cultures of a patient with the SMS phenotype at 850 band level of resolution revealed a low level mosaicism (11%) for the deletion. Examination of fibroblasts at relatively low resolution revealed the deletion in all cells. In a second study, we reported molecular evidence for mosaicism in the unaffected mother of an SMS patient who demonstrated mosaicism (55%) for the deletion at a resolution level of < 500 bands. We now report a different SMS patient who was initially diagnosed as mosaic del(17)(p11.2) in two different cytogenetic laboratories. A third blinded cytogenetic study yielded a questionable diagnosis. Fluorescence in situ hybridization (FISH) conducted in two different laboratories with two different markers shown to be within the deletion region and a control marker from chromosome 17 demonstrated a deletion in 20/20 and 25/25 metaphases scored, respectively. It appears the latter patient may harbor a very small deletion and that FISH is a more reliable test for the Smith-Magenis deletion. Furthermore, FISH should be used to confirm or refute mosaicism seen in routine cytogenetics studies.

  1. Fluorescent in situ hybridization of human sperm: diagnostics, indications, and therapeutic implications.

    PubMed

    Ramasamy, Ranjith; Besada, Stefan; Lamb, Dolores J

    2014-12-01

    Male factor infertility is a relatively common condition, affecting at least 6% of men of reproductive age. Typically, men with unknown genetic abnormalities resort to using assisted reproductive techniques (ART) to achieve their reproductive goals. Infertile men who father biological children using ART could have a higher incidence of aneuploidy, which is a deviation from the normal haploid or diploid chromosomal state. Aneuploidy can be evaluated using fluorescent in situ hybridization (FISH), a cytogenetic assay that gives an estimate of the frequencies of chromosomal abnormalities. The chromosomes that are generally analyzed in FISH (13, 18, 21, X, and Y) are associated with aneuploidies that are compatible with life. The technique is indicated for various reasons but primarily in [1] men who despite normal semen parameters suffer recurrent pregnancy loss, and [2] men with normal semen parameters, who are undergoing in vitro fertilization but still experiencing recurrent implantation failure. As a screening tool, the technique can help in reproductive and genetic counseling of affected couples, or those who have previously experienced failure of ART. A qualitative analysis of FISH study results allows couples to make informed reproductive choices. Given the increasing clinical use of FISH in various infertility diagnoses, and the development of novel adjunct technologies, one can expect much progress in the areas of preimplantation genetic screening, diagnostics, and therapeutics. PMID:25439797

  2. Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine

    PubMed Central

    2014-01-01

    Extensive studies of the genetic aberrations related to human diseases conducted over the last two decades have identified recurrent genomic abnormalities as potential driving factors underlying a variety of cancers. Over the time, a series of cutting-edge high-throughput genetic tests, such as microarrays and next-generation sequencing, have been developed and incorporated into routine clinical practice. Although it is a classical low-throughput cytogenetic test, fluorescence in situ hybridization (FISH) does not show signs of fading; on the contrary, it plays an increasingly important role in detecting specific biomarkers in solid and hematologic neoplasms and has therefore become an indispensable part of the rapidly developing field of personalized medicine. In this article, we have summarized the recent advances in FISH application for both de novo discovery and routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies. In addition, we have reviewed the recent developments in FISH methodology as well. PMID:24499728

  3. A new approach to screen transgenic offspring using fluorescence in situ hybridization

    SciTech Connect

    Swiger, R.R.; Tucker, J.D. [Lawrence Livermore National Laboratory, CA (United States); Heddle, J.A. [York Univ., Ontario (Canada)

    1995-11-01

    In order to identify the presence of vector, specifically Lacl or LacZ, in putative transgenic mice rapidly and reliably, we developed a new method which utilizes fluorescence in situ hybridization (FISH). Transgenic mouse models are being used with increasing frequency for mutational and toxicological studies. By applying our method, an investigator can reliably determine the presence and the number of integration sites of a transgenic vector in numerous samples with less effort compared to conventional methods. This approach involves gazing a tail with a scalpel to obtain a blood smear on a microscope slide. After fixing the slide in 3:1 methanol: acetic acid, typical FISH analysis using biotinylated lambda DNA as the probe in performed. Our method eliminates the need for DNA extraction, blotting, and PCR, and yields results from a large number of individually identifiable cells from each animal. This assay is more accurate, reliable and easier to perform than conventional schemes presently used for screening transgenic animals. A particular advantage of this assay is the ability to discriminate between animals that are heterozygous and homozygous, something that eludes the PCR-based methods and Southern blotting. We have successfully analyzed over 95 samples with this method. Based on these results, we believe our system is more sensitive and accurate than conventional means of screening.

  4. Potential clinical impact of three-dimensional visualization for fluorescent in situ hybridization image analysis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Li, Shibo; Bin, Zheng; Zhang, Roy; Li, Yuhua; Tian, Huimin; Chen, Wei; Liu, Hong

    2012-05-01

    Chromosomal translocation is strong indication of cancers. Fluorescent in situ hybridization (FISH) can effectively detect this translocation and achieve high accuracy in disease diagnosis and prognosis assessment. For this purpose, whole chromosome paint probes are utilized to image the configuration of DNA fragments. Although two-dimensional (2-D) microscopic images are typically used in FISH signal analysis, we present a case where the translocation occurs in the depth direction where two probed FISH signals are overlapped in the projected image plane. Thus, the translocation cannot be identified. However, when imaging the whole specimen with a confocal microscope at 27 focal planes with 0.5-?m step interval, the translocation can be clearly identified due to the free rotation capability by the three-dimensional (3-D) visualization. Such a translocation detection error of using 2-D images might be critical in detecting and diagnosing early or subtle disease cases where detecting a small number of abnormal cells can make diagnostic difference. Hence, the underlying implication of this report suggests that utilizing 3-D visualization may improve the overall accuracy of FISH analysis for some clinical cases. However, the clinical efficiency and cost of using 3-D versus 2-D imaging methods are also to be assessed carefully.

  5. Analysis of human invasive cytotrophoblasts using multicolor fluorescence in situ hybridization.

    PubMed

    Weier, Jingly F; Hartshorne, Christy; Nguyen, Ha Nam; Baumgartner, Adolf; Polyzos, Aris A; Lemke, Kalistyn H; Zeng, Hui; Weier, Heinz-Ulrich G

    2013-12-01

    Multicolor fluorescence in situ hybridization, or FISH, is a widely used method to assess fixed tissues or isolated cells for numerical and structural chromosome aberrations. Unlike other screening procedures which provide average chromosome numbers for heterogeneous samples, FISH is a sensitive cell-by-cell method to analyze the distribution of abnormal cells in complex tissues. Here, we applied FISH to characterize chromosomal composition of a rare, but very important class of human cells that stabilize the fetal-maternal interface connecting the placenta to the uterine wall during early pregnancy, called invasive cytotrophoblasts (iCTBs). Combining differently-labeled, chromosome-specific DNA probes, we were able to unambiguously determine the number of up to six different autosomes and gonosomes in individual cell nuclei from iCTBs selected on the basis of their invasive behavior. In this manuscript, we describe a method for generation of iCTBs from placental villi, and provide the complete workflow of our FISH experiments including a detailed description of reagents and a trouble-shooting guide. We also include an in-depth discussion of the various types and sources of DNA probes which have evolved considerably in the last two decades. Thus, this communication represents both a complete guide as well as a valuable resource, intended to allow an average laboratory to reproduce the experiments and minimize the amount of specialized, and often costly, equipment. PMID:23748112

  6. Molecular cytogenetic characterization of the DiGeorge syndrome region using fluorescence in situ hybridization

    SciTech Connect

    Lindsay, E.A. (St. Mary's Hosptial, London (United Kingdom) Imperial Cancer Research Fund, London (United Kingdom)); Halford, S.; Wadey, R.; Scambler, P.J. (St. Mary's Hospital, London (United Kingdom)); Baldini, A. (Instituto di Genetica Molecolare del CNR, Alghero (Italy))

    1993-08-01

    DiGeorge syndrome (DGS) is a developmental defect characterized by cardiac defects, facial dysmorphism, and mental retardation. Several studies have described a critical region for DGS at 22q11, within which the majority of DGS patients have deletions. The authors have isolated nine cosmid and three YAC clones using previously described and newly isolated probes that have been shown to be deleted in many DGS patients. Using fluorescence in situ hybridization and digital imaging, they have mapped and ordered these clones relative to the breakpoints of two balanced translocations at 22q11 (one in a DGS patient and one in the unaffected parent of a DGS child). The data indicate that the breakpoint in the unaffected individual distally limits the DGS critical region (defined as the smallest region of overlap), while proximally the region is limited by repeat-rich DNA. The critical region includes the balanced translocation breakpoint of the DGS patient that presumably disrupts the gene causing this syndrome.

  7. Human chromosome 19p: A fluorescence in situ hybridization map with genomic distance estimates for 79 intervals spanning 20 Mb

    SciTech Connect

    Brandriff, B.F.; Gordon, L.A.; Fertitta, A. [Lawrence Livermore National Lab., CA (United States)] [Lawrence Livermore National Lab., CA (United States); [Univ. of California, Livermore, CA (United States)] [and others

    1994-10-01

    A physical map of human chromosome 19p has been constructed by fluorescence in situ hybridization of cosmids to metaphase chromosomes and sperm pronuclear interphases. The map spans {approximately}20 Mb and was generated with 141 multiple, partially overlapping estimates of genomic distances for 79 intervals separating 80 sequentially ordered cosmid reference points. The average distance separating pairs of cosmids was 250 kb, with a range from 50 to 700 kb; 75% of the intervals were estimated to be less than or equal to 300 kb and only 8 intervals were between 500 and 700 kb. Cosmids positive for 33 genes or gene families and 5 polymorphic markers were included among the mapped elements. The fluorescence in situ hybridization map will be useful for furthering the integration of the physical and genetic maps of 19p and for placing newly identified markers within a few hundred kb of their neighbors. 42 refs., 3 figs.

  8. Fluorescence in situ hybridization (FISH) signal analysis using automated generated projection images

    PubMed Central

    Wang, Xingwei; Chen, Xiaodong; Li, Yuhua; Liu, Hong; Li, Shibo; Zhang, Roy R.; Zheng, Bin

    2012-01-01

    Fluorescence in situ hybridization (FISH) tests provide promising molecular imaging biomarkers to more accurately and reliably detect and diagnose cancers and genetic disorders. Since current manual FISH signal analysis is low-efficient and inconsistent, which limits its clinical utility, developing automated FISH image scanning systems and computer-aided detection (CAD) schemes has been attracting research interests. To acquire high-resolution FISH images in a multi-spectral scanning mode, a huge amount of image data with the stack of the multiple three-dimensional (3-D) image slices is generated from a single specimen. Automated preprocessing these scanned images to eliminate the non-useful and redundant data is important to make the automated FISH tests acceptable in clinical applications. In this study, a dual-detector fluorescence image scanning system was applied to scan four specimen slides with FISH-probed chromosome X. A CAD scheme was developed to detect analyzable interphase cells and map the multiple imaging slices recorded FISH-probed signals into the 2-D projection images. CAD scheme was then applied to each projection image to detect analyzable interphase cells using an adaptive multiple-threshold algorithm, identify FISH-probed signals using a top-hat transform, and compute the ratios between the normal and abnormal cells. To assess CAD performance, the FISH-probed signals were also independently visually detected by an observer. The Kappa coefficients for agreement between CAD and observer ranged from 0.69 to 1.0 in detecting/counting FISH signal spots in four testing samples. The study demonstrated the feasibility of automated FISH signal analysis applying a CAD scheme to the automated generated 2-D projection images. PMID:22935778

  9. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    Microsoft Academic Search

    Susan Rigby; Gary W. Procop; Gerhard Haase; Deborah Wilson; Cletus Kurtzman; Kenneth Oliveira; Sabina Von Oy; Jens J. Hyldig-Nielsen; James Coull; Henrik Stender

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C.

  10. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    Microsoft Academic Search

    Kenneth Oliveira; Gary W. Procop; Deborah Wilson; James Coull; Henrik Stender

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein- labeled PNA probe that targets a species-specific sequence of the 16S rRNA of

  11. Characterization by fluorescence and electron microscopy in situ hybridization of a double Y isochromosome

    SciTech Connect

    Fetni, R.; Lemieux, N.; Richer, C.L. [Universite de Montreal, Quebec (Canada)] [and others] [Universite de Montreal, Quebec (Canada); and others

    1996-06-14

    A patient with mixed gonadal dysgenesis and Y isochromosomes I(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X, + mar1/46,X, + mar2/47,X, + mar1x2/47,X + mar2x2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (ENT), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp). 24 refs., 3 figs., 1 tab.

  12. A Case of Renal Primitive Neuroectodermal Tumor Confirmed by Fluorescence in situ Hybridization

    PubMed Central

    Etani, Toshiki; Naiki, Taku; Ando, Ryosuke; Iida, Keitaro; Naiki-Ito, Aya; Takahashi, Satoru; Kobayashi, Daichi; Kawai, Noriyasu; Tozawa, Keiichi; Yasui, Takahiro; Kohri, Kenjiro

    2015-01-01

    Primitive neuroectodermal tumor (PNET) is a member of the Ewing's sarcoma family of tumors (ESFT). We report a case of PNET in a 66-year-old male who presented with a large solid tumor within the parenchyma of the middle pole of the left kidney with metastases to the left adrenal gland and right ischium. A fine-needle biopsy was performed and showed a small round cell tumor. Results of immunohistochemical staining suggested this tumor belonged to ESFT. Preoperative VDC-IE (combined vincristine, doxorubicin and cyclophosphamide followed by another combination of ifosfamide and etoposide) chemotherapy and left radical nephrectomy and adrenalectomy were performed. The histopathological findings of the resected tumor were similar to those in the biopsy specimen, but the results of AE1/AE3 were different. For the diagnosis, fluorescence in situ hybridization was performed. Split signals of the EWSR1 gene were detected, and transmission electron microscopy showed neuroendocrine granules and microtubules. The final diagnosis of this tumor was PNET of the kidney. PMID:26034481

  13. Metaphase FISH on a chip: miniaturized microfluidic device for fluorescence in situ hybridization.

    PubMed

    Vedarethinam, Indumathi; Shah, Pranjul; Dimaki, Maria; Tumer, Zeynep; Tommerup, Niels; Svendsen, Winnie E

    2010-01-01

    Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3 5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies. PMID:22163442

  14. Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH)

    Microsoft Academic Search

    Xiao-Bo Zhong; J. Hans de Jong; Pim Zabel

    1996-01-01

    Fluorescencein situ hybridization (FISH) is an increasingly powerful tool with a variety of applications in both basic and applied research. With excellent genetic, cytogenetic and molecular maps available, the tomato genome provides a good model to benefit from the full potential of FISH. Tomato chromosomes at mitotic metaphase are small and not particularly suitable for high-resolution FISH. In contrast, chromosomes

  15. Visualization of episomal and integrated Epstein-Barr virus DNA by fiber fluorescence in situ hybridization.

    PubMed

    Reisinger, Jürgen; Rumpler, Silvia; Lion, Thomas; Ambros, Peter F

    2006-04-01

    For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV. PMID:16217752

  16. Investigation of chromosomal aberrations in Egyptian hepatocellular carcinoma patients by fluorescence in situ hybridization

    PubMed Central

    Aly, Magdy S.; Bahnassy, Abeer A.; Abdel-Rahman, Zekri N.

    2010-01-01

    BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a very common and highly malignant tumor, associated mainly with chronic viral hepatitis, cirrhosis of any cause, aflatoxin exposure and ethanol consumption. Cytogenetic analysis on HCC has been limited because of poor hepatocyte growth in vitro. Conventional cytogenetic studies have demonstrated frequent abnormalities of specific chromosomes in HCC. Molecular cytogenetic approaches have been applied only rarely in the characterization of HCC. The main aim of this study was to evaluate genetic aberrations of different chromosomes in HCC. The study included 35 patients with HCC, who have been diagnosed and treated at National Cancer Institute, Cairo University, Egypt. The clinico-pathologic features of the studied patient were collected from patient’s files. MATERIALS AND METHODS: Interphase cytogenetics by fluorescence in situ hybridization with the use of a panel of centromere-associated DNA probes for chromosomes 1, 4, 8, 9, 13, 17, 20 and Y were performed on paraffin-embedded HCC specimens. RESULTS: The most common chromosomal aberrations detected were gain of chromosomes 8 in 12 cases (34.28%), 17 in 6 cases (17.14%). Loss of chromosome Y was detected in 6 of male cases (30%). Monosomy 4 was also detected in 5 cases (14.28%). Negative correlation could be detected only between chromosome 4 and 8. (r = -0.381, P < 0.05). Correlations between gain or loss of chromosomes and the different clinicopathologic parameters in the patients investigated, indicated negative correlation between: chromosome Y and age and chromosome 1 and cirrhosis. CONCLUSION: Gains and losses of DNA found in this study probably involve oncogenes and tumor suppressor genes that play a role in the puzzle of hepatocarcinogenesis. PMID:21031057

  17. Non-enzymatic, low temperature fluorescence in situ hybridization of human chromosomes with a repetitive alpha-satellite probe.

    PubMed

    Durm, M; Haar, F M; Hausmann, M; Ludwig, H; Cremer, C

    1997-01-01

    In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence, in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 degrees C, hybridization was performed at 52 degrees C for 30 min, i.e. at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 degrees C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF). PMID:9090071

  18. Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum.

    PubMed

    Strepparava, Nicole; Wahli, Thomas; Segner, Helmut; Polli, Bruno; Petrini, Orlando

    2012-01-01

    F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum. PMID:23152887

  19. Fluorescence in situ hybridization for detecting urothelial carcinoma: A clinicopathological study

    PubMed Central

    Caraway, Nancy P.; Khanna, Abha; Fernandez, Ricardo L.; Payne, Linda; Bassett, Roland L.; Zhang, Hua-Zhong; Kamat, Ashish; Katz, Ruth L.

    2010-01-01

    BACKGROUND Because urothelial carcinoma (UC) is associated with a significant high risk of recurrence and progression, patients with UC require long-term surveillance. Fluorescence in situ hybridization (FISH) has been shown to be more sensitive than cytology in the detection of UC. This study evaluated the use of FISH for detecting UC. METHODS We used a pathology database to identify patients who had urine cytology and FISH performed at our institution between 2004 and 2006. Urinary specimens were analyzed using UroVysion FISH probes for abnormalities in centromeric chromosomes 3, 7 and 17 and locus specific 9p21. FISH results were correlated with cytologic findings and a minimal clinical follow-up of 24 months. RESULTS We identified 1006 consecutive urinary specimens from 600 patients (448 men and 152 women) who were monitored for recurrent UC (915 specimens) or evaluated for urinary symptoms (91 specimens). On FISH analysis, 669 specimens were negative for UC and 272 specimens were positive for UC. Sixty-five (6%) specimens were insufficient for FISH analysis. The sensitivity and specificity of FISH for UC were 58% and 66%, respectively, and 59% and 63% when FISH and cytology results were combined. Factors contributing to decreased FISH sensitivity included the paucity or absence of tumor cells, low-grade tumors, degenerated cells, method of specimen collection, type of specimen, and obscuring inflammatory cells or lubricant. CONCLUSIONS We found UroVysion FISH had good sensitivity and specificity for detecting UC in urinary specimens. It is important to correlate the FISH results with the cytologic findings. PMID:20665656

  20. Differential distribution of long and short interspersed element sequences in the mouse genome: chromosome karyotyping by fluorescence in situ hybridization.

    PubMed Central

    Boyle, A L; Ballard, S G; Ward, D C

    1990-01-01

    Fluorescence in situ hybridization has been used to demonstrate the differential distribution of interspersed repetitive elements in the genome of Mus musculus domesticus. Hybridization with a mouse long interspersed element sequence results in a sharp, highly reproducible banding pattern on metaphase chromosomes, which is quite similar to Giemsa banding for all chromosomes except 7 and X. The families of short interspersed elements, B1 and B2, preferentially cluster in the R, or reverse, bands. There is no evidence of any interspersed repeat present in the centromeric heterochromatic regions. Both the long interspersed element and B2 probes give banding patterns suitable for karyotype analysis. Simultaneous hybridization of the biotinylated long interspersed element probe and a digoxigenin-labeled cosmid to metaphase spreads allows rapid localization of a probe of interest to a particular cytogenetic band on a chromosome. Images PMID:2170987

  1. Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology

    PubMed Central

    Lee, Natuscka; Nielsen, Per Halkjær; Andreasen, Kjær Holm; Juretschko, Stefan; Nielsen, Jeppe Lund; Schleifer, Karl-Heinz; Wagner, Michael

    1999-01-01

    A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities. PMID:10049895

  2. Standardization criteria for the detection of BCR\\/ABL fusion in interphase nuclei of chronic myelogenous leukemia patients by fluorescence in situ hybridization

    Microsoft Academic Search

    Ninette Cohen; Ilya Novikov; Izhar Hardan; Arif Esa; Frida Brok-Simoni; Ninette Amariglio; Gideon Rechavi; Isaac Ben-Bassat; Luba Trakhtenbrot

    2000-01-01

    Fluorescence in situ hybridization (FISH), as a new clinical test, is not presently standardized. For practical reasons, each laboratory must build its own criteria. In this work, we present our standardization criteria for clinical practice, which include not only the methods for cell fixation, specimen preparation, and hybridization conditions, but mainly the definition of false-positive range and the scoring criteria

  3. Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres.

    PubMed

    Knight, S J; Horsley, S W; Regan, R; Lawrie, N M; Maher, E J; Cardy, D L; Flint, J; Kearney, L

    1997-01-01

    We report an innovative fluorescence in situ hybridization technique which exploits a unique resource of 41 telomere-specific probes and allows the simultaneous analysis of the subtelomeric region of every chromosome for deletion, triplication and balanced translocation events. This technique requires only a single microscope slide per patient and is expected to be a useful diagnostic tool with applications in the fields of idiopathic mental retardation, the detection of congenital abnormalities and in some forms of cancer. This will lead to more accurate genetic counselling of patients and their families and will provide the basis for future diagnostic, therapeutic and preventative measures. PMID:9156314

  4. Sex chromosome differentiation in Humulus japonicus Siebold & Zuccarini, 1846 (Cannabaceae) revealed by fluorescence in situ hybridization of subtelomeric repeat

    PubMed Central

    Alexandrov, Oleg S.; Divashuk, Mikhail G.; Yakovin, Nikolay A.; Karlov, Gennady I.

    2012-01-01

    Abstract Humulus japonicus Siebold et Zucc (Japanese hop) is a dioecious species of the family Cannabaceae. The chromosome number is 2n = 16 = 14 + XX for females and 2n = 17 = 14 + XY1Y2 for male. To date, no fluorescence in situ hybridization (FISH) markers have been established for the identification of Humulus japonicus sex chromosomes. In this paper, we report a method for the mitotic and meiotic sex chromosome differentiation in Humulus japonicus by FISH for HJSR, a high copy subtelomeric repeat. The signal is present in the subtelomeric region of one arm of the X chromosome. We demonstrate that males have two Y chromosomes that differ in FISH signal with the HJSR probe. Indeed, the HJSR probe hybridizes to a subtelomeric region on both arms of chromosome Y1 but not of chromosome Y2. The orientation and position of pseudoautosomal regions (PAR1 and PAR2) were also determined. PMID:24260665

  5. Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

    PubMed

    Egger, D; Bolten, R; Rahner, C; Bienz, K

    1999-04-01

    Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing. PMID:10219632

  6. Identification of Neurofibromatosis 1 ( NF1) Homologous Loci by Direct Sequencing, Fluorescence in Situ Hybridization, and PCR Amplification of Somatic Cell Hybrids

    Microsoft Academic Search

    Smita M. Purandare; Heidi Huntsman Breidenbach; Ying Li; Xiao Lin Zhu; Shun'ichi Sawada; Shannon M. Neil; Arthur Brothman; Ray White; Richard Cawthon; David Viskochil

    1995-01-01

    Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci

  7. Improved signal recognition for interphase fluorescent in-situ hybridization using a non-ionic detergent (NP-40) pretreatment

    SciTech Connect

    Zhu, H.M.; Day-Salvatore, D.L.; Sciorra, L.J. [Univ. of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ (United States)] [and others

    1994-09-01

    We have reported that the non-ionic detergent ethylphenolpoly (ethyleneglycolether)x known as Noniet-P40 (Shell International Petroleum) can gently disrupt cell membranes, resulting in cells with varying degrees of free chromatin release. The extent of this phenomena is dependent upon the concentration of NP-40 and the detergent`s exposure time to the cells. Treated cells can range from halos of DNA around the cells to fully extended free chromatin configurations. We have demonstrated that these treated cells are excellent targets for many different fluorescently labelled probes used for in situ hybridization studies. Recently, we have compared NP-40 harvested lymphocytes with normally harvested cells to see if we could improve upon the number of cells showing discreet signals in interphase fluorescent in situ hybridization. Preliminary work has shown that using a trisomy 21 cell line, one can get a statistically significant improvement with NP-40 pretreatment cells over control levels, in the number of cells having three discreet signals in interphase {open_quotes}FISH{close_quotes}. Such a pretreatment is simple to perform and may be of value when the number of cells available for analysis is low, as in the search for fetal cells from maternal circulation.

  8. A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis

    PubMed Central

    Cartwright, Ian M.; Genet, Matthew D.; Kato, Takamitsu A.

    2013-01-01

    Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80–90°C) step followed by 1–3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well. PMID:23161278

  9. Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

    SciTech Connect

    Park, June-Woo [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Tompsett, Amber [Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Zhang, Xiaowei [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Newsted, John L. [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); ENTRIX, Inc., Okemos, MI 48823 (United States); Jones, Paul D.; Au, Doris [Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Kong, Richard; Wu, Rudolf S.S. [School of Environmental Science, Nanjing University, Nanjing (China); Giesy, John P. [Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); School of Environmental Science, Nanjing University, Nanjing (China); ENTRIX, Inc., Saskatoon, Saskatchewan (Canada)], E-mail: JGiesy@aol.com; Hecker, Markus [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); ENTRIX, Inc., Saskatoon, Saskatchewan (Canada)

    2008-10-15

    The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 {mu}g/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.

  10. Towards a cellular multi-parameter analysis platform: fluorescence in situ hybridization (FISH) on microhole-array chips.

    PubMed

    Kurz, Christian M; Moosdijk, Stefan V D; Thielecke, Hagen; Velten, Thomas

    2011-01-01

    Highly-sensitive analysis systems based on cellular multi-parameter are needed in the diagnostics. Therefore we improved our previously developed chip platform for another additional analysis method, the fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) is a technique used in the diagnostics to determine the localization and the presence or absence of specific DNA sequence. To improve this labor- and cost-intensive method, we reduced the assay consumption by a factor of 5 compared to the standard protocol. Microhole chips were used for making the cells well addressable. The chips were fabricated by semiconductor technology on the basis of a Silicon wafer with a thin deposited silicon nitride layer (Si(3)N(4)). Human retina pigment epithelia (ARPE-19) cells were arrayed on 5-?m holes of a 35 × 35 microhole-array by a gently negative differential pressure of around 5 mbar. After 3 hours of incubation the cells were attached to the chip and the FISH protocol was applied to the positioned cells. A LabView software was developed to simplify the analysis. The software automatically counts the number of dots (positive labeled chromosome regions) as well as the distance between adjacent dots. Our developed platform reduces the assay consumption and the labor time. Furthermore, during the 3 hours of incubation non-invasive or minimal-invasive methods like Raman- and impedance-spectroscopy can be applied. PMID:22256298

  11. Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization.

    PubMed

    Falistocco, E

    2000-01-01

    Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed. PMID:10833055

  12. Pallister-Killian syndrome: A mild case diagnosed by fluorescence in situ hybridization. Review of the literature and expansion of the phenotype

    SciTech Connect

    Bielanska, M.M.; Khalifa, M.M.; Duncan, A.M.V. [Queen`s Univ., Kingston, Ontario (Canada)] [Queen`s Univ., Kingston, Ontario (Canada)

    1996-10-16

    Pallister-Killian syndrome (PKS) is a rare disorder characterized by a specific combination of anomalies, mental retardation and mosaic presence of a supernumerary isochromosome 12p which is tissue-limited. We report an atypical case of PKS with a mild phenotype. Fluorescence in situ hybridization (FISH) was used to demonstrate that the supernumerary marker chromosome identified in the patient`s fibroblasts was an isochromosome 12p. This study broadens the spectrum of PKS phenotype. It also illustrates the usefulness of fluorescence in situ hybridization in diagnosis of patients with chromosomal abnormalities and mild or atypical clinical findings. 40 refs., 2 figs., 1 tab.

  13. Analysis of lymphocytes from uranium mineworkers in Namibia for chromosomal damage using fluorescence in situ hybridization (FISH).

    PubMed

    Zaire, R; Griffin, C S; Simpson, P J; Papworth, D G; Savage, J R; Armstrong, S; Hultèn, M A

    1996-11-01

    Workers in the open pit uranium mine in Namibia appear to suffer from health problems including malignant diseases at a much higher prevalence when compared with the general population. The objective of the present study was to determine whether long-term exposure to low-dose uranium increases the risk of biological radiation damage which could lead to malignant diseases. In order to investigate this risk, we measured the relative frequency of chromosome alterations using Fluorescence in situ hybridization (FISH). A representative cohort of 11 non-smoking miners, were compared to a control group of 9 individuals with no occupational history in mining. We determined a significant increase in chromosome aberrations in the circulating lymphocytes of miners versus the non-smoking controls (p = 0.0000096). Therefore, we concluded that these uranium exposed miners are at an increased risk to acquire genetic damage, which may be associated with an increased risk for malignant transformation. PMID:8950356

  14. Detection of aneuploidy in sperm of an ataxia telangiectasia patient using three-chromosome fluorescence in situ hybridization

    SciTech Connect

    Lowe, X.R.; Baulch, J.E. [Lawrence Livermore National Lab., CA (United States); Arnheim, N. [USC, Los Angeles, CA (United States)] [and others

    1994-09-01

    Ataxia telangiectasia (A-T) is an inherited, recessive, cancer-prone disorder. Fluorescence in situ hybridization (FISH) with DNA probes specific for three chromosomes was applied to sperm of an A-T patient to determine if there may be an increased germinal risk for aneuploidy. Air-dried sperm smears were treated with proteinase K and were decondensed with DTT and LIS. The slides were then hybridized with fluorescently labeled repetitive DNA probes specific for chromosomes X, Y and 8, and a total of 11,825 sperm cells were scored. The ratio of sperm bearing X-8 and Y-8 was 1:1, as predicted. The frequencies of hyperhaploidy were 3.9, 1.0, 17.6 and 7.8 per 10,000 cells for categories X-X-8, Y-Y-8, X-Y-8 and 8-8-(X or Y), respectively, In addition, the frequency of diploidy (X-Y-8-8) was 18.6 and auto-diploidies (X-X-8-8 and Y-Y-8-8) were 1.0 and 2.0, respectively. These frequencies were not significantly different when compared with levels in healthy men (p > 0.1). Our finding suggests that chromosome X, Y and 8 aneuploidies are not elevated in the sperm of A-T patients, but studies with additional patients and chromosomes are needed.

  15. Detection of Y chromosome sequences in a 45,X\\/46,XXq - patient by Southern blot analysis of PCR-amplified DNA and fluorescent in situ hybridization (FISH)

    Microsoft Academic Search

    Mirjana Kocova; Selma Feldman Siegel; Sharon L. Wenger; Peter A. Lee; Michael Nalesnik; Massimo Trucco

    1995-01-01

    In some cases of gonadal dysgenesis, cytogenetic analysis seems to be discordant with the phenotype of the patients. We have applied techniques such as Southern blot analysis and fluorescent in situ hybridization (FISH) to resolve the phenotype\\/genotype discrepancy in a patient with ambiguous genitalia in whom the peripheral blood karotype was 45,X. Gonadectomy at age 7 months showed the gonadal

  16. Peptide nucleic acid fluorescence in-situ hybridization for identification of Vibrio spp. in aquatic products and environments.

    PubMed

    Zhang, Xiaofeng; Li, Ke; Wu, Shan; Shuai, Jiangbing; Fang, Weihuan

    2015-08-01

    A peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for specific detection of the Vibrio genus. In silico analysis by BLAST and ProbeCheck showed that the designed PNA probe targeting the 16S rRNAs was suitable for specific identification of Vibrio. Specificity and sensitivity of the probe Vib-16S-1 were experimentally verified by its reactivity against 18 strains of 9 Vibrio species and 14 non-Vibrio strains of 14 representative species. The PNA-FISH assay was able to identify 47 Vibrio positive samples from selectively enriched cultures of 510 samples of aquatic products and environments, comparable with the results obtained by biochemical identification and real-time PCR. We conclude that PNA-FISH can be an alternative method for rapid identification of Vibrio species in a broad spectrum of seafood or related samples. PMID:25955286

  17. Rapid Identification of Staphylococcus aureus in Blood Cultures by a Combination of Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes and Flow Cytometry

    Microsoft Academic Search

    Hanna Hartmann; Henrik Stender; Andrea Schafer; Ingo B. Autenrieth; Volkhard A. J. Kempf

    2005-01-01

    Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

  18. Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

    Microsoft Academic Search

    Markku J. Lehtola; Eila Torvinen; Ilkka T. Miettinen; C. William Keevil

    2006-01-01

    Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybrid- ization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive

  19. Visualization and quantification of archaeal and bacterial metabolically active cells in soil using fluorescence in situ hybridization method

    NASA Astrophysics Data System (ADS)

    Semenov, Mikhail; Manucharova, Natalia; Stepanov, Alexey

    2015-04-01

    The method of in situ hybridization using fluorescent labeled 16S rRNA-targeted oligonucleotide probes (FISH - fluorescence in situ hybridization) combines identification and quantification of groups of microorganisms at different phylogenetic levels, from domain to species. The FISH method enables to study the soil microbial community in situ, avoiding plating on nutrient media, and allows to identify and quantify living, metabolically active cells of Bacteria and Archaea. The full procedure consists of the following steps: desorption of the cells from the soil particles, fixation of cells, coating a fixed sample on the glass slide, hybridization with the specific probes and, finally, microscopic observation and cell counting. For the FISH analysis of Bacteria and Archaea, the paraformaldehyde-fixed samples were hybridized with Cy3-labeled Archaea-specific probe(Arc915) and 6-carboxyfluorescein (FAM)-labeled Bacteria-specific probe(EUB338). When a molecular probe is incorporated into a cell, it can hybridize solely with a complementary rRNA sequence. The hybridization can be visualized under the fluorescent microscope and counted. The application of FISH will be demonstrated by the abundance of metabolically active cells of Archaea and Bacteria depending on soil properties, depth and land use. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of Chernozem and Kastanozem with distinct land use. Quantification of metabolically active cells in virgin and arable Chernozem revealed that the abundance of Archaea in topsoil of virgin Chernozem was doubled as compared with arable soil, but it leveled off in the deeper horizons. Plowing of Chernozem decreased an amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. In Kastanozem, a significant change in the abundance of metabolically active cells due to plowing was detected only within 40 cm soil layer, and this effect disappeared in lower horizons. The abundance of Archaea was higher in the upper horizons of arable soil as compared to virgin. Conversely, the abundance of Bacteria in the upper layers of arable Kastanozem decreased versus virgin soil. A relationship between soil organic carbon and the amount of soil metabolically active Bacteria and Archaea cells revealed that distribution of both Bacteria and Archaea throughout the soil profile was governed mostly by the organic matter content. Thus, the organic matter was a main factor of declining the Bacteria:Archaea ratio with the soil depth (from 7.1 to 4.2 for virgin soil and from 5 to 3.9 for arable soil). As a result, Archaea out-compete Bacteria under conditions of reduced energy supply. Thus, the FISH method combines classical microscopic and modern phylogenetic microbiological approaches and can be considered as an effective tool for ecological, diagnostic and environmental research in microbiology.

  20. Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

    Microsoft Academic Search

    Michael Lefmann; Birgitta Schweickert; Petra Buchholz; Ulf B. Gobel; Timo Ulrichs; Peter Seiler; Dirk Theegarten; Annette Moter

    2006-01-01

    With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify

  1. A Study of the Relative Dominance of Selected Anaerobic Sulfate-Reducing Bacteria in a Continuous Bioreactor by Fluorescence in Situ Hybridization

    Microsoft Academic Search

    B. Icgen; S. Moosa; S. T. L. Harrison

    2007-01-01

    The diversity and the community structure of sulfate-reducing bacteria (SRB) in an anaerobic continuous bioreactor used for\\u000a treatment of a sulfate-containing wastewater were investigated by fluorescence in situ hybridization. Hybridization to the 16S rRNA probe EUB338 for the domain Bacteria was performed, followed by a nonsense probe\\u000a NON338 as a control for nonspecific staining. Sulfate-reducing consortia were identified by using

  2. Detection of Ralstonia solanacearum, which causes brownrot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes

    Microsoft Academic Search

    B. A. WULLINGS; Beuningen van A. R; J. D. Janse; A. D. L. Akkermans

    1998-01-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacea- rum strains and one Ralstonia pickettii strain were PCR amplified,

  3. Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Khaled K. Mahmoud; Damian McNeely; Chelsea Elwood; Susan F. Koval

    2007-01-01

    A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment. Bdellovibrio and like organisms (BALOs) are predatory gram-negative bacteria that are ubiquitous

  4. Midkine gene (MDK), a gene for prenatal differentiation and neuroregulation, maps to band 11p11. 2 by fluorescence in situ hybridization

    SciTech Connect

    Kaname, Tadashi; Uehara, Kazuyoshi; Muramatsu, Taskashi (Kagoshima Univ. (Japan)); Kuwano, Akira; Murano, Ichiro; Kajii, Tadashi (Yamaguchi Univ. School of Medicine, Ube, Yamaguchi (Japan))

    1993-08-01

    Midkine (MDK) is a retinoic acid-responsive gene concerned with prenatal development and neurite growth. The authors mapped the gene to band p11.2 of chromosome 11 through fluorescence in situ hybridization analysis and using a 4.5-kb fragment of its human genomic DNA. 11 refs., 1 fig.

  5. Chromosomal assignment of human nuclear envelope protein genes LMNA, LMNB1, and LBR by fluorescence in situ hybridization

    SciTech Connect

    Wydner, K.L.; McNeil, J.A. [Univ. of Masssachusetts Medical Center, Worcester, MA (United States)] [Univ. of Masssachusetts Medical Center, Worcester, MA (United States); Lin, Feng [Columbia Univ., New York, NY (United States)] [and others] [Columbia Univ., New York, NY (United States); and others

    1996-03-05

    We have used fluorescence in situ hybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2-q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3-q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments. 30 refs., 2 figs., 1 tab.

  6. Pre-implantation genetic screening using fluorescence in situ hybridization in couples of Indian ethnicity: Is there a scope?

    PubMed Central

    Saxena, Shailaja Gada; Desai, Kundanbala; Shewale, Lata; Ranjan, Prabhat

    2014-01-01

    CONTEXT: There is a high incidence of numerical chromosomal aberration in couples with repeated in vitro fertilization (IVF) failure, advanced maternal age, repeated unexplained abortions, severe male factor infertility and unexplained infertility. Pre-implantation genetic screening (PGS), a variant of pre-implantation genetic diagnosis, screens numerical chromosomal aberrations in couples with normal karyotype, experiencing poor reproductive outcome. The present study includes the results of the initial pilot study on 9 couples who underwent 10 PGS cycles. AIM: The aim of the present study was to evaluate the beneficial effects of PGS in couples with poor reproductive outcome. SETTINGS AND DESIGN: Data of initial 9 couples who underwent 10 PGS for various indications was evaluated. SUBJECTS AND METHODS: Blastomere biopsy was performed on cleavage stage embryos and subjected to two round fluorescence in situ hybridization (FISH) testing for chromosomes 13, 18, 21, X and Y as a two-step procedure. RESULTS: Six of the 9 couples (10 PGS cycles) conceived, including a twin pregnancy in a couple with male factor infertility, singleton pregnancies in a couple with secondary infertility, in three couples with adverse obstetric outcome in earlier pregnancies and in one couple with repeated IVF failure. CONCLUSION: In the absence of availability of array-comparative genomic hybridization in diagnostic clinical scenario for PGS and promising results with FISH based PGS as evident from the current pilot study, it is imperative to offer the best available services in the present scenario for better pregnancy outcome for patients. PMID:24829527

  7. Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis.

    PubMed

    Nikolakakis, K; Lehnert, E; McFall-Ngai, M J; Ruby, E G

    2015-07-15

    The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. PMID:25956763

  8. Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

    SciTech Connect

    Martin, R.H. [Univ. of Calgary, Alberta (Canada)]|[Alberta Children`s Hospital, Calgary (Canada); Rademaker, A. [Northwestern Univ. Medical School, Chicago, IL (United States)

    1994-09-01

    Aneuploidy estimates for individual chromosomes in human sperm have varied more than 10-fold in different laboratories using fluorescence in situ hybridization (FISH). These laboratories use different scoring criteria in the assessment of a disomic sperm. In order to determine reliable estimates of aneuploidy, we have investigated whether scoring criteria affect the aneuploidy frequency in human sperm. Aneuploidy estimates for chromosomes 1(pUC1.77), 12(pBR12), X(XC) and Y(DYZ3Z) were obtained in human sperm from five donors using multicolor FISH analysis to provide an internal control to differentiate between nullisomy and lack of hybridization and between disomy and diploidy. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one scoring criterion used one-half a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other scoring criterion set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half domain as the scoring criterion and 113,478 were scored using one domain as the criterion. The mean percent disomy for chromosomes 1, 12, X, Y and XY was .18, .16, .15, .19, .25 respectively using the one-half domain criterion and .08, .17, .07, .12, .16 respectively using the one domain criterion. The percent disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X and Y split into more than one domain in decondensed interphase sperm and use of the one-half domain criterion leads to an overestimate of aneuploidy frequencies.

  9. Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J. [Lawrence Livermore National Lab., CA (United States); Robbins, W.A. [Lawrence Livermore National Lab., CA (United States)]|[Univ. of California, Berkeley, CA (United States); Pinkel, D.; Weier, H.U. [Univ. of California, San Francisco, CA (United States); Mehraein, Y. [Univ. of California, San Francisco, CA (United States)]|[Philipps Universitat, Marburg (Germany)

    1994-10-15

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of {approximately} 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of {approximately} 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring. 44 refs., 1 fig., 3 tabs.

  10. Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

    Microsoft Academic Search

    Mahesh Choolani; Hilary O'Donnell; Cesare Campagnoli; Sailesh Kumar; Irene Roberts; Phillip R. Bennett; Nicholas M. Fisk

    2010-01-01

    Isolating fetal erythroblasts from mater- nal blood offers a promising noninvasive alternative for prenatal diagnosis. The current immunoenzymatic methods of identifying fetal cells from background maternal cells postenrichment by label- ing g-globin are problematic. They are nonspecific because maternal cells may produce g-globin, give poor hybridization efficiencies with chromosomal fluores- cence in situ hybridization (FISH), and do not permit simultaneous

  11. Mapping of the human centromere protein B gene (CENPB) to chromosome 20p13 by fluorescence in situ hybridization

    SciTech Connect

    Naohiko, Seki [Kazusa DNA Research Institute, Chiba (Japan); Toshiyuki, Saito; Katsumi, Kitagawa [National Institute of Radiological Science, Chiba (Japan)] [and others

    1994-11-01

    Using a panel of rodent/human hybrid DNA, the two centromere protein genes (CENPB and CENPC) have been mapped to human chromosomes 20 and 12, respectively. We report here a regional assignment of the CENP-B-coding gene (CENPB) on human chromosomes using fluorescene in situ hybridization techniques.

  12. Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance

    Microsoft Academic Search

    Yoon-Kyoung Cho; Sunhee Kim; Young A Kim; Hee Kyun Lim; Kyusang Lee; DaeSung Yoon; Geunbae Lim; Y. Eugene Pak; Tai Hwan Ha; Kwan Kim

    2004-01-01

    We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage

  13. Medical devices; hematology and pathology devices; classification of early growth response 1 gene fluorescence in-situ hybridization test system for specimen characterization. Final order.

    PubMed

    2014-09-01

    The Food and Drug Administration (FDA) is classifying early growth response 1 (EGR1) gene fluorescence in-situ hybridization (FISH) test system for specimen characterization into class II (special controls). The special controls that will apply to this device are identified in this order and will be part of the codified language for the early growth response 1 (EGR1) gene fluorescence in-site hybridization (FISH) test system for specimen characterization classification. The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. PMID:25195217

  14. ZebraFISH: fluorescent in situ hybridization protocol and three-dimensional imaging of gene expression patterns.

    PubMed

    Welten, Monique C M; de Haan, Simon B; van den Boogert, Niels; Noordermeer, Jasprien N; Lamers, Gerda E M; Spaink, Herman P; Meijer, Annemarie H; Verbeek, Fons J

    2006-01-01

    We present a method and protocol for fluorescent in situ hybridization (FISH) in zebrafish embryos to enable three-dimensional imaging of patterns of gene expression using confocal laser scanning microscopy. We describe the development of our protocol and the processing workflow of the three-dimensional images from the confocal microscope. We refer to this protocol as zebraFISH. FISH is based on the use of tyramide signal amplification (TSA), which results in highly sensitive and very localized fluorescent staining. The zebraFISH protocol was extensively tested and here we present a panel of five probes for genes expressed in different tissues or single cells. FISH in combination with confocal laser scanning microscopy provides an excellent tool to generate three-dimensional images of patterns of gene expression. We propose that such three-dimensional images are suitable for building a repository of gene expression patterns, complementary to our previously published three-dimensional anatomical atlas of zebrafish development (bio-imaging.liacs.nl/). Our methodology for image processing of three-dimensional confocal images allows an analytical approach to the definition of gene expression domains based on the three-dimensional anatomical atlas. PMID:18377226

  15. Fluorescent in situ hybridization of pre-incubated blood culture material for the rapid diagnosis of histoplasmosis.

    PubMed

    da Silva, Roberto Moreira; da Silva Neto, João Ricardo; Santos, Carla Silvana; Cruz, Kátia Santana; Frickmann, Hagen; Poppert, Sven; Koshikene, Daniela; de Souza, João Vicente Braga

    2015-02-01

    Fluorescence in situ hybridization (FISH) has been shown to be useful for the detection of Candida and Cryptococcus species in blood culture materials. FISH procedures for the detection of Histoplasma capsulatum var. capsulatum have not been reported so far. This study describes the development and evaluation of fluorescently labeled rRNA-targeting FISH probes to detect and identify H. capsulatum in blood cultures. All three analyzed H. capsulatum reference strains and clinical isolates showed positive signals with the newly designed specific oligonucleotide probes for H. capsulatum, whereas negative reactions were observed for all three nontarget yeast species and the two nontarget bacteria. The assay was also successfully applied for detections of H. capsulatum cells in pre-incubated blood culture samples of patients with clinical suspicion of histoplasmosis (n = 33). The described FISH-based assay was shown to be easy to apply, sensitive, and specific (compared to polymerase chain reaction) for the detection and identification of H. capsulatum in this proof-of-principle analysis. Larger multicentric assessments are recommended for a thorough diagnostic evaluation of the procedure. PMID:25537280

  16. Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.

    PubMed Central

    Simon, N; LeBot, N; Marie, D; Partensky, F; Vaulot, D

    1995-01-01

    Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features. PMID:7618862

  17. Karyotype analysis and chromosomal distribution of repetitive DNA sequences of cucumis metuliferus using fluorescence in situ hybridization.

    PubMed

    Yagi, Kouhei; Siedlecka, Ewa; Pawe?kowicz, Magdalena; Wojcieszek, Micha?; Przybecki, Zbigniew; Tagashira, Norikazu; Hoshi, Yoshikazu; Malepszy, Stefan; Pl?der, Wojciech

    2014-01-01

    Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4',6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram. PMID:25402685

  18. The human sorbitol dehydrogenase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization

    SciTech Connect

    Lee, F.K.; Chung, S. (Univ. of Hong Kong (Hong Kong)); Cheung, M.C. (Univ. of California, San Francisco, CA (United States))

    1994-05-15

    The cDNA for human sorbitol dehydrogenase (SORD) has been cloned and sequenced. It translates into a peptide of 356 amino acid residues, one more than the sequence previously reported from peptide analysis. An extra alanine was found at the acetyl-blocked N-terminal, between positions 1 and 4. This matches the rat cDNA, which also has 356 amino acids, with an extra proline at position 3. Four other mismatches were also observed, but these are all amino acid substitutions that occur outside proposed functionally important regions. Further work must be performed to determine whether these discrepancies represent polymorphic forms of the enzyme. The SORD gene was mapped by fluorescence in situ hybridization and found to occupy a single site on chromosome 15q15, indicating that it is a single-copy gene. This was confirmed by Southern blot hybridization. SORD is thought to be involved in the etiology of diabetic complications, and its deficiency has been linked to congenital cataracts. The cloned gene could be used as a probe to study the role of this enzyme in the pathogenesis of these diseases. 24 refs., 4 figs.

  19. Assignment of the human aggrecan gene (AGC1) to 15q26 using fluorescence in situ hybridization analysis

    SciTech Connect

    Korenberg, J.R.; Chen, X.N. [Univ. of California, Los Angeles, CA (United States)] [Univ. of California, Los Angeles, CA (United States); Doege, K. [Shriners Hospital for Crippled Children, Portland, OR (United States)] [Shriners Hospital for Crippled Children, Portland, OR (United States); Grover, J.; Roughley, P.J. [McGill Univ., Montreal, Quebec (Canada)] [McGill Univ., Montreal, Quebec (Canada)

    1993-05-01

    The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No other sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.

  20. Detection of chromosomal abnormalities in chronic lymphocytic leukemia increased by interphase fluorescence in situ hybridization in tetradecanoylphorbol acetate–stimulated peripheral blood cells

    Microsoft Academic Search

    Jana Sánchez; Anna Aventín

    2007-01-01

    Interphase fluorescent in situ hybridization on unstimulated peripheral blood mononuclear cells (I-FISH-PBMC) is used to detect chromosomal abnormalities such as 11q?, 13q?, 17p?, and trisomy 12 in chronic lymphocytic leukemia (CLL). A total of 56 samples from 49 patients with CLL were studied using commercially available probes for chromosome regions 11q22.3 (ATM), 13q14 (13S272), 17p13 (p53) and 12 centromere (D12Z3).

  1. Nitrification in Freshwater Sediments as Influenced by Insect Larvae: Quantification by Microsensors and Fluorescence in Situ Hybridization

    Microsoft Academic Search

    D. Altmann; P. Stief; R. Amann; D. de Beer

    2004-01-01

    Sediment-reworking macrofauna can stimulate nitrification by increasing the O 2 penetration into sediments or it can reduce nitrification by grazing on nitrifying bacteria. We investigated the influence of Chironomus riparius larvae (Insecta: Diptera) on the in situ activity, abundance, and distribution of NH 4 +-oxidizing (AOB) and NO 2 ?-oxidizing bacteria (NOB) in two freshwater sediments with microsensors and fluorescence

  2. Detection of aneuploid human sperm by fluorescence in situ hybridization: Evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y

    SciTech Connect

    Robbins, W.A. (Univ. of California, Berkeley (United States) Lawrence Livermore National Lab., CA (United States)); Segraves, R.; Pinkel, D. (Univ. of California, San Francisco (United States)); Wyrobek, A.J. (Lawrence Livermore National Lab., CA (United States))

    1993-04-01

    Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2[+-]2.4/10,000 and 5.6[+-]1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated [approximately]2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. The authors conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm. 43 refs., 1 fig., 4 tabs.

  3. Radiation-induced damage, repair and exchange formation in different chromosomes of human fibroblasts determined by fluorescence in situ hybridization

    SciTech Connect

    Kovacs, M.S.; Evans, J.W.; Johnstone, I.M.; Brown, J.M. [Stanford Univ., CA (United States)

    1994-01-01

    We have used fluorescence in situ hybridization with whole-chromosome probes for human chromosomes 1, 4, 8 and 13 to investigate the extent to which the induction of damage and its repair after exposure to ionizing radiation is distributed randomly among these chromosomes. All the studies were performed with AG1522 human fibroblasts irradiated with 6 Gy and maintained in a nondividing state for at least 6 h after irradiation except for the measurements of initial damage. The extent of initial damage was determined by fusion of the cells immediately after irradiation with metaphase HeLa cells to obtain premature chromosome condensation (PCC). Breaks and exchanges were also scored by PCC 24 h after irradiation and in metaphase spreads at the first division after irradiation. The data obtained were consistent with random breakage and repair in these chromosomes. Comparing PCC 24 h after irradiation with first metaphase, there was a deficit in aberrations at metaphase, particularly in unrejoined breaks, implying loss or slowing of cells containing aberrations prior to the first division. An analysis of dicentrics and translocations in chromosome 4 at first and in subsequent divisions showed that there was an equal number of dicentrics and translocations at first metaphase with loss of dicentrics, but no loss of translocations in subsequent divisions. These data are supportive of the hypothesis that the total number of chromosome aberrations in cells can be estimated from a single chromosome pair. 34 refs., 5 figs., 2 tabs.

  4. Paternal-age effects on sperm aneuploidy investigated in mice and humans by three-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J.; Lowe, X. [Lawrence Livermore National Lab., Livermore, CA (United States); Holland, N.T. [Unvi. of California, Berkeley, CA (United States)] [and others

    1994-09-01

    We conducted a cross-species comparison of the effects of paternal age on sperm aneuploidy in mice and humans. A new murine assay was developed to detect sperm hyperhaploidy and polyploidy for chromosomes X, Y, and 8 using fluorescence in situ hybridization with chromosome-specific DNA probes, to serve as a direct corollate to the three-chromosome method developed early for human sperm. Sperm aneuploidy was evaluated in eight male B6C3F1 male mice (aged 22.5-30.5 mo) and compared to young controls (2.4 mo). The aged group showed significant ({approximately}2.0-fold) increases in hyperhaploidies involving chromosomes X, Y and 8, with the greatest effects seen in the oldest animals. Sperm aneuploidy was also evaluated in two groups of healthy men who differed in mean age [46.8{plus_minus}3.1 (n=4) vs. 28.5{plus_minus}5.0 (n=10) yrs], using the three-chromosome method. The older group showed a statistically significant increase in hyperhaploid sperm for both sex chromosomes. Additional controlled human studies are planned. Taken together, the murine and human data are consistent with a positive effect of paternal age on sperm aneuploidy. In both species, the strongest age effect was observed for hyperhaploidies of chromosome Y. Future studies are needed to investigate the shape of the age-effect curve and to evaluate chromosomal differences, especially for humans in their late reproductive years.

  5. Interphase fluorescence in situ hybridization improves the detection of malignant cells in effusions from breast cancer patients.

    PubMed Central

    Zojer, N.; Fiegl, M.; Angerler, J.; Müllauer, L.; Gsur, A.; Roka, S.; Pecherstorfer, M.; Huber, H.; Drach, J.

    1997-01-01

    In diagnostic evaluation of effusions, difficulties are encountered when atypical reactive mesothelial cells have to be differentiated from malignant cells. We tested the impact of fluorescence in situ hybridization (FISH) to identify metastatic cells in breast cancer effusions by detection of numerical chromosomal changes. Pleural and ascitic fluid samples (n=57) from 41 breast cancer patients were concomitantly evaluated by routine cytology and FISH, using centromere-specific probes representing chromosomes 7, 11, 12, 17 and 18. After setting stringent cut-off levels deduced from non-malignant control effusions (n=9), the rates of cells with true aneuploidy were determined in each effusion sample from breast cancer patients. The occurrence of aneuploid cells, as detected by FISH and indicative of malignancy, was correlated with the cytological findings. Routine cytology revealed malignancy in 60% of effusions. Using FISH, aneuploid cell populations could be observed in 94% of cytologically positive and in 48% of cytologically negative effusions, thus reverting diagnosis to malignancy. To confirm malignancy in cases with a low frequency of aneuploid cells, two-colour FISH was additionally performed and indeed showed heterogeneous chromosomal aneuploidy within single nuclei. We conclude that FISH is a valuable tool in the diagnosis of malignancy and may serve as an adjunct to routine cytological examination, as demonstrated here for breast cancer effusions. Images Figure 1 PMID:9020486

  6. Triplex in-situ hybridization

    DOEpatents

    Fresco, Jacques R. (Princeton, NJ); Johnson, Marion D. (East Windsor, NJ)

    2002-01-01

    Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

  7. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage

    SciTech Connect

    Nicomrat, D.; Dick, W.A.; Tuovinen, O.H. [Ohio State University, Wooster, OH (United States). Environmental Science Graduate Programme

    2006-07-15

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant.

  8. Monitoring of chimerism using fluorescence in situ hybridization in a child with severe combined immune deficiency following bone marrow transplant

    SciTech Connect

    Wenger, S.L.; Chen, X.O.; Katz, A.J. [Children`s Hospital of Pittsburgh, PA (United States)]|[Univ. of Pittsburgh, PA (United States)

    1994-09-01

    A boy with severe combined immunodeficiency received a bone marrow transplant from his sister when he was approximately 3 years of age. His peripheral blood karyotype at age 3 and 4 years was 46,XX (20 cells analyzed). Because of a decline in antibody production at 19 years of age, the patient`s peripheral blood was analyzed again for suspected chimerism. His karyotype in phytohemagglutinin (PHA)-stimulated culture was 46,XX in 49 cells and 46,XY in one cell. Both metaphase and interphase cells were examined for sex chromosome constitution using X and Y dual-color alpha-satellite probes for fluorescence in situ hybridization (FISH). FISH results for metaphase cells showed 1/50 XY cells, but 38% of interphase cells showed the presence of both X and Y centromere. Pokeweed mitogen (PWM)-stimulated cultures grew poorly and were therefore analyzed using FISH only: 81% of interphase cells were 46,XX. The discrepancy between metaphase and interphase in the PHA-stimulated cultures most likely represents a failure of this boy`s own XY T-cells to be stimulated.

  9. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage.

    PubMed

    Nicomrat, Duongruitai; Dick, Warren A; Tuovinen, Olli H

    2006-01-01

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant. Heterotrophs in the Acidiphilium genus totaled 20% of the bacterial population. Leptospirillum ferrooxidans was below the level of detection in the bacterial community. The results from the FISH technique from this field study are consistent with results from other experiments involving enumeration by most probable number, dot-blot hybridization, and denaturing gradient gel electrophoresis analyses and with the geochemistry of the site. PMID:16825452

  10. Quantitative Fluorescence In Situ Hybridization Analysis of Microbial Consortia from a Biogenic Gas Field in Alaska's Cook Inlet Basin

    PubMed Central

    Str?po?, Dariusz; Huizinga, Brad; Lidstrom, Ulrika; Ashby, Matt; Macalady, Jennifer L.

    2012-01-01

    Filter-collected production water samples from a methane-rich gas field in the Cook Inlet basin of Alaska were investigated using whole-cell rRNA-targeted fluorescence in situ hybridization (FISH) and 16S rRNA tag pyrosequencing. Both techniques were consistent in determining the microbial community composition, including the archaeal or bacterial dominance of samples. The archaeal community is dominated by the obligate methylotrophic methanogen genus Methanolobus as well as the nutritional generalist methanogen genus Methanosarcina, which is capable of utilizing acetate, CO2, and methyl-bearing compounds. The most-abundant bacterial groups are Firmicutes, notably of the Acetobacterium genus, and Cytophaga-Flexibacter-Bacteroides species (CFBs) affiliated with the order Bacteroidales. We observed spatial variation among samples in both the percentage of members of Archaea compared to that of members of Bacteria and the dominant members of the bacterial community, differences which could not be explained with the available geochemical data. Based upon the microbial community composition and the isotopic signature of methane associated with the Cook Inlet basin site, we propose a simplified reaction network beginning with the breakdown of coal macromolecules, followed by fermentation and methylotrophic and acetoclastic methane production. PMID:22427501

  11. HER2\\/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: Comparison of results and correlation with survival

    Microsoft Academic Search

    Sanjay Kakar; Nick Puangsuvan; Jane M. Stevens; Romualda Serenas; George Mangan; Shalini Sahai; Michael L. Mihalov

    2000-01-01

    Background: HER-2\\/neu immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results guide breast cancer therapy; however, few studies compared the results and no published studies have correlated them with patient outcome. Methods and Results: We compared results, cost, and turnaround time in 117 archival, invasive breast carcinomas and compared 50-month survival in 65 of these cases using commercial HER-2\\/neu IHC

  12. Detection of sex chromosomal aneuploidies XX, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    Microsoft Academic Search

    Andrew J. Wyrobek; Wendie A. Robbins; D. Pinkel; H. U. Weier; Y. Mehraein

    1994-01-01

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with

  13. Analysis of the nitrifying bacterial community in BioCube sponge media using fluorescent in situ hybridization (FISH) and microelectrodes.

    PubMed

    Chae, Kyu-Jung; Rameshwar, T; Jang, Am; Kim, Sung H; Kim, In S

    2008-09-01

    There is growing interest in the development of more cost-effective and retrofit technologies for the upgrade and expansion of existing wastewater treatment plants with extreme space constraints. A free-floating sponge media (BioCube) process, using a 24 L lab scale reactor, was operated to study the nitrification profiles and microbial community. The COD removal efficiencies were maintained, at an average of 95%, with the mixed liquor suspended solids (MLSS) inside the BioCube sponge media maintained at 12,688 mg/L. The nitrification removal efficiencies were between 92% and 100%, with an average value of 99%. From the results of microelectrode measurements, the ammonium ion concentration was found to rapidly decrease from the surface of the BioCube sponge media to a depth of 2mm due to chemical reactions carried out by ammonia oxidizing bacteria (AOB) species. Multi-fluorescence in situ hybridization (FISH) has been used to investigate the spatial distributions of various microbial activities within reactors. Microbial communities were targeted using different oligonucleotide probes specific to AOB and nitrite oxidizing bacteria (NOB). There were a large number of AOB populations, but these were not uniformly distributed in the biofilm compared to the NOB populations. PMID:17765389

  14. Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study.

    PubMed

    van der Logt, Elise M J; Kuperus, Deborah A J; van Setten, Jan W; van den Heuvel, Marius C; Boers, James E; Schuuring, Ed; Kibbelaar, Robby E

    2015-01-01

    HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (? = 0.94) and 93.8% (? = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer. PMID:25844540

  15. Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fluorescence in situ hybridization

    Microsoft Academic Search

    O. Schrader; H. Budahn; R. Ahne

    2000-01-01

    Different ribosomal RNA (5S and 25S) genes were investigated simultaneously by fluorescence in situ hybridization (FISH) in\\u000a Sinapis alba, Raphanus sativus and Brassica napus. The chromosomes of S. alba carried four 5S and six 25S gene sites, and those of R. sativus four sites of each gene, respectively. These two species have one chromosome pair with both rDNA genes; the

  16. Multitarget Fluorescence In Situ Hybridization Assay Detects Transitional Cell Carcinoma in the Majority of Patients with Bladder Cancer and Atypical or Negative Urine Cytology

    Microsoft Academic Search

    MAREK SKACEL; MONA FAHMY; JENNIFER A. BRAINARD; JAMES D. PETTAY; CHARLES V. BISCOTTI; LOUIS S. LIOU; GARY W. PROCOP; J. STEPHEN JONES; JAMES ULCHAKER; CRAIG D. ZIPPE; RAYMOND R. TUBBS

    2003-01-01

    PurposeThe multitarget fluorescence in situ hybridization (FISH) probe set UroVysion (Vysis, Downers Grove, Illinois), containing probes to chromosomes 3, 7 and 17, and to the 9p21 band, has been recently shown to have high sensitivity and specificity for detecting transitional cell carcinoma. In this study we retrospectively tested 120 urine samples from patients with atypical, suspicious and negative cytology for

  17. Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures

    Microsoft Academic Search

    HENRIK STENDER; KAARE LUND; KENNETH H. PETERSEN; OLE F. RASMUSSEN; POONPILAS HONGMANEE; HÅKAN MIORNER; SVEN E. GODTFREDSEN

    1999-01-01

    TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontubercu- lous mycobacteria (NTM) in acid-fast bacillus-positive (AFB1) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears

  18. Visualization of alternative Epstein-Barr virus expression programs by fluorescent in situ hybridization at the cell level.

    PubMed

    Szeles, A; Falk, K I; Imreh, S; Klein, G

    1999-06-01

    Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). They regularly express six virally encoded nuclear proteins (EBNA1 to EBNA6) and three membrane proteins (LMP1, LMP2A, and LMP2B). In contrast, EBV-carrying Burkitt lymphoma (BL) cells in vivo and derived type I cell lines that maintain the BL phenotype express only EBNA1. During prolonged in vitro culturing, most EBV-carrying BL lines drift toward a more immunoblastic (type II or III) phenotype. Their viral antigen expression is upregulated in parallel. We have used fluorescent in situ hybridization to visualize viral transcripts in type I and III BL lines and LCLs. In type I cells, EBNA1 is encoded by a monocistronic message that originates from the Qp promoter. In type III cells, the EBNA1 transcript is spliced from a giant polycistronic message that originates from one of several alternative Wp or Cp promoters and encodes all six EBNAs. We have obtained a "track" signal with a BamHI W DNA probe that could hybridize with the polycistronic but not with the monocistronic message in two type III BL lines (Namalwa-Cl8 and MUTU III) and three LCLs (LCL IB4-D, LCL-970402, and IARC-171). A BamHI K probe that can hybridize to both the monocistronic and the polycistronic message visualized the same pattern in the type III BLs and the LCLs as the BamHI W probe. A positive signal was obtained with the BamHI K but not the BamHI W probe in the type I BL lines MUTU I and Rael. The RNA track method can thus distinguish between cells that use a type III and those that use a type I program. The former cells hybridize with both the W and the K probes, but the latter cells hybridize with only the K probe. Our findings may open the way for studies of the important but still unanswered question of whether cells with type I latency arise from immunoblasts with a full type III program or are generated by a separate pathway during primary infection. PMID:10233969

  19. Frequent Amplification of C-erbB2 (HER2\\/Neu) Oncogene in Cervical Carcinoma as Detected by Non-Fluorescence in situ Hybridization Technique on Paraffin Sections

    Microsoft Academic Search

    A. Sharma; M. Pratap; V. Malhotra Sawhney; I. U. Khan; S. Bhambhani; A. B. Mitra

    1999-01-01

    Sixty primary untreated squamous cell carcinomas were studied for C-erbB2 gene amplification by non-fluorescence in situ hybridization technique. Amplified tumour cells showed intranuclear dark brown, often paired signals under light microscopy. Twenty-two out of 60 tumours (36.6%) showed signs of amplification of different degree (+ to ++++). Considerable heterogeneity of C-erbB2 amplification per cell was seen within each amplified tumour.

  20. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    SciTech Connect

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  1. [Rapid enrichment and cultivation of denitrifying phosphate-removal bacteria and its identification by fluorescence in situ hybridization technology].

    PubMed

    Liu, Li; Tang, Bing; Huang, Shao-Song; Fu, Feng-Lian; Zhang, Qi-Qin; Li, Jian-Bin; Luo, Jian-Zhong

    2013-07-01

    The present work focused on a rapid enrichment and cultivation of denitrifying phosphate-removal bacteria (DPB) in a membrane bio-reactor(MBR) by using A2/O anaerobic sludge from a wastewater treatment plant as seed, as well as providing an identification method. In the experiments, sodium acetate was used as the carbon source and a certain amount of nitrate was added to the MBR in the anoxic stage. Results showed that, with the efficient trap of the hollow-fiber membrane module, the proportion of DPB in all the phosphate-accumulating organisms (PAOs) increased from 24% to 93% within 35 days after two-stage's cultivation including anaerobic/aerobic and anaerobic/anoxic, during which the removal efficiency of nitrogen and phosphorus reached more than 90%. The activated sludge was identified by combining a regular method and the fluorescence in situ hybridization (FISH) technique, which demonstrated that Pseudomonas sp. and Rhodocyclus sp. were the dominant bacteria in the used bioreactor. PMID:24028025

  2. Molecular cytogenetics of the genus Artemisia (Asteraceae, Anthemideae): fluorochrome banding and fluorescence in situ hybridization. I. Subgenus Seriphidium and related taxa

    Microsoft Academic Search

    M. Torrell; M. Cerbah; S. Siljak-Yakovlev; J. Vallès

    2003-01-01

    The distributional pattern of AT- and GC-rich regions and the physical mapping of ribosomal DNA (location of 18S-5.8S-26S and 5S rDNA) in the chromosomes of seven Artemisia species have been established by means of fluorochrome banding and fluorescence in situ hybridization (FISH). This is the first study in the large genus Artemisia using FISH. Five species ( A. barrelieri, A.

  3. A Case of Acute Lymphoblastic Leukemia, Near-Triploidy, and Poor Outcome: Characterization by Fluorescence In Situ Hybridization Using Chromosome-Specific Libraries from All Human Chromosomes

    Microsoft Academic Search

    Ann Nordgren; Magnus Nordenskjöld; Stefan Söderhäll; Anna Porwit-MacDonald; Elisabeth Blennow

    1997-01-01

    We have applied fluorescence in situ hybridization (FISH) using chromosome-specific libraries from all 24 chromosomes on metaphase spreads from bone marrow cells, in order to resolve the chromosomal changes in leukemic cells from a 10-year-old boy with acute lymphoblastic leukemia (ALL), near-triploidy, and a subsequent poor outcome. The FISH analysis revealed a pattern of chromosome gains and losses that differed

  4. Molecular analysis of hematopoietic colonies derived from chronic myeloid leukemia patients: interphase fluorescence in situ hybridization compared with RT-PCR

    Microsoft Academic Search

    SFT Thijsen; GJ Schuurhuis; JW van Oostveen; AP Theijsmeijer; MMAC Langenhuijsen; GJ Ossenkoppele

    1997-01-01

    In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte–macrophage (CFU-GM) colonies. One half was analyzed with

  5. Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization

    Microsoft Academic Search

    Darren K. Griffin; Alan H. Handyside; Joyce C. Harper; Leeanda J. Wilton; Glenn Atkinson; Iakovos Soussis; Dagan Wells; Elena Kontogianni; Juan Tarin; Selmo Geber; Asangla Ao; Robert M. L. Winston; Joy D. A. Delhanty

    1994-01-01

    Purpose  \\u000a Our purpose was to assess the clinical application of dual fluorescent in situhybridization (FISH) for the diagnosis of sex in the human preimplantation embryo.\\u000a \\u000a \\u000a \\u000a Results  \\u000a Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis\\u000a of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in

  6. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    NASA Astrophysics Data System (ADS)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  7. Fluorescent in situ hybridization (FISH) and high resolution karyotype analysis reveal a novel inversion duplication of 10q

    SciTech Connect

    Czarnecki, P.; Dyke, D.L. Van [Henry Ford Hospital, Detroit, MI (United States); Dowling, P.K. [MetPath, Inc., Teterboro, NJ (United States)] [and others

    1994-09-01

    A white male born with dysmorphic features, including upslanting palpebral fissures, bilateral simian creases, posteriorly rotated ears, bitemporal narrowing, frontal bossing, camptodactyly and head circumference and weight less than the 5th percentile was found to have a de novo add(10)(q26.1). High resolution karyotype analysis revealed a novel chromosomal abnormality: 46,XY,inv dup(10)(q26.3-q25.1). Fluorescent in situ hybridization using a chromosome 10-specific painting probe (Oncor, Inc.) confirmed that the extra material was derived from chromosome 10. Duplication of 10q24 or 10q25 is associated with characteristic craniofacial malformations, minor malformations of the hands and feet, major malformations of the heart, skeleton, and kidneys and severe mental retardation. Our patient, currently 7 months old, has many of the skeletal and craniofacial manifestations of other patients, but is developmentally normal at this early age. This is the first FISH confirmation of a 10q duplication and demonstrates the utility of this technology in addition to karyotype analysis. Molecular studies to determine the parental origin and extent of the duplication are in progress, since the apparent lack of developmental delay was unexpected. Identification of the origin of duplicated material will help assist in genetic counseling by further delineating new genetic syndromes.

  8. 3D-catFISH: a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity imaged by fluorescence in situ hybridization

    Microsoft Academic Search

    Monica K. Chawla; Gang Lin; Kathy Olson; Almira Vazdarjanova; Sara N. Burke; Bruce L. McNaughton; Paul F. Worley; John F. Guzowski; Badrinath Roysam; Carol A. Barnes

    2004-01-01

    Fluorescence in situ hybridization (FISH) of neural activity-regulated, immediate-early gene (IEG) expression provides a method of functional brain imaging with cellular resolution. This enables the identification, in one brain, of which specific principal neurons were active during each of two distinct behavioral epochs. The unprecedented potential of this differential method for large-scale analysis of functional neural circuits is limited, however,

  9. Smith-Magenis syndrome deletion: A case with equivocal cytogenetic findings resolved by fluorescence in situ hybridization

    SciTech Connect

    Juyal, R.C.; Patel, P.I.; Greenberg, F. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-09-11

    The availability of markers for the 17p11.2 region has enabled the diagnosis of Smith-Magenis syndrome (SMS) by fluorescence in situ hybridization (FISH). SMS is typically associated with a discernible deletion of band 17p11.2 upon cytogenetic analysis at a resolution of 400-550 bands. We present a case that illustrates the importance of using FISH to confirm a cytogenetic diagnosis of del(17)(p11.2). Four independent cytogenetic analyses were performed with different conclusions. Results of low resolution analyses of amniocytes and peripheral blood lymphocytes were apparently normal, while high resolution analyses of peripheral blood samples in two laboratories indicated mosaicism for del(17)(p11.2). FISH clearly demonstrated a 17p deletion on one chromosome of all peripheral blood cells analyzed and ruled out mosaicism unambiguously. The deletion was undetectable by flow cytometric quantitation of chromosomal DNA content, suggesting that it is less than 2 Mb. We conclude that FISH should be used to detect the SMS deletion when routine chromosome analysis fails to detect it and to verify mosaicism. 23 refs., 3 figs., 1 tab.

  10. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    PubMed Central

    Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

  11. Chromosomal Constitution of Embryos Derived from Tripronuclear Zygotes Studied by Fluorescence in situ Hybridization Using Probes for Chromosomes 4, 13, 18, 21, X, and Y

    Microsoft Academic Search

    Myung-Geol Pang; Byung-Chul Jee; Seok-Hyun Kim; Buom-Yong Ryu; Sun-Kyung Oh; Chang-Suk Suh; Shin-Yong Moon

    2005-01-01

    This study was designed to assess the chromosomal constitution and segregation patterns of cleaving embryos derived from tripronuclear zygotes. Thirty-two embryos obtained from 19 conventional IVF patients were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 4, 13, 18, 21, X, and Y. Sixteen embryos (50.0%) exhibited uniform, non-mosaic patterns. These embryos showed pure triploid (n =

  12. Differentiation of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacterial Liquid Cultures by Using Peptide Nucleic Acid-Fluorescence In Situ Hybridization Probes

    Microsoft Academic Search

    F. A. DROBNIEWSKI; P. G. MORE; G. S. HARRIS

    2000-01-01

    A blinded comparison of peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) with routine identification methods was performed on 74 consecutively positive mycobacterial liquid cultures. All Mycobac- terium tuberculosis cultures (48 of 48) and 22 of 27 (81.5%) nontuberculous cultures were correctly identified (including one mixed culture). Five isolates yielded no reaction with either probe and were identified as Mycobacterium xenopi,

  13. Utility of fluorescence in situ hybridization to detect MDM2 amplification in liposarcomas and their morphological mimics

    PubMed Central

    Kimura, Hiroaki; Dobashi, Yoh; Nojima, Takayuki; Nakamura, Hiroyuki; Yamamoto, Norio; Tsuchiya, Hiroyuki; Ikeda, Hiroko; Sawada-Kitamura, Seiko; Oyama, Takeru; Ooi, Akishi

    2013-01-01

    The atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLS) and the de-differentiated liposarcoma (DDLS) represent the most common category of liposarcomas. ALT/WDLSs and DDLSs are often difficult to distinguish from other tumors with similar morphological characteristics. In this study, we investigated whether the detection of amplified or overexpressed murine double-minute 2 (MDM2) can be a useful diagnostic ancillary aid. We used fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) to detect MDM2 amplification and protein overexpression, respectively, in 49 WDLSs, 5 DDLSs, 23 myxoid liposarcomas, 25 benign lipomatous tumors, and 75 spindle and pleomorphic sarcomas. MDM2 amplification was detected in 48 of 49 WDLSs, 5 of 5 DDLSs, 2 of 9 malignant peripheral nerve sheath tumors, and 2 of 10 myxofibrosarcomas. We did not detect MDM2 amplification in any of the benign lipomatous tumors. FISH-mediated detection of MDM2 amplification was the most valuable diagnostic aid for ALT/WDLS, as determined by using the Fisher exact test to compare two different diagnoses of 19 biopsies. On the contrary, unequivocal nuclear overexpression of MDM2 was found in only 10 of 50 ALT/WDLSs. The sensitivity and specificity of MDM2 amplification in distinguishing a DDLS from spindle and pleomorphic sarcomas were 100% and 95%, respectively, while those of MDM2 overexpression were 100% and 87%, respectively. In conclusion, our results indicate that FISH-mediated detection of MDM2 amplification is the most useful adjunct in the diagnosis of both ALT/WDLS and DDLS. However, IHC-mediated detection of MDM2 protein is useful only for the diagnosis of DDLS. PMID:23826411

  14. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques

    Microsoft Academic Search

    Rudolf Amann; Bernhard M. Fuchs

    2008-01-01

    The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can

  15. Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization(1).

    PubMed

    Jurgens; Glöckner; Amann; Saano; Montonen; Likolammi; Münster

    2000-10-01

    We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel 'non-extreme' environmental Archaea in the pelagic water of a boreal forest lake. PMID:11053735

  16. Fluorescence in situ hybridization for detection of MAML2 rearrangements in oncocytic mucoepidermoid carcinomas: utility as a diagnostic test.

    PubMed

    García, Joaquin J; Hunt, Jennifer L; Weinreb, Ilan; McHugh, Jonathan B; Barnes, E Leon; Cieply, Kathleen; Dacic, Sanja; Seethala, Raja R

    2011-12-01

    Oncocytic mucoepidermoid carcinoma poses diagnostic challenge because of its histologic overlap with other oncocytic salivary gland lesions, including Warthin tumor. Although the prognostic value of the t(11;19) MECT1-MAML2 fusion gene has been established in mucoepidermoid carcinoma, its diagnostic use in discriminating oncocytic mucoepidermoid carcinoma from histologic mimics is unexplored. We evaluated the translocation status in 14 cases of oncocytic mucoepidermoid carcinoma using a MAML2-11q21 break-apart probe spanning the entire chromosome region of the MAML2 gene and correlated these findings with clinicopathologic parameters including age, sex, stage, predominant growth pattern, grade, and p63 immunostaining pattern. All oncocytic mucoepidermoid carcinomas were parotid tumors with a mean patient age of 54.6 years (range, 9-85) and a female to male ratio of 5:2. Grade distribution was as follows: low grade, 9; intermediate grade, 2; and high grade, 3. The histologic patterns observed were as follows: solid, 4; cystic, 8 (of these, 5 had Warthin-like lymphoid stroma); and mixed, 2. Solid oncocytic mucoepidermoid carcinomas showed a diffuse p63 staining pattern, whereas cystic oncocytic mucoepidermoid carcinomas showed staining of the outer layer of intermediate cells ranging from a bilayer to areas of complex multilayering and plaque-like proliferation. Ten (71%) of the 14 cases showed a MAML2 rearrangement by fluorescence in situ hybridization. No correlation was seen between rearrangement status and histologic grade, growth pattern, or p63 staining pattern. However, we demonstrate that the presence of MAML2 rearrangement can be used as supportive evidence to distinguish oncocytic mucoepidermoid carcinoma from other oncocytic lesions. PMID:21777943

  17. Paternally inherited chromosomal structural aberrations detected in mouse first-cleavage zygote metaphases by multicolour fluorescence in situ hybridization painting

    Microsoft Academic Search

    Francesco Marchetti; Xiu Lowe; Dan H. Moorell; Jack Bishop; Andrew J. Wyrobek

    1996-01-01

    We describe a fluorescencein situ hybridization (FISH) procedure for assessing zygotic risk of paternal exposure to endogenous or exogenous agents. The procedure employs multicolour FISH with chromosome-specific DNA painting probes plus DAPI staining for detecting both balanced and unbalanced chromosomal aberrations in mouse first-cleavage (1-Cl) zygote metaphases. Four composite probes specific for chromosomes 1, 2, 3 or X, each labelled

  18. Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

    Microsoft Academic Search

    C. Gutierrez-Mateo; J. Benet; D. Wells; P. Colls; M. G. Bermudez; J. F. Sanchez-Garcia; J. Egozcue; J. Navarro; S. Munne ´

    2004-01-01

    BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by com- parative genomic hybridization (CGH), while the corresponding metaphase II (MII)

  19. Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

    PubMed Central

    Liu, Fang; Ling, Jian; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

    2012-01-01

    Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established. PMID:22442728

  20. Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids

    SciTech Connect

    Purandare, S.M.; Neil, S.M.; Brothman, A. [Univ. of Utah, Salt Lake City (United States)] [Univ. of Utah, Salt Lake City (United States); [Jikei Univ., Tokyo (Japan)] [and others

    1995-12-10

    Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1-homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5{prime} end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template. 41 refs., 3 figs., 3 tabs.

  1. Tubulocystic carcinoma of the kidney with poorly differentiated foci: a series of 3 cases with fluorescence in situ hybridization analysis.

    PubMed

    Al-Hussain, Turki O; Cheng, Liang; Zhang, Shaobo; Epstein, Jonathan I

    2013-07-01

    We identified 3 consult cases of tubulocystic renal cell carcinoma with poorly differentiated areas. Two lesions measuring 9.5 and 3.8 cm were described as partly solid and cystic. One case was grossly a 14.0-cm cyst with a granular lining. Microscopically, all had classic areas of circumscribed tubulocystic renal cell carcinoma occupying 30%, 80%, and 90% of the tumor; 2 cases had small components of papillary renal cell carcinoma, and 1 case had a central large cystic component. In 2 cases, proliferations of small tubules infiltrated away from the main mass with typical features of collecting duct carcinoma. In the third case, a focus of poorly differentiated carcinoma was seen adjacent to the tubulocystic renal cell carcinoma. In 2 cases, tumor invaded perirenal tissue. The third case was organ confined with vascular invasion. One patient died 9 months postoperatively with metastases to the abdominal wall and femur. The second case developed a recurrence in the renal bed 3 years postoperatively. The third patient was lost to follow-up. Fluorescence in situ hybridization studies results showed some features overlapping with papillary renal cell carcinoma in both the tubulocystic and collecting duct-like components and with 1 exception showed identical cytogenetic findings between the 2 components. Morphologically, in 2 cases, the collecting duct-like areas were also indistinguishable from collecting duct carcinoma suggesting a relationship between the 2 entities. This is the first series and only the second report of tubulocystic renal cell carcinoma with poorly differentiated components and documents the increased the risk of aggressive behavior above that of usual tubulocystic renal cell carcinoma. PMID:23427871

  2. Trisomy 10p resulting from an inv dup of 10p defined by fluorescence in situ hybridization

    SciTech Connect

    Clement, S.J.; Easterling, T.R.; Leppig, K.A. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    De novo cases of trisomy for the entire short arm of chromosome 10 are infrequently reported and are most commonly the result of translocation of 10p to an acrocentric chromosome. Most reported cases of trisomy 10p are not trisomy for the complete short arm of chromosome 10, but are duplication, deficiency syndromes that result from either inheritance of an unbalanced translocation from a parent possessing a balanced reciprocal translocation, or from a recombinant chromosome derived from a parental pericentric inversion of chromosome 10. Here, we report a case of a de novo trisomy 10p that resulted from an inverted duplication of the entire short arm of chromosome 10. A 42 year old G7,P5,SAB1 woman was referred for amniocentesis because of advanced maternal age. Ultrasound examination at 17 weeks demonstrated a fetus of normal size with no apparent anatomic abnormalities. Cytogenetic evaluation demonstrated one homologue of chromosome 10 had a tandem inverted duplication of the short arm. The fetal karyotype was interpreted to be 46,XX,inv dup (10) (peter-cen::cen-p15::q11-pter). Parental karyotype are normal. Fluorescence in situ hybridization (FISH) using a chromosome 10 paint, chromosome 10 centromere, and all telomere probe, confirmed the inverted duplication involved the entire short arm of chromosome 10. Termination of pregnancy was performed at 20 weeks gestation. Autopsy revealed multiple anomalies including low-set posteriorly rotated ears, cleft of the soft palate, ocular hypertelorism, small upturned nose, agenesis of the gallbladder, sacral hemivertebrae, and abnormal flexion of the thumbs. The fetal karyotype was confirmed by cytogenetic analysis in lung and kidney. This is the second reported case of a de novo tandem duplication of 10p of which we are aware, and the first using FISH technology to characterize the abnormality.

  3. Identification of sex chromosome molecular markers using RAPDs and fluorescent in situ hybridization in rainbow trout

    Microsoft Academic Search

    P. Iturra; J. F. Medrano; M. Bagley; N. Lam; N. Vergara; J. C. Marin

    1997-01-01

    The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode\\u000a of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified\\u000a that generated polymorphic bands amplifying preferentially in males of the Mount Lassen and Scottish strains of rainbow trout.\\u000a Chromosomal

  4. Assessment of ERBB2 and EGFR gene amplification and protein expression in gastric carcinoma by immunohistochemistry and fluorescence in situ hybridization

    Microsoft Academic Search

    Wang YK; Gao CF; Yun T; Chen Z; Zhang XW; Lv XX; Meng NL; Zhao WZ

    2011-01-01

    Background  The goal of this study was to investigate ERBB2(HER2) and EGFR gene amplification and protein expression in gastric cancer. Fluorescence in situ hybridization (FISH) and immunohistochemistry were used to analyze ERBB2 and EGFR gene amplification and protein expression in 69 cases of gastric cancer.\\u000a \\u000a \\u000a \\u000a \\u000a Results  FISH analysis revealed that 20.3% of the cases exhibited ERBB2 gene amplification. Increases in ERBB2 copy

  5. Comparison between fluorescence in situ hybridization and conventional cytogenetics for dicentric scoring: a first-step validation for the use of FISH in biological dosimetry.

    PubMed

    Roy, L; Sorokine-Durm, I; Voisin, P

    1996-12-01

    In this study the suitability of fluorescence in situ hybridization (FISH) for dicentric detection using a commercially available alpha-satellite probe (Oncor) to label centromeres was compared with the conventional technique for the detection of unstable aberrations. A standard FISH protocol was applied for centromere labelling. Dose-response curves using blood samples irradiated in vitro with gamma-rays (60Co) at a dose-rate of 0.1 Gy/min were established using both techniques and compared. No statistical difference was observed between either method. The FISH technique thus allows a correct detection of unstable aberrations when an alpha-satellite DNA probe is used. PMID:8980663

  6. Novel karyotype in the Ullrich-Turner syndrome - 45,X/46,X,r(X)/46,X,dic(X) - investigated with fluorescence in situ hybridization

    SciTech Connect

    Robson, L.; Jackson, J.; Cowell, C.; Sillence, D.; Smith, A. [Children`s Hospital, Camperdown (Australia)

    1994-04-15

    A 10-year-old girl with Ullrich-Turner syndrome was found to have the novel karyotype 45,X/46,X,r(X)(p11q11)/46,X,dic(X)(p11). Fluorescence in situ hybridization (FISH) with the {alpha} satellite X centromere probe established the origin of the small ring chromosome. Scanning a large number of cells by interphase FISH showed that the dicentric (X) was the least prevalent cell line. The common breakpoint of Xp11 suggests a sequence of errors as the mechanism whereby these 3 distinct cell lines have arisen. 11 refs., 4 figs., 1 tab.

  7. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos

    E-print Network

    Ji, Ni

    In C. elegans, the expression pattern of a gene provides important clues to understanding its biological function. To accurately depict endogenous transcriptional activity, a highly sensitive method is required to measure ...

  8. Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization

    SciTech Connect

    Lucas, J.N.

    2000-03-28

    A method is provided for determining a clastogenic signature of a sample of chromosomes by quantifying a frequency of a first type of chromosome aberration present in the sample; quantifying a frequency of a second, different type of chromosome aberration present in the sample; and comparing the frequency of the first type of chromosome aberration to the frequency of the second type of chromosome aberration. A method is also provided for using that clastogenic signature to identify a clastogenic agent or dosage to which the cells were exposed.

  9. Enhanced Two-Color Fluorescent In Situ Hybridization for Low-Abundance Genes

    E-print Network

    @ RT. *Wash slides with TNT - 1 x 5 min. *To quench peroxidase activity, incubate in 3% H2O2 for 1hr-2 µl of DIG and/or FITC probe (see section below for probe synthesis; can adjust amount of probe used of the template DNA is the major determinant of successful RNA synthesis. This protocol therefore takes special

  10. Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Jakob Pernthaler; Annelie Pernthaler; Rudolf Amann

    2003-01-01

    We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine

  11. Interphase study by fluorescence in situ hybridization of spermatozoa of a paracentric inversion heterozygote

    SciTech Connect

    Brock, J.K.; Best, R.G. [Univ. of South Carolina School of Medicine, Columbia, SC (United States)

    1994-09-01

    Cytogenetic studies of peripheral lymphocytes were initiated on a couple with a history of three spontaneous 1st trimester losses and oligospermia. The results revealed the woman to have a normal female karyotype. The man had a karyotype of 46,XY,inv(2)(q14.2q24.3). Interphase sperm studies were offered as an attempt to quantify the relative proportion of sperm with acentric and dicentric chromosome No. 2 in response to the couple`s concern that chromosomally unbalanced sperm resulting from recombination of the inversion was the primary cause of pregnancy losses. Slides were prepared and processed as previously described. A two-color probe cocktail consisting of a biotinylated {alpha}-satellite probe for No. 2 and a digoxigenin-labeled probe for {alpha}-satellite No. 17 as an internal control was employed. Among 496 cells with a single signal for chromosome No. 17, we observed 2 with no signal for chromsome No. 2, 492 with a single signal for No. 2, and 2 cells with 2 signals for No. 2. These findings indicate that the proportion of unbalanced recombinant sperm is probably under 1%, and that other factors are likely to be involved in the etiology of this couple`s pregnancy losses.

  12. Partial trisomy 11q involving chromosome 1 detected by fluorescence in situ hybridization

    SciTech Connect

    McCorquodale, M.; Bereziouk, O.; McCorquodale, D.J. [Univ. of Illinois College of Medicine, Chicago, IL (United States)] [and others

    1994-09-01

    Partial trisomy 11q was detected in an infant delivered 3-4 weeks prematurely. The phenotype included slanted palpebral fissures, high arched palate, developmental delay, microcephaly, and cardiac defects, all of which occur in the majority of cases with this syndrome. Other features included a column-shaped skull, preauricular pit, single palmar crease, short, broad great toes, flat occiput, unilateral kidney agenesis, and strabismus. Chromosomes obtained from peripheral blood cells revealed the presence of extra material on the long arm of chromosome 1. The G-banding pattern of this extra material indicated that it might be derived from chromosome 1 or 11. Chromosomal {open_quotes}paints{close_quotes} showed that it was not chromosome 1 material, but was chromosome 11 material extending from band q21 to qter. Partial trisomy 11q arising from translocation of the 11q material to chromosome 2, 3, 4, 5, 6, 9, 10, 13, 17, 21, 22, and X has been reported previously, whereas translocation to chromosome 1 has not. The chromosome to which the 11q material is translocated does not alter the most frequent features of the partial trisomy 11q syndrome, but may influence other less common features.

  13. Comparison of culture, polymerase chain reaction, and fluorescent in situ hybridization for detection of Brachyspira hyodysenteriae and "Brachyspira hampsonii" in pig feces.

    PubMed

    Wilberts, Bailey L; Warneke, Hallie L; Bower, Leslie P; Kinyon, Joann M; Burrough, Eric R

    2015-01-01

    Swine dysentery is characterized by mucohemorrhagic diarrhea and can occur following infection by Brachyspira hyodysenteriae or "Brachyspira hampsonii?". A definitive diagnosis is often based on the isolation of strongly beta-hemolytic spirochetes from selective culture or by the application of species-specific polymerase chain reaction (PCR) assays directly to feces. While culture is highly sensitive, it typically requires 6 or more days to complete, and PCR, although rapid, can be limited by fecal inhibition. Fluorescent in situ hybridization (FISH) has been described in formalin-fixed tissues; however, completion requires approximately 2 days. Because of the time constraints of available assays, a same-day FISH assay was developed to detect B. hyodysenteriae and "B. hampsonii?" in pig feces using previously described oligonucleotide probes Hyo1210 and Hamp1210 for B. hyodysenteriae and "B. hampsonii", respectively. In situ hybridization was simultaneously compared with culture and PCR on feces spiked with progressive dilutions of spirochetes to determine the threshold of detection for each assay at 0 and 48 hr. The PCR assay on fresh feces and FISH on formalin-fixed feces had similar levels of detection. Culture was the most sensitive method, detecting the target spirochetes at least 2 log-dilutions less when compared to other assays 48 hr after sample preparation. Fluorescent in situ hybridization also effectively detected both target species in formalin-fixed feces from inoculated pigs as part of a previous experiment. Accordingly, FISH on formalin-fixed feces from clinically affected pigs can provide same-day identification and preliminary speciation of spirochetes associated with swine dysentery in North America. PMID:25525136

  14. Detection of chromosome changes in pathology archives: an application of microwave-assisted fluorescence in situ hybridization to human carcinogenesis studies.

    PubMed

    Sugimura, Haruhiko

    2008-04-01

    Pathology archives provide unique and abundant opportunities to investigate human carcinogenesis and identify potential targets for cancer therapy. Microwaving was introduced into various procedures used in histopathology two decades ago, although the precise mechanisms underlying its effectiveness in any of the procedures, including antigen retrieval, acceleration of fixation and nucleic acid hybridization, are not known. Since microwaving was first applied to fluorescence in situ hybridization (FISH), many pathologists and researchers have enjoyed the benefits of excellent preservation of histological structures as well as good retrieval of FISH signals by this method. Microwave-assisted fluorescence in situ hybridization (MW-FISH) has proved to be especially useful in retrospective investigations of tissues fixed and preserved for long periods of time, and the success rates in the randomly selected pathology archives have been greater (70-95%) than by the conventional protocol (in human tumor tissue, even in the incipient stage. In practice, this protocol is very useful for retrospective surveillance of amplicons in tumor tissue by using hundreds of bacterial artificial chromosome clones and many specimens in the form of a tissue microarray. Effective retrieval of specific genome-wide amplicon profiles from human tumors stored unaware in ordinary pathology laboratories would help to further stratify tumors so that individually tailored treatment strategies would become feasible in clinical settings. PMID:18283042

  15. Fluorescence in situ hybridization and sequential catalyzed reporter deposition (2C-FISH) for the flow cytometric sorting of freshwater ultramicrobacteria.

    PubMed

    Neuenschwander, Stefan M; Salcher, Michaela M; Pernthaler, Jakob

    2015-01-01

    Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH) it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb), is still challenging. Current FISH protocols, even in combination with signal amplification by catalyzed reporter deposition (CARD), are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labeled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates. PMID:25873914

  16. Fluorescence In Situ Hybridization Using 16S rRNA-Targeted Oligonucleotides Reveals Localization of Methanogens and Selected Uncultured Bacteria in Mesophilic and Thermophilic Sludge Granules

    PubMed Central

    Sekiguchi, Yuji; Kamagata, Yoichi; Nakamura, Kazunori; Ohashi, Akiyoshi; Harada, Hideki

    1999-01-01

    16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35°C) and thermophilic (55°C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655–2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges. PMID:10049894

  17. Multicolor fluorescence in situ hybridization with centromeric DNA probes as a new approach to distinguish chromosome breakage from aneuploidy in interphase cells and micronuclei

    SciTech Connect

    Eastmond, D.A.; Rupa, D.S.; Chen, H.W.; Hasegawa, L. [Univ. of California, Riverside, CA (United States)

    1993-12-31

    Chromosomal abnormalities are believed to contribute significantly to human reproductive failure, carcinogenesis and other pathophysiological conditions. For example, approximately 15% of recognized pregnancies terminate in spontaneous abortion, and of these approximately 30% have been shown to be chromosomally abnormal. The contribution of chromosomal abnormalities to early embryonic and fetal death appears to decrease with gestational age, suggesting that as many as 67% of the aborted embryos in early embryonic deaths are chromosomally abnormal. Furthermore, clinically significant chromosomal abnormalities can also be found to be present in approximately 0.58 to 0.67% of live births. These figures indicate that within a given year, hundreds of thousands of chromosomally abnormal babies will be born throughout the world and additional millions of chromosomally abnormal embryos will have been spontaneously aborted. For the past several years, our research has focused on utilizing new molecular cytogenetic techniques to develop assays for detecting aneuploidy-inducing agents in mammalian cells. One approach that we have sucessfully employed involves the use of fluorescence in situ hybridization with chromosome-specific DNA probes to determine the number of copies of a representative chromosome present within the nucleus following chemical exposure. DNA sequences (probes) which hybridize to blocks of repetitive centromeric DNA on specific chromosomes have been developed for most of the human chromosomes. In situ hybridization with these probes results in the staining of a compact chromosomal region which can be easily detected in interphase nuclei. The presence of 3 (or more) hybridization domains in an interphase nucleus indicates the presence of three centromeric regions and has been presumed to indicate that three copies of the entire chromosome were present in the nucleus.

  18. In situ preparation and fluorescence quenching properties of polythiophene/ZnO nanocrystals hybrids through atom-transfer radical polymerization and hydrolysis

    NASA Astrophysics Data System (ADS)

    Peng, Xiaoming; Zhang, Lin; Chen, Yiwang; Li, Fan; Zhou, Weihua

    2010-02-01

    In this paper, a new approach for in situ preparing nanocomposites of conjugated polymers (CPs) and semiconductor nanocrystals was developed. Polythiophene grafted poly(zinc methacrylate) (PTh-g-PZMA) copolymer was synthesized by atom-transfer radical polymerization (ATRP) of zinc methacrylate (ZMA) initiated from the macroinitiator poly(2,5-(3-(bromoisopropyl-carbonyl-oxymethylene) thiophene)) (PTh-Br) with pendant initiator groups. Subsequently, the polythiophene grafted poly(methacrylate)/ZnO (PTh-g-PMA/ZnO) hybrid heterojunction nanocomposites were successfully prepared by in situ hydrolysis of PTh-g-PZMA casting films in alkaline aqueous solution. The structures of PTh-Br, PTh-g-PZMA and PTh-g-PMA/ZnO were confirmed by the proton nuclear magnetic resonance ( 1H NMR) spectra, Fourier transform infrared (FTIR) spectra and X-ray photoelectron spectroscopy (XPS). The morphologies of PTh-g-PMA/ZnO films prepared for different hydrolysis time were observed in the cross-sections by scanning electron microscope (SEM). The SEM images revealed that ZnO nanocrystals were uniformly dispersed in polymers without any aggregation and the appearances of ZnO nanocrystals changed from nanoparticles to nanorods with the hydrolysis treatment time increasing. The optical properties of these nanocomposites were studied by ultraviolet-visible (UV-vis) absorption and fluorescence spectroscopy. UV-vis absorption spectroscopy showed that the adsorption band of PTh-g-PMA/ZnO hybrids was broader than that of PTh-Br, implying that the existence of ZnO nanocrystals increased the optical absorption region of hybrids. The photoluminescence (PL) spectra of the hybrids showed that fluorescence quenching occurred in PTh-g-PMA/ZnO blends and a maximum of 85% of the fluorescence intensity quenched in the PTh-g-PMA/ZnO obtained from treatment in NaOH aqueous solution for 2 h, which revealed the existence of photo-induced charge transfer between the polythiophene chains and ZnO. These results indicated that the hybrid heterojunction nanocomposites could be promising candidates for photovoltaic applications.

  19. Allelic loss at SMAD4 in polyps from juvenile polyposis patients and use of fluorescence in situ hybridization to demonstrate clonal origin of the epithelium.

    PubMed

    Woodford-Richens, K; Williamson, J; Bevan, S; Young, J; Leggett, B; Frayling, I; Thway, Y; Hodgson, S; Kim, J C; Iwama, T; Novelli, M; Sheer, D; Poulsom, R; Wright, N; Houlston, R; Tomlinson, I

    2000-05-01

    Juvenile polyposis syndrome (JPS; Online Mendelian Inheritance in Man2 174900) is a rare Mendelian disorder in which individuals have typical hamartomatous polyps within the gastrointestinal tract. The stromal element of the polyps has classically been thought to be the proliferative component, although epithelial malignancies (largely gastrointestinal cancers) occur more frequently than expected in JPS patients. Germ-line mutations in SMAD4 (DPC4) account for about a third of JPS cases. It has been postulated that the apparent paradox of a stromal lesion predisposing to epithelial malignancy can be resolved by the "landscaper" effect: an abnormal stromal environment affects the development of adjacent epithelial cells, and the resulting regeneration of damaged epithelium leads to an increased risk of cancer. We have found allele loss at the SMAD4 locus on 18q in polyps from JPS individuals with a germ-line SMAD4 mutation, showing that SMAD4 is acting as a tumor suppressor gene in JPS polyps, as it does in sporadic cancers of the gastrointestinal tract. Interphase fluorescence in situ hybridization showed deletion of one copy of SMAD4 in the epithelial component of JPS polyps, but not in the inflammatory infiltrate. Fluorescence in situ hybridization also suggested that a single copy of SMAD4 was present in stromal fibroblasts of JPS polyps. Thus, biallelic inactivation of SMAD4 occurs in both the epithelium and some of the stromal cells in these lesions, suggesting a common clonal origin. Epithelial malignancies almost certainly develop in juvenile polyposis through direct malignant progression of the epithelial component of the hamartomas. SMAD4/DPC4 probably acts as a "gatekeeper" tumor suppressor in juvenile polyps, and there is no need to invoke a "landscaper hypothesis." PMID:10811127

  20. Clinical and cytogenetic findings in seven cases of inverted duplication of 8p with evidence of a telomeric deletion using fluorescence in situ hybridization

    SciTech Connect

    Guo, Wen-Jun; Callif-Daley, F.; Zapata, M.C.; Miller, M.E. [Children`s Medical Center, Dayton, OH (United States)

    1995-09-11

    We report on the clinical and cytogenetic findings in 7 cases of inverted duplication of region 8p11.2-p23. The phenotype of inv dup (8p) compiled from this series and the literature (N = 29) consists of severe mental retardation (100%), minor facial alterations (97%), agenesis of the corpus callosum (80%), hypotonia (66%), orthopedic abnormalities (58%), scoliosis/kyphosis (40%), and congenital heart defect (26%). A telomeric deletion of region 8p23.3-pter was confirmed in 3 of our cases studied using fluorescent in situ hybridization with a telomeric probe for 8p. Thus, these karyotypes are inv dup del(8) (qter{r_arrow} p23.1::p23.1{r_arrow}p11.2:). Our findings suggest that most cases of inv dup(8p) probably have a telomeric deletion. 20 refs., 4 figs., 2 tabs.

  1. Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

    1998-01-01

    A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

  2. Use of fluorescence in situ hybridization (fish) to study chromosomal damage induced by radiation and bromodeoxyuridine in human colon cancer cells

    SciTech Connect

    Wilt, S.R.; Burgess, A.C.; Lawrence, T.S. [ Univ. of Michigan Medical Center, Ann Arbor, MI (United States)] [and others

    1994-11-15

    Although the thymidine analog radiation sensitizer bromodeoxyuridine (BrdUrd) increases radiation-induced chromosomal aberrations, it is not known whether these aberrations are uniformly distributed among chromosomes. Using fluorescence in situ hybridization, we carried out a study to test the hypothesis that BrdUrd-induced radiosensitization may be mediated by nonuniform chromsomal damage. Log phase HT29 human colon cancer cells were exposed to 10 {mu}M BrdUrd (or media alone) for one cell cycle, and the G1 cells were separated by centrifugal elutriation. Half of the control and BrdUrd samples were irradiated with 8 Gy. Cells were then incubated for 24-28 h, and metaphase spreads were prepared. Fluorescence in situ hybridization was performed using paint probes for chromosomes 1 and 4. We found that radiation induced 0.20 aberrations per chromosome in chromosome 4. Based on the ratio of the relative lengths of chromosome 1-4(1.34), it was predicted that chromosome 1 would have {approx}0.26 aberrations per chromosome. However, we observed 0.39 aberrations per chromosome 1, which was significantly greater than the predicted (p<0.001 by chi-square). Incubation with BrdUrd prior to irradiation significantly increased the aberrations found in chromosome 1 (by a factor of 1.4) and chromosome 4 (by a factor of 1.9) compared to radiation alone (p<0.001 for both chromosome 1 and 4). This study demonstrates that individual chromosomes in human colon cancer cells show significantly different rates of aberration after irradiation. Furthermore, the BrdUrd-mediated increase in radiation-induced chromosomal aberrations may not be uniform among chromosomes. 20 refs., 4 figs., 1 tab.

  3. Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions.

    PubMed

    Valle, Edith R; Henderson, Gemma; Janssen, Peter H; Cox, Faith; Alexander, Trevor W; McAllister, Tim A

    2015-06-01

    In this study, methanogen-specific coenzyme F420 autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis. PMID:25924182

  4. Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes.

    PubMed

    Wullings, B A; Van Beuningen, A R; Janse, J D; Akkermans, A D

    1998-11-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

  5. The Hybrid Origin of Two Cultivars of Crocus (Iridaceae) Analysed by Molecular Cytogenetics including Genomic Southern and in situ Hybridization

    Microsoft Academic Search

    M. Ørgaard; N. Jacobsen; J. S. Heslop-Harrison

    1995-01-01

    The origin of the two common cultivars of Crocus, C. 'Stellaris' (2n = 2x = 10) and C. 'Golden Yellow' (2n = 3x = 14) was investigated by fluorescent in situ hybridization using both total genomic DNA and cloned DNA sequences as probes. The clear differentiation between the chromosomes after genomic in situ hybridization supports the proposals of a hybrid

  6. Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.

    PubMed

    Xia, Xiujin; Rasmussen, Terri; Alvarez, Xavier; Taguchi, Takahiro; Li, Marilyn; La Russa, Vincent F

    2007-11-01

    To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues. PMID:17595337

  7. Determination of HER2 Gene Amplification by Fluorescence In situ Hybridization and Concordance with the Clinical Trials Immunohistochemical Assayin Women with Metastatic Breast Cancer Evaluated for Treatment with Trastuzumab

    Microsoft Academic Search

    Noël Dybdal; Grazyna Leiberman; Steven Anderson; Bryan McCune; Alex Bajamonde; Robert L. Cohen; Robert D. Mass; Corsee Sanders

    2005-01-01

    Summary Purpose. To evaluate the concordance between HER2 gene amplification, determined by fluorescence in situ hybridization (FISH), and HER2 protein overexpression assessed by an immunohistochemical (IHC) assay. The IHC protocol used was a research assay, known as the Clinical Trial Assay (CTA), developed to select women with metastatic breast cancer (MBC) for three pivotal clinical trials of trastuzumab therapy.

  8. Prenatal diagnosis with use of fetal cells isolated from maternal blood: Five-color fluorescent in situ hybridization analysis on flow-sorted cells for chromosomes X, Y, 13, 18, and 21

    Microsoft Academic Search

    Farideh Z. Bischoff; Dorothy E. Lewis; Dianne D. Nguyen; Sarah Murrell; Wendy Schober; Jeffrey Scott; Joe Leigh Simpson; Sherman Elias

    1998-01-01

    OBJECTIVE: Currently, prenatal diagnosis of chromosome abnormalities requires invasive techniques such as amniocentesis and chorionic villus sampling that carry small but finite risks of fetal loss. A noninvasive approach is to isolate fetal cells from maternal blood by flow sorting followed by genetic interphase analysis with fluorescence in situ hybridization. Because the ratio of fetal to maternal cells is relatively

  9. Cross-Species Bacterial Artificial Chromosome–Fluorescence in Situ Hybridization Painting of the Tomato and Potato Chromosome 6 Reveals Undescribed Chromosomal Rearrangements

    PubMed Central

    Tang, Xiaomin; Szinay, Dóra; Lang, Chunting; Ramanna, Munikote S.; van der Vossen, Edwin A. G.; Datema, Erwin; Lankhorst, René Klein; de Boer, Jan; Peters, Sander A.; Bachem, Christian; Stiekema, Willem; Visser, Richard G. F.; de Jong, Hans; Bai, Yuling

    2008-01-01

    Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC–FISH as a unique tool for comparative genetic studies across Solanum species. PMID:18791231

  10. Comparison of conventional culture method and fluorescent in situ hybridization technique for detection of Listeria spp. in ground beef, turkey, and chicken breast fillets in ?zmir, Turkey.

    PubMed

    Baysal, Ayse Handan

    2014-12-01

    The occurrence of Listeria species in refrigerated fresh chicken breast fillet, turkey breast fillet, and ground beef was evaluated, comparing the conventional culture method and fluorescent in situ hybridization (FISH). FISH uses hybridization of a nucleic acid sequence target of a microorganism with a specific DNA probe labeled with a fluorochrome and imaging by a fluorescence microscope. First, Listeria was inoculated in chicken breast fillet, turkey breast fillet, or ground beef, and the applicability of the FISH method was evaluated. Second, Listeria was detected in fresh chicken breast fillet, turkey breast fillet, and ground beef by culture and FISH methods. Listeria was isolated from 27 (37.4%) of 216 samples by the standard culture method, whereas FISH detected 25 (24.7%) preenriched samples. Of these isolates, 17 (63%) were L. innocua, 6 (22%) L. welshimeri, and 4 (14.8%) L. seeligeri. Overall, the prevalences of Listeria spp. found with the conventional culture method in chicken breast fillet, turkey breast fillet, and ground beef were 9.7, 6.9, and 20.8%, whereas with the FISH technique these values were 11.1, 6.9, and 16.7%, respectively. The molecular FISH technique appears to be a cheap, sensitive, and time-efficient procedure that could be used for routine detection of Listeria spp. in meat. This study showed that retail raw meats are potentially contaminated with Listeria spp. and are, thus, vehicles for transmitting diseases caused by foodborne pathogens, underlining the need for increased precautions, such as implementation of hazard analysis and critical control points and consumer food safety education. PMID:25474046

  11. Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

    PubMed

    Vašut, Radim J; Vijverberg, Kitty; van Dijk, Peter J; de Jong, Hans

    2014-11-01

    Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed. PMID:25760668

  12. Numerical and Structural Genomic Aberrations Are Reliably Detectable in Tissue Microarrays of Formalin-Fixed Paraffin-Embedded Tumor Samples by Fluorescence In-Situ Hybridization

    PubMed Central

    Horn, Heike; Bausinger, Julia; Staiger, Annette M.; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M. Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. PMID:24733537

  13. Detection of the genome and transcripts of a persistent DNA virus in neuronal tissues by fluorescent in situ hybridization combined with immunostaining.

    PubMed

    Catez, Frédéric; Rousseau, Antoine; Labetoulle, Marc; Lomonte, Patrick

    2014-01-01

    Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue. PMID:24514006

  14. Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis

    SciTech Connect

    Marrone, B.L.; Campbell, E.W.; Anzick, S.L.; Shera, K.; Campbell, M.; Yoshida, T.M.; McCormick, M.K.; Deaven, L. (Los Alamos National Lab., NM (United States))

    1994-05-01

    Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (Flpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome. The chromosome FL maps have a 1- to 2-Mb resolution, and the FL measurement of each probe has a typical standard error of 0.5-1 Mb. 14 refs., 3 figs., 3 tabs.

  15. Chromosome analysis by fluorescence in situ hybridization: further indications for a non-DNA-proportional involvement of single chromosomes in radiation-induced structural aberrations.

    PubMed

    Knehr, S; Zitzelsberger, H; Braselmann, H; Nahrstedt, U; Bauchinger, M

    1996-10-01

    The frequencies of symmetrical complete and incomplete translocations and dicentrics induced in human lymphocytes after in vitro irradiation with 3Gy X-rays were analysed by the use of fluorescence in situ hybridization (FISH). Single whole chromosome painting (WCP) probes, specific for chromosomes 1-4, 6-10, 12, 14 and X were hybridized separately. A human pancentromeric DNA-probe was used simultaneously for unequivocal centromere detection. For both aberration types, symmetrical translocations and dicentrics, a significant deviation from a DNA-proportional distribution was found. In general, chromosomes with a higher DNA content (chromosomes 1-3, 6 and 7) were less frequently involved in the formation of symmetrical translocations and dicentrics than expected according to their DNA-content, whereas smaller chromosomes were more frequently involved. The only exception was chromosome 4, exhibiting the highest translocation frequency of all chromosomes analysed. Ratios of the yields of symmetrical translocations to the yields of dicentrics varied between 0.9 and 1.8 for the single chromosomes. The present results substantiate our previous data obtained with identical chromosomes but examined in four different triple combinations. PMID:8862449

  16. Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence In Situ Hybridization and Flow Cytometry

    PubMed Central

    Shepard, Alicia K.; Raes, Eric J.; Waite, Anya M.; Quigg, Antonietta

    2014-01-01

    Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and 14C/15N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean. PMID:25172848

  17. Gene protein detection platform-a comparison of a new human epidermal growth factor receptor 2 assay with conventional immunohistochemistry and fluorescence in situ hybridization platforms.

    PubMed

    Stålhammar, Gustav; Farrajota, Pedro; Olsson, Ann; Silva, Cristina; Hartman, Johan; Elmberger, Göran

    2015-08-01

    Human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are widely used semiquantitative assays for selecting breast cancer patients for HER2 antibody therapy. However, both techniques have been shown to have disadvantages. Our aim was to test a recent automated technique of combined IHC and brightfield dual in situ hybridization-gene protein detection platform (GPDP)-in breast cancer HER2 protein, gene, and chromosome 17 centromere status evaluations, comparing the results in accordance to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from both 2007 and 2013. The GPDP technique performance was evaluated on 52 consecutive whole slide invasive breast cancer cases with HER2 IHC 2/3+ scoring results. Applying in turns the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from 2007 and 2013 to both FISH and GPDP DISH assays, the HER2 gene amplification results showed 100% concordance among amplified/nonamplified cases, but there was a shift in 4 cases toward positive from equivocal results and toward equivocal from negative results. This might be related to the emphasis on the average HER2 copy number in the 2013 criteria. HER2 expression by IVD market IHC kit (Pathway®) has a strong correlation with GPDP HER2 protein, including a full concordance for all cases scored as 3+ and a reduction from 2+ to 1+ in 7 cases corresponding to nonamplified cases. Gene protein detection platform HER2 protein "solo" could have spared the need for 7 FISH studies. In addition, the platform offered advantages on interpretation reassurance including selecting areas for counting gene signals paralleled with protein IHC expression, on heterogeneity detection, interpretation time, technical time, and tissue expense. PMID:25921313

  18. High-resolution cytogenetic mapping of 342 new cosmid markers including 43 RFLP markers on human chromosome 17 by fluorescence in situ hybridization

    SciTech Connect

    Inazawa, Johji; Ariyama, Takeshi; Abe, Tatsuo (Kyoto Prefectual Univ. of Medicine (Japan)); Saito, Hiroko; Nakamura, Yusuke (Cancer Institute, Tokyo (Japan))

    1993-07-01

    The authors have constructed a high-resolution cytogenetic map of human chromosome 17 with 342 cosmid markers, each newly isolated from a cosmid library constructed from a human-mouse hybrid cell line containing a single human chromosome 17. Direct mapping on R- and/or G-banded (pro)metaphase chromosomes by fluorescence in situ hybridization localized these markers throughout the chromosome, although density was highest in the R-band-dominant regions of 17p13, 17p11.2, 17q11.2-q12, 17q21.3, 17q23, and 17q25. By screening some of the cosmid clones, they identified 71 polymorphic systems with 43 markers; 11 of these are VNTRs. As the high-resolution cytogenetic map contains a large number of markers, it can provide useful landmarks for a contig map of chromosome 17. Furthermore, the map will contribute to positional cloning of aberrant genes responsible for inherited diseases such as Miller-Dieker syndrome (MDS), Smith-Magenis syndrome (SMS), and familial early-onset breast cancer, as well as putative tumor suppressor genes on this chromosome. 47 refs., 2 figs., 2 tabs.

  19. Alignment of the Genomes of Brachypodium distachyon and Temperate Cereals and Grasses Using Bacterial Artificial Chromosome Landing With Fluorescence in Situ Hybridization

    PubMed Central

    Hasterok, Robert; Marasek, Agnieszka; Donnison, Iain S.; Armstead, Ian; Thomas, Ann; King, Ian P.; Wolny, Elzbieta; Idziak, Dominika; Draper, John; Jenkins, Glyn

    2006-01-01

    As part of an initiative to develop Brachypodium distachyon as a genomic “bridge” species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice. PMID:16489232

  20. Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

    PubMed Central

    Yilmaz, Özlem; Demiray, Ebru

    2007-01-01

    H pylori is etiologically associated with gastritis, gastric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradication therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue sections. This technique is helpful for determining the bacterial density and the results of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro. PMID:17278188

  1. Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy.

    PubMed

    Yilmaz, Ozlem; Demiray, Ebru

    2007-02-01

    H pylori is etiologically associated with gastritis, gastric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradication therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue sections. This technique is helpful for determining the bacterial density and the results of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro. PMID:17278188

  2. Comparison of Vertical Distributions of Prokaryotic Assemblages in the Anoxic Cariaco Basin and Black Sea by Use of Fluorescence In Situ Hybridization

    PubMed Central

    Lin, Xueju; Wakeham, Stuart G.; Putnam, Isabell F.; Astor, Yrene M.; Scranton, Mary I.; Chistoserdov, Andrei Y.; Taylor, Gordon T.

    2006-01-01

    Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria, Crenarchaeota, and Euryarchaeota were more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances of Archaea in both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group I Crenarchaeota (median, 5.5%) to prokaryotic communities were significantly higher (P < 0.001; n = 20) than those of group II Euryarchaeota (median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4?,6?-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select. PMID:16597973

  3. Combining fluorescent in situ hybridization data with ISS staging improves risk assessment in myeloma: an International Myeloma Working Group collaborative project.

    PubMed

    Avet-Loiseau, H; Durie, B G M; Cavo, M; Attal, M; Gutierrez, N; Haessler, J; Goldschmidt, H; Hajek, R; Lee, J H; Sezer, O; Barlogie, B; Crowley, J; Fonseca, R; Testoni, N; Ross, F; Rajkumar, S V; Sonneveld, P; Lahuerta, J; Moreau, P; Morgan, G

    2013-03-01

    The combination of serum ?2-microglobulin and albumin levels has been shown to be highly prognostic in myeloma as the International Staging System (ISS). The aim of this study was to assess the independent contributions of ISS stage and cytogenetic abnormalities in predicting outcomes. A retrospective analysis of international studies looking at both ISS and cytogenetic abnormalities was performed in order to assess the potential role of combining ISS stage and cytogenetics to predict survival. This international effort used the International Myeloma Working Group database of 12?137 patients treated worldwide for myeloma at diagnosis, of whom 2309 had cytogenetic studies and 5387 had analyses by fluorescent in situ hybridization (iFISH). Comprehensive analyses used 2642 patients with sufficient iFISH data available. Using the comprehensive iFISH data, combining both t(4;14) and deletion (17p), along with ISS stage, significantly improved the prognostic assessment in terms of progression-free survival and overall survival. The additional impact of patient age and use of high-dose therapy was also demonstrated. In conclusion, the combination of iFISH data with ISS staging significantly improves risk assessment in myeloma. PMID:23032723

  4. Effects of Compost on Colonization of Roots of Plants Grown in Metalliferous Mine Tailings, as Examined by Fluorescence In Situ Hybridization?

    PubMed Central

    Iverson, Sadie L.; Maier, Raina M.

    2009-01-01

    The relationship between compost amendment, plant biomass produced, and bacterial root colonization as measured by fluorescence in situ hybridization was examined following plant growth in mine tailings. Mine tailings can remain devoid of vegetation for decades after deposition due to a combination of factors that include heavy metal toxicity, low pH, poor substrate structure and water-holding capacity, and a severely impacted heterotrophic microbial community. Research has shown that plant establishment, a desired remedial objective to reduce eolian and water erosion of such tailings, is enhanced by organic matter amendment and is correlated with significant increases in rhizosphere populations of neutrophilic heterotrophic bacteria. Results show that for the acidic metalliferous tailings tested in this study, compost amendment was associated with significantly increased bacterial colonization of roots and increased production of plant biomass. In contrast, for a Vinton control soil, increased compost had no effect on root colonization and resulted only in increased plant biomass at high levels of compost amendment. These data suggest that the positive association between compost amendment and root colonization is important in the stressed mine tailings environment where root colonization may enhance both microbial and plant survival and growth. PMID:19047384

  5. Assignment of the human diacylglycerol kinase gene (DAGK) to 12q13.3 using fluorescence in situ hybridization analysis

    SciTech Connect

    Hart, T.C.; Zhou, J.; Champagne, C. [Eastman Dental Center, Rochester, NY (United States)] [and others] [Eastman Dental Center, Rochester, NY (United States); and others

    1994-07-01

    A 1-kb cDNA probe specific for the human DAGK gene was prepared by polymerase chain reaction and used to assign it to human chromosome 12 using a human x hamster somatic cell hybrid mapping panel. To determine the chromosomal sublocalization of DAGK, used the 1-kg DAGK cDNA probe to isolate a DAGK-specific phage clone from a human chromosome 12 library by Southern blot techniques. This clone (phEDCDAGK) contained a 17-kb human genomic insert in the phage Charon 40 that hybridized to the 1-kb DAGK cDNA previously characterized.

  6. Deletions of the region 17p11-13 in advanced melanoma revealed by cytogenetic analysis and fluorescence in situ hybridization

    PubMed Central

    Okamoto, I; Pirc-Danoewinata, H; Ackermann, J; Drach, J; Wadl, H Schlagbauer; Jansen, B; Wolff, K; Pehamberger, H; Marosi, C

    1999-01-01

    The significance of the p53 tumour-suppressor gene in the oncogenesis of a variety of malignant tumours has been demonstrated over recent years. However, the role of p53 in human malignant melanoma is still unclear. Therefore, we investigated melanoma metastases from 11 patients cytogenetically and with fluorescence in situ hybridization (FISH) after short-term culture, employing a p53 region-specific probe for 17p13.1 and a probe detecting the centromere of chromosome 17. Furthermore, paraffin-embedded tissue samples from nine of these patients were investigated immunohistochemically for expression of the p53 protein. Deletions of the short arm of chromosome 17 were seen in six melanomas in cytogenetic analysis. With FISH, three malignant melanomas had clones with only one p53-allele and an additional four malignant melanomas showed a reduced number of signals at the p53 tumour-suppressor gene locus compared with signals for the centromeric region of chromosome 17. This was confirmed by immunohistochemistry. Our results suggest that the 17p11–13 region is frequently deleted in malignant melanomas and that p53 or other genes located on this band might contribute to the malignant potential of advanced melanoma. © 1999 Cancer Research Campaign PMID:10408704

  7. Isodicentric (X)(q13) in haematological malignancies: presentation of five new cases, application of fluorescence in situ hybridization (FISH) and review of the literature.

    PubMed

    Dierlamm, J; Michaux, L; Criel, A; Wlodarska, I; Zeller, W; Louwagie, A; Michaux, J L; Mecucci, C; Van den Berghe, H

    1995-12-01

    Idic(X)(q13) represents a rare but recurrent chromosomal abnormality in haematological malignancies. We present five new cases characterized by this particular aberration and review the literature on this subject. The patients were elderly females with a diagnosis of refractory anaemia (1/5), refractory anaemia with ringed sideroblasts (2/5), chronic myelomonocytic leukaemia (1/5), and Philadelphia chromosome-negative chronic myeloid leukaemia (1/5). Three out of the five patients demonstrated an increased proportion of bone marrow ringed sideroblasts. After a follow-up period of 30-57 months all patients but one are alive. Idic(X)(q13) always occurred as the sole chromosomal abnormality, either in one or in two copies. We confirmed the dicentric nature of the aberration by fluorescence in situ hybridization (FISH) on metaphases as well as interphase nuclei using an X-chromosome-specific alpha-satellite probe, and performed chromosome painting to visualize possible additional chromosomal changes involving the X chromosomes. Our findings and the data of 17 previously published cases indicate that idic(X)(q13): (1) may play a significant pathogenetic role in haematological malignancies affecting exclusively females and deriving predominantly from early progenitor cells; (2) is frequently associated with a pathological iron accumulation; (3) indicates a variable prognosis. PMID:8547134

  8. Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR.

    PubMed

    Koudelakova, Vladimira; Berkovcova, Jitka; Trojanec, Radek; Vrbkova, Jana; Radova, Lenka; Ehrmann, Jiri; Kolar, Zdenek; Melichar, Bohuslav; Hajduch, Marian

    2015-07-01

    Administration of drugs targeting HER2 (official symbol ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. Its limit of detection was determined with breast cancer cell lines and validated with 223 breast cancer patient samples. On the basis of comparisons with fluorescent ISH (FISH) and IHC data, the sensitivity of the new method was 94.2% and 95.1%, its specificity was 100% and 99.1%, and overall concordance between results obtained with the quantitative real-time PCR method and FISH/IHC was 97.6% for both methods. The quantitative real-time PCR method was then used to evaluate the HER2 status of 198 of 3696 breast cancer tissues that yielded indeterminate FISH results. The HER2 copy number was successfully determined in 69.2% of these indeterminate samples. Thus, the DNA-based technique appears to be a specific, sensitive method for determining HER2 copy numbers when the FISH assay fails, which may complement IHC tests. PMID:25956448

  9. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. (Universite de Montreal (Canada)); Malfoy, B. (Institut Curie Section de Biologie, Paris (France)); Forrest, G.L. (Beckman Research Institute at the City of Hope, Duarte, CA (United States))

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  10. Aneuploidy in 165,330 human sperm; results of two- and three-colour fluorescence in situ hybridization for chromosomes 1, 12, 15, 18, X, and Y

    SciTech Connect

    Spriggs, E.L.; Martin, R.H. [Alberta Childrens Hospital, Alberta (Canada)]|[Univ. of Calgary, Alberta (Canada)

    1994-09-01

    To understand the mechanisms that affect aneuploidy, fluorescence in situ hybridization (FISH), using chromosome-specific centromeric probes, was employed to screen a large population of human sperm for numerical errors. To determine the true rate of disomy for chromosomes 1, 12, 15, 18, two-color FISH was performed, and for the gonosomes, three-color FISH. The use of multiple, differently-colored probes allows one to distingish a true disomic sperm from a diploid cell. For each centromeric probe, a minimum of 10,000 sperm nuclei for each of five donors was scored, giving a total count of 165,330 sperm nuclei. The incidence of disomic sperm for the sex chromosomes was significantly increased as compared to the frequency for the autosomes ({chi}{sup 2}=232.3, p<0.001), confirming the results observed in studies of sperm karyotypes and spontaneous abortions. The disomy frequencies for autosomes 1, 12, 15, and 18 were found to be uniform. Inter-donor heterogeneity for disomy frequencies was found to exist for the sex chromosomes and for chromosomes 1 and 15, suggesting significant variation among normal men.

  11. Evaluation of a Fluorescence In Situ Hybridization Assay for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Lowenstein-Jensen and Mycobacteria Growth Indicator Tube Cultures Using Peptide Nucleic Acid Probes

    Microsoft Academic Search

    POONPILAS HONGMANEE; HENRIK STENDER; OLE F. RASMUSSEN

    A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of Mycobacterium tuber- culosis complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evalu- ated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as

  12. Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH)

    PubMed Central

    Custódio, Adriana Regina; Seijo, Guillermo; Valls, José Francisco Montenegro

    2013-01-01

    The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species. PMID:24130444

  13. Assignment of the human homologue of the mTRiC-P5 gene (TRIC5) to band 1q23 by fluorescence in situ hybridization

    SciTech Connect

    Sevigny, G.; Joly, E.C.; Bibor-Hardy, V. [Institut du Cancer de Montreal, Quebec (Canada)] [and others] [Institut du Cancer de Montreal, Quebec (Canada); and others

    1994-08-01

    The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t-complex locus on mouse chromosome 17. This locus is involved in several embryonic defects, male sterility, and the transmission ratio distortion. In humans, the t-complex genes map to chromosome 6. Other components of TRiC are thought to be TCP1-related proteins. Recently, a mouse cDNA coding for one of these proteins has been cloned and named mTRiC-P5. Here we report the cloning of a partial human cDNA clone, homologous to mTRiC-P5, and its chromosome localization by fluorescence in situ hybridization. The human TRiC-P5 gene (TRIC5) maps to human chromosome 1q23, a region known to be a preferential chromosomal breakpoint involved in leukemia. Therefore, even if TCP1 and TRiC-P5 are related proteins and are found in the same protein complex, they are not coded by syntenic genes in humans. 16 refs., 2 figs.

  14. Reagent and labor cost optimization through automation of fluorescence in situ hybridization (FISH) with the VP 2000: an Italian case study.

    PubMed

    Zanatta, Lucia; Valori, Laura; Cappelletto, Eleonora; Pozzebon, Maria Elena; Pavan, Elisabetta; Dei Tos, Angelo Paolo; Merkle, Dennis

    2015-02-01

    In the modern molecular diagnostic laboratory, cost considerations are of paramount importance. Automation of complex molecular assays not only allows a laboratory to accommodate higher test volumes and throughput but also has a considerable impact on the cost of testing from the perspective of reagent costs, as well as hands-on time for skilled laboratory personnel. The following study tracked the cost of labor (hands-on time) and reagents for fluorescence in situ hybridization (FISH) testing in a routine, high-volume pathology and cytogenetics laboratory in Treviso, Italy, over a 2-y period (2011-2013). The laboratory automated FISH testing with the VP 2000 Processor, a deparaffinization, pretreatment, and special staining instrument produced by Abbott Molecular, and compared hands-on time and reagent costs to manual FISH testing. The results indicated significant cost and time saving when automating FISH with VP 2000 when more than six FISH tests were run per week. At 12 FISH assays per week, an approximate total cost reduction of 55% was observed. When running 46 FISH specimens per week, the cost saving increased to 89% versus manual testing. The results demonstrate that the VP 2000 processor can significantly reduce the cost of FISH testing in diagnostic laboratories. PMID:25395292

  15. Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

    PubMed Central

    Gu, JunGang; Li, LiHua; Zhou, ZhiGang

    2012-01-01

    Background There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (?=?Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4?, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future. PMID:23166593

  16. Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH)

    PubMed Central

    Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng; Golding, Ido

    2014-01-01

    We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis. PMID:23680982

  17. 3D-catFISH: a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity imaged by fluorescence in situ hybridization.

    PubMed

    Chawla, Monica K; Lin, Gang; Olson, Kathy; Vazdarjanova, Almira; Burke, Sara N; McNaughton, Bruce L; Worley, Paul F; Guzowski, John F; Roysam, Badrinath; Barnes, Carol A

    2004-10-15

    Fluorescence in situ hybridization (FISH) of neural activity-regulated, immediate-early gene (IEG) expression provides a method of functional brain imaging with cellular resolution. This enables the identification, in one brain, of which specific principal neurons were active during each of two distinct behavioral epochs. The unprecedented potential of this differential method for large-scale analysis of functional neural circuits is limited, however, by the time-intensive nature of manual image analysis. A comprehensive software tool for processing three-dimensional, multi-spectral confocal image stacks is described which supports the automation of this analysis. Nuclei counterstained with conventional DNA dyes and FISH signals indicating the sub-cellular distribution of specific, IEG RNA species are imaged using different spectral channels. The DNA channel data are segmented into individual nuclei by a three-dimensional multi-step algorithm that corrects for depth-dependent attenuation, non-isotropic voxels, and imaging noise. Intra-nuclear and cytoplasmic FISH signals are associated spatially with the nuclear segmentation results to generate a detailed tabular/database and graphic representation. Here we present a comprehensive validation of data generated by the automated software against manual quantification by human experts on hippocampal and parietal cortical regions (96.5% concordance with multi-expert consensus). The high degree of reliability and accuracy suggests that the software will generalize well to multiple brain areas and eventually to large-scale brain analysis. PMID:15351517

  18. Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker.

    PubMed

    Balakrishnan, S; Payawal, J; Schuler, M J; Hasegawa, L; Eastmond, D A

    2002-11-26

    Aneuploidy is associated with spontaneous abortions, birth defects, and many types of human cancers. Currently there are few assays developed for the efficient detection of aneuploidy in vivo. However, with the recent availability of chromosome-specific DNA probes for the rat, fluorescence in situ hybridization (FISH) techniques could be used for the rapid and sensitive detection of aneuploidy in different tissue and cell types. In order to develop a system that can detect alterations in chromosome number in rat cells in vitro, we treated cultured rat lymphocytes with three aneugens-noscapine hydrochloride (0-150 microM) and vincristine and vinblastine sulfate (0-0.06 microM). 5-Bromo-2-deoxyuridine (BrdU; 1 microM) was added to the culture medium to allow proliferating and non-proliferating cells to be distinguished. To test this assay under in vivo conditions, 21-day-old male Sprague-Dawley rats were subcutaneously implanted with osmotic pumps that delivered BrdU (approximately 12 mg/kg per day) continuously. These rats were administered vinblastine sulfate (0, 0.5 and 1mg/kg) by intraperitoneal injection. The rat lymphocytes and hepatocytes incorporating BrdU were detected by immuno-fluorescent labeling, and FISH with a rat chromosome 4 probe was performed on the labeled and unlabeled cells. Highly significant increases in hyperdiploidy were seen in the replicating rat lymphocytes treated with noscapine, vincristine or vinblastine in vitro and in the rat hepatocytes treated with vinblastine in vivo. In contrast, no significant increase in hyperdiploidy was observed in the non-replicating cells. These results demonstrate that this BrdU-enhanced FISH assay with chromosome-specific rat probes can be used to efficiently detect numerical chromosomal aberrations in vitro and in vivo in slowly or moderately replicating rat tissues. The combination of BrdU-labeling and FISH allows the scoring of hyperdiploidy to be focused on the actively replicating cells, thereby increasing the sensitivity of the FISH technique. PMID:12438006

  19. Assignment of the gene for the human proliferating cell nucleolar protein P120 (NOL1) to chromosome 12p13 by fluorescence in situ hybridization and polymerase chain reaction with somatic cell hybrids

    SciTech Connect

    Baens, M.; Chaffanet, M.; Cassiman, J.J.; Marynen, P. (Center for Human Genetics, Leuven (Belgium)); Aerssens, J. (Univ. of Leuven (Belgium))

    1994-05-01

    The human NOL1 protein is a proliferation associated antigen that is temporally regulated during the cell cycle with a dramatic increase in protein expression at the G1-S boundary, suggesting that NOL1 may play a role in the regulation of the cell cycle and the increased nucleolar activity that is associated with the cell proliferation. The authors have isolated a cosmid (LL12NCO1N147G1) for NOL1 from the chromosome 12-specific cosmid library LL12NCO1, constructed at the Biomedical Sciences Division, Lawrence Livermore Laboratory (Livermore, CA) and obtained from P. de Jong. The identity of the cosmid was confirmed by restriction mapping and PCR. Fluorescence in situ hybridization (FISH) was performed on metaphase chromosomes of normal lymphocytes with the cosmid 147G1 and a probe for the alphoid centromeric region of chromosome 12, developed by U. Weier. Probe labeling and in situ hybridization were performed as described. The FISH results showed that NOL1 maps to the distal part of chromosome 12p. 6 refs., 1 fig.

  20. Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

    PubMed

    Ooi, Akishi; Oyama, Takeru; Nakamura, Ritsuko; Tajiri, Ryosuke; Ikeda, Hiroko; Fushida, Sachio; Nakamura, Hiroyuki; Dobashi, Yoh

    2015-06-01

    The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with 'gain' or 'amplified' status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy. PMID:25743022

  1. Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer.

    PubMed

    Schildhaus, Hans-Ulrich; Heukamp, Lukas C; Merkelbach-Bruse, Sabine; Riesner, Katharina; Schmitz, Katja; Binot, Elke; Paggen, Ellen; Albus, Kerstin; Schulte, Wolfgang; Ko, Yon-Dschun; Schlesinger, Andreas; Ansén, Sascha; Engel-Riedel, Walburga; Brockmann, Michael; Serke, Monika; Gerigk, Ulrich; Huss, Sebastian; Göke, Friederike; Perner, Sven; Hekmat, Khosro; Frank, Konrad F; Reiser, Marcel; Schnell, Roland; Bos, Marc; Mattonet, Christian; Sos, Martin; Stoelben, Erich; Wolf, Jürgen; Zander, Thomas; Buettner, Reinhard

    2012-11-01

    We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ?2.0, or average number of FGFR1 signals per tumor cell nucleus ?6, or the percentage of tumor cells containing ?15 FGFR1 signals or large clusters ?10%) was detected at a frequency of 16% and low-level amplification (as defined by ?5 FGFR1 signals in ?50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials. PMID:22684217

  2. Detection of Y chromosome sequences in a 45,X/46,XXq - patient by Southern blot analysis of PCR-amplified DNA and fluorescent in situ hybridization (FISH)

    SciTech Connect

    Kocova, M.; Siegel, S.F.; Wenger, S.L. [Univ. of Pittsburgh School of Medicine, Pittsburgh, PA (United States)

    1995-02-13

    In some cases of gonadal dysgenesis, cytogenetic analysis seems to be discordant with the phenotype of the patients. We have applied techniques such as Southern blot analysis and fluorescent in situ hybridization (FISH) to resolve the phenotype/genotype discrepancy in a patient with ambiguous genitalia in whom the peripheral blood karotype was 45,X. Gonadectomy at age 7 months showed the gonadal tissue to be prepubertal testis on the left side and a streak gonad on the right. The karyotype obtained from the left gonad was 45,X/46,XXq- and that from the right gonad was 45,X. Three different techniques, PCR amplification, FISH, and chromosome painting for X and Y chromosomes, confirmed the presence of Y chromosome sequences. Five different tissues were evaluated. The highest percentage of Y chromosome positive cells were detected in the left gonad, followed by the peripheral blood lymphocytes, skin fibroblasts, and buccal mucosa. No Y chromosomal material could be identified in the right gonad. Since the Xq- chromosome is present in the left gonad (testis), it is likely that the Xq- contains Y chromosomal material. Sophisticated analysis in this patient showed that she has at least 2 cell lines, one of which contains Y chromosomal material. These techniques elucidated the molecular basis of the genital ambiguity for this patient. When Y chromosome sequences are present in patients with Ullrich-Turner syndrome or gonadal dysgenesis, the risk for gonadal malignancy is significantly increased. Hence, molecular diagnostic methods to ascertain for the presence of Y chromosome sequences may expedite the evaluation of patients with the ambiguous genitalia. 21 refs., 4 figs., 2 tabs.

  3. Rat karyotyping by fluorescence in situ hybridization (FISH): Localization of oncogene c-raf to 4q42, retinoblastoma antioncogene to 15q12, and mitochondrial D-loop-like sequences to the Y chromosome

    SciTech Connect

    Zullo, S. [Yale Univ. School of Medicine, New Haven, CT (United States)] [Yale Univ. School of Medicine, New Haven, CT (United States); [NIMH Neuroscience Center, Washington, DC (United States); Upender, M. [Yale Univ. School of Medicine, New Haven, CT (United States)] [Yale Univ. School of Medicine, New Haven, CT (United States)

    1995-02-10

    Fluorescence in situ hybridization (FISH) has been used to karyotype the rat genome with long interspersed repetitive elements (LINEs). Two-color FISH experiments were used to localize the rat c-raf oncogene to 4q42 and the rat retinoblastoma anti-oncogene to 15q12. In addition, sequences similar to the rat mitochondrial origin of replication (D-loop-like sequences) have been found to be concentrated among repetitive element sequences on the Y chromosome. This report extends hybridization-based karyotype technology to the rat, thus facilitating the application of FISH to rat gene mapping. 13 refs., 1 fig.

  4. Reliability Evaluation of Fluorescence In Situ Hybridization (FISH) and G-Banding on Bone Marrow and Peripheral Blood Cells in Chronic Myelogenous Leukemia Patients.

    PubMed

    Manaflouyan Khajehmarjany, Soheila; Rahmani, Seyed Ali; Chavoshi, Seyed Hadi; Esfahani, Ali; Movassaghpour Akbari, Ali Akbar

    2015-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disease. The cytogenetic hallmark of CML is Philadelphia (Ph) chromosome. This study aimed to diagnose suspected CML patients, to monitor CML patients under therapy using cytogenetic and fluorescence in situ hybridization (FISH) techniques to analyze their bone marrow (BM) and peripheral blood (PB) samples, and finally to compare their obtained results for both specimens. This study was conducted during one-year period (2012-2013). The participants were recruited from the Hematology and Oncology Clinic of Shahid Gazi (Emam Reza) Hospital of Tabriz University of Medical Sciences, Tabriz, East Azerbaijan Province, Iran. We analyzed 90 samples from 60 suspected CML patients (30 BM and 60 PB samples). All samples were analyzed using G-banding, 5 samples using dual fusion FISH (DF-FISH) probes, as well as 30 samples using both FISH and G-banding. Among the 90 analyzed samples of 60 patients, 25 (41.66%) were Ph+ using karyotyping, whereas five cases were not analyzable, so FISH was applied and the results confirmed that only two individuals were BCR-ABL+. In the comparison between 25 BM and 25 PB samples using karyotyping, 15 (60%) and 10 (40%) were ph+, respectively. The comparison of FISH and karyotyping on 30 samples showed that 9 (30%) and 8 (26.66%) were Ph+, respectively, and only 18.18% of Ph+ patients showed atypical patterns. In the comparison between BM-cytogenetic and PB- interphase-FISH (I-FISH), BM-cytogenetic was more reliable than PB-I-FISH in detecting Ph. Our data demonstrate that FISH analysis is a rapid, reliable and sensitive technique. The comparison between BM and PB showed that PB can not be replaced by BM, even in detecting by FISH. PMID:25870848

  5. Identification of a microdeletion at 7q21.3 with fluorescence in situ hybridization in a patient with split hand/split foot (ectrodactyly)

    SciTech Connect

    Hudgins, L. [Children`s Hospital and Medical Center, Seattle, WA (United States); Massa, H.; Disteche, C. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    Split hand/split foot (SHSF), often referred to as ectrodactyly or lobster claw deformity, is a human developmental disorder characterized by a deep median cleft of the hands and feet, missing digits, and fusion of remaining digits. This anomaly can be seen alone, frequently autosomal dominant, or in association with other abnormalities. One locus for this defect has been localized to chromosome 7q21.3-q22.1. We report a patient with SHSF plus mental retardation, short stature and dysmorphic features who was found to have a microdeletion at this locus detected only with the aid of fluorescence in situ hybridization (FISH). T.H. is a 7 y.o. male who was referred for evaluation of foot anomalies and mild mental retardation. History was remarkable for growth retardation of postnatal onset and hypotonia. Renal ultrasound and audiology evaluation were normal. Physical exam revealed dysplastic ears, micrognathia, long philtrum, high narrow palate, and malformations of the feet consistent with SHSF. Family history was negative for limb abnormalities and mental retardation. A number of patients with SHSF and other anomalies have been found to have deletions involving chromosome 7q21-q22; therefore, high resolution chromosome analysis was performed in T.H. but was inconclusive. Cosmids and yeast artificial chromosomes which we had previously mapped to the SHSF critical region were used as FISH probes and a microdeletion was detected. We were thus able to determine the etiology of this child`s abnormalities and provide accurate genetic counseling, which would not have been possible with standard cytogenetic techniques. This technique also allowed us to further refine the SHSF critical region. This case illustrates the utility of FISH for the rapid identification of suspect microdeletions in SHSF. This approach should also be useful as an expeditious way of defining the critical regions for the location of genes which give rise to other developmental malformations.

  6. Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH)

    PubMed Central

    2013-01-01

    Background Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. Results We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. Conclusion We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species. PMID:24373333

  7. In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

    Microsoft Academic Search

    SATOSHI OKABE; HISASHI SATOH; YOSHIMASA WATANABE

    1999-01-01

    We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in do- mestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these tech- niques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In

  8. The human gene for xeroderma pigmentosum complementation group G (XPG) maps to 13q33 by fluorescence in situ hybridization

    SciTech Connect

    Samec, S.; Corlet, J.; Scherly, D.; Clarkson, S.G. (Centre Medical Universitaire, Geneva (Switzerland)); Jones, T.A.; Sheer, D. (Imperial Cancer Research Fund, London (United Kingdom)); Wood, R.D. (Clare Hall Labs., Hertfordshire (United Kingdom))

    1994-05-01

    Recently, a human cDNA was isolated that restores normal levels of UV resistance and DNA repair synthesis when expressed in vivo in a lymphoblastoid cell line representing XP group G. The XP-G complementing gene (XPG) generates an mRNA of [approximately]4 kb and encodes a protein (XPGC) with homology to the RAD2 DNA repair protein of Saccharomyces cerevisiae. One hundred twenty nanograms of labeled probe was mixed with 2 [mu]g of human C[sub 0]t-1 DNA as a competitor for repetitive elements. Denaturation, dehydration, hybridization, and washing were performed as described. Probe detection was achieved by incubating metaphase spreads sequentially with 2 [mu]g/ml avidin-Texas Red, 5 [mu]g/ml biotinylated goat anti-avidin antibody, and 2 [mu]g/ml avidin-Texas Red. R-banding was revealed after incubation with fluorescein-labeled anti-BrdU mouse monoclonal antibody. Chromosomes were counter-stained with 0.06 [mu]g/ml DAPI in Citifluor. Analysis of 40 metaphase spreads showed paired signals on both copies of chromosome 13 at band 13q33. No other paired signals were seen consistently on any other chromosome. 9 refs., 1 fig.

  9. Diagnostic utility of NCOA2 fluorescence in situ hybridization and Stat6 immunohistochemistry staining for soft tissue angiofibroma and morphologically similar fibrovascular tumors.

    PubMed

    Sugita, Shintaro; Aoyama, Tomoyuki; Kondo, Kei; Keira, Yoshiko; Ogino, Jiro; Nakanishi, Katsuya; Kaya, Mitsunori; Emori, Makoto; Tsukahara, Tomohide; Nakajima, Hisaya; Takagi, Masayuki; Hasegawa, Tadashi

    2014-08-01

    Soft tissue angiofibroma (STA), a recently suggested new histologic entity, is a benign fibrovascular soft tissue tumor composed of bland spindle-shaped tumor cells with abundant collagenous to myxoid stroma and branching small vessels. The lesion has a characteristic AHRR-NCOA2 fusion gene derived from chromosomal translocation of t(5;8)(p15;q13). However, morphologically similar tumors containing abundant fibrovascular and myxoid stroma can complicate diagnosis. We designed an original DNA probe for detecting NCOA2 split signals on fluorescence in situ hybridization (FISH) and estimated its utility with 20 fibrovascular tumors: 4 each of STAs, solitary fibrous tumors (SFTs), and cellular angiofibromas and 3 each of low-grade myxofibrosarcomas, myxoid liposarcomas, and low-grade fibromyxoid sarcomas. We also performed FISH for 13q14 deletion and immunohistochemistry (IHC) staining for estrogen receptor, progesterone receptor, retinoblastoma protein, and MUC-4 expression. Furthermore, IHC for Stat6 was conducted in the 20 cases analyzed by FISH and in an additional 26 SFTs. We found moderate to strong nuclear Stat6 expression in all SFTs but no expression in the other tumors. Both estrogen receptor and progesterone receptor expressions were observed in STAs, SFTs, and cellular angiofibromas. Expression of retinoblastoma protein was found in less than 10% of cells in all tumor types except myxoid liposarcoma. The low-grade fibromyxoid sarcomas were strongly positive for MUC-4. All STAs showed NCOA2 split signals on FISH. All tumors, regardless of histologic type, had 13q14 deletion. The NCOA2 FISH technique is a practical method for confirming STA diagnosis. The combination of NCOA2 FISH and Stat6 IHC proved effective for the differential diagnosis of STA, even when using small biopsy specimens. PMID:24856853

  10. Comparative study between quantitative digital image analysis and fluorescence in situ hybridization of breast cancer equivocal human epidermal growth factor receptors 2 score 2+ cases

    PubMed Central

    Ayad, Essam; Mansy, Mina; Elwi, Dalal; Salem, Mostafa; Salama, Mohamed; Kayser, Klaus

    2015-01-01

    Background: Optimization of workflow for breast cancer samples with equivocal human epidermal growth factor receptors 2 (HER2)/neu score 2+ results in routine practice, remains to be a central focus of the on-going efforts to assess HER2 status. According to the College of American Pathologists/American Society of Clinical Oncology guidelines equivocal HER2/neu score 2+ cases are subject for further testing, usually by fluorescence in situ hybridization (FISH) investigations. It still remains on open question, whether quantitative digital image analysis of HER2 immunohistochemistry (IHC) stained slides can assist in further refining the HER2 score 2+. Aim of this Work: To assess utility of quantitative digital analysis of IHC stained slides and compare its performance to FISH in cases of breast cancer with equivocal HER2 score 2+. Materials and Methods: Fifteen specimens (previously diagnosed as breast cancer and was evaluated as HER 2- score 2+) represented the study population. Contemporary new cuts were prepared for re-evaluation of HER2 immunohistochemical studies and FISH examination. All the cases were digitally scanned by iScan (Produced by BioImagene [Now Roche-Ventana]). The IHC signals of HER2 were measured using an automated image analyzing system (MECES, www.Diagnomx.eu/meces). Finally, a comparative study was done between the results of the FISH and the quantitative analysis of the virtual slides. Results: Three out of the 15 cases with equivocal HER2 score 2+, turned out to be positive (3+) by quantitative digital analysis, and 12 were found to be negative in FISH too. Two of these three positive cases proved to be positive with FISH, and only one was negative. Conclusions: Quantitative digital analysis is highly sensitive and relatively specific when compared to FISH in detecting HER2/neu overexpression. Therefore, it represents a potential reliable substitute for FISH in breast cancer cases, which desire further refinement of equivocal IHC results. PMID:26110098

  11. Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

    PubMed Central

    Almeida, C.; Sousa, J. M.; Rocha, R.; Cerqueira, L.; Fanning, S.; Azevedo, N. F.

    2013-01-01

    Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10?2 to 1 × 102 CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day. PMID:23934486

  12. Genomic In Situ Hybridization (GISH) as a Tool to Identify Chromosomes of Parental Species in Sunflower Interspecific Hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interspecific hybridization has been widely used to transfer genes from wild species into cultivated sunflower. Fluorescent genomic in situ hybridization (GISH) has been used to identify alien chromosomes or segments in other crops, but an equivalent technique for sunflower is lacking. The objective...

  13. Rapid physical mapping of the human trk protooncogene (NTRK1) to human chromosome 1q21-q22 by P1 clone selection, fluorescence in situ hybridization (FISH), and computer-assisted microscopy

    SciTech Connect

    Weier, H.U.G.; Rhein, A.P.; Shadravan, F. [Lawrence Berkeley Lab., CA (United States)] [and others] [Lawrence Berkeley Lab., CA (United States); and others

    1995-03-20

    Physical mapping of small genomic DNA fragments or expressed sequences by in situ hybridization is typically limited by the size of the target DNA sequence. Isolation of large insert DNA clones from libraries containing the target DNA sequence facilitates physical mapping by fluorescence in situ hybridization and allows rapid assignment of genes to cytogenetic bands. Here, we demonstrate the scheme by mapping the human protooncogene trk (NTRK1), a tyrosine kinase receptor type I gene that has earlier been assigned to two different cytogenetic loci. Large DNA insert library screening was carried out by in vitro DNA amplification using oligonucleotide primers flanking exon 4 of trk. The scheme presented here can easily be generalized to map physically very small nonrepetitive genomic DNA fragments or incomplete cDNAs. 17 refs., 2 figs.

  14. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

  15. Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry

    PubMed Central

    Salido, Marta; Tusquets, Ignasi; Corominas, Josep M; Suarez, Marta; Espinet, Blanca; Corzo, Cristina; Bellet, Meritxell; Fabregat, Xavier; Serrano, Sergi; Solé, Francesc

    2005-01-01

    Introduction One of the most common genetic aberrations associated with breast cancer is the amplification and overexpression of the ERBB2 proto-oncogene located at chromosome 17, bands q12-21. The amplification/overexpression occurs in 25 to 30% of all breast cancers. In breast cancer, aneusomy of chromosome 17, either monosomy or polysomy, is frequently observed by conventional cytogenetics and fluorescence in situ hybridization (FISH). The aim of this study was to discover whether or not numerical aberrations on chromosome 17 have a correlation to the amplification or overexpression of the ERBB2 gene and to analyze their clinical implications in subgroups showing 2+ or 3+ positive scores by immunohistochemistry (IHC). Methods We used FISH on a series of 175 formalin-fixed paraffin-embedded breast carcinomas to detect ERBB2 amplification, using a dual-probe system for the simultaneous enumeration of the ERBB2 gene and the centromeric region of chromosome 17, as well as using IHC to detect overexpression. We analyzed clinical and pathological variables in a subgroup of patients with 2+ and 3+ IHC scores (147 patients), to describe any differences in clinicopathological characteristics between polysomic and non-polysomic cases with the use of the ?2 test. Results We found 13% of cases presenting polysomy, and three cases presented monosomy 17 (2%). According to the status of the ERBB2 gene, instances of polysomy 17 were more frequently observed in non-amplified cases than in FISH-amplified cases, suggesting that the mechanism for ERBB2 amplification is independent of polysomy 17. Polysomy 17 was detected in patients with 2+ and 3+ IHC scores. We found that nodal involvement was more frequent in polysomic than in non-polysomic cases (P = 0.046). Conclusions The determination of the copy number of chromosome 17 should be incorporated into the assesment of ERBB2 status. It might also be helpful to differentiate a subgroup of breast cancer patients with polysomy of chromosome 17 and overexpression of ERBB2 protein that probably have genetic and clinical differences. PMID:15743507

  16. Localization of the gene for DNA polymerase [epsilon] (POLE) to human chromosome 12q24. 3 and rat chromosome 12 by somatic cell hybrid panels and fluorescence in situ hybridization

    SciTech Connect

    Szpirer, J.; Riviere, M.; Szpirer, C. (Universite Libre de Bruxelles, Rhode-St-Genese (Belgium)); Pedeutour, F.; Turc-Carel, C. (Universite de Nice-Sophia Antipolis (France)); Kesti, T.; Syvaeoja, J.E. (Univ. of Oulu, Linnanmaa (Finland))

    1994-03-15

    DNA polymerase [epsilon] [DNA pol [epsilon] (EC 2.7.7.7)] is one of the four nuclear DNA polymerases in eukaryotic cells. The mammalian enzyme is involved in DNA repair and possibly also in replication of chromosomal DNA. The gene encoding pol [epsilon] (POLE) was assigned to human and rat chromosomes 12 by Southern blot analysis of genomic dNA from mouse-human and mouse-rat somatic cell hybrid panels using human cDNA probe. The human gene was then regionally localized to band 12q24.3 by fluorescence in situ hybridization of metaphase spreads of chromosomes from human lymphocytes using genomic DNA probe. POLE is closely linked to HNF1A, a gene encoding a liver-enriched transcription factor, HNF1[alpha]. The two genes thus define a new synteny group retained on human and rat chromosomes 12. Another gene mapping to the same or close-by region as human POLE is a gene for the inherited disorder tuberous sclerosis 3, TSC3. 19 refs., 3 figs., 1 tab.

  17. Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer.

    PubMed

    Li, Jiao; Su, Wei; Zhang, Sheng; Hu, Yunhui; Liu, Jingjing; Zhang, Xiaobei; Bai, Jingchao; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lei, Zhenmin; Zhang, Jin

    2015-05-01

    The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes. PMID:25702787

  18. Specific and Rapid Detection by Fluorescent In Situ Hybridization of Bacteria in Clinical Samples Obtained from Cystic Fibrosis Patients

    Microsoft Academic Search

    MICHAEL HOGARDT; KARLHEINZ TREBESIUS; ANNA M. GEIGER; MATHIAS HORNEF; JOSEF ROSENECKER; JURGEN HEESEMANN

    bacteria within sputum samples by FISH was approximately 4 3 105 CFU\\/ml of sputum (resulting in a 90% sen- sitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, and P. aeruginosa exacerbations. Therefore, FISH is

  19. Analysis of lymphocytes from uranium mineworkers in Namibia for chromosomal damage using Fluorescence in situ Hybridization (FISH)

    Microsoft Academic Search

    Carol S. Griffin; Paul J. Simpson; David G. Papworth; John R. K. Savage; Sue Armstrong; Maj A. Hultèn

    1996-01-01

    Workers in the open pit uranium mine in Namibia appear to suffer from health problems including malignant diseases at a much higher prevalence when compared with the general population. The objective of the present study was to determine whether long-term exposure to low-dose uranium increases the risk of biological radiation damage which could lead to malignant diseases. In order to

  20. Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae)

    PubMed Central

    Lakshmanan, Prabhu Shankar; Van Laere, Katrijn; Eeckhaut, Tom; Van Huylenbroeck, Johan; Van Bockstaele, Erik; Khrustaleva, Ludmila

    2015-01-01

    Abstract Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthurium andraeanum Linden ex André, 1877, Monstera deliciosa Liebmann, 1849, Philodendron scandens Koch & Sello, 1853, Spathiphyllum wallisii Regel, 1877, Syngonium auritum (Linnaeus, 1759) Schott, 1829 and Zantedeschia elliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendron scandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllum wallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthurium andraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschia elliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.

  1. Analysis of the nitrifying bacterial community in BioCube sponge media using fluorescent in situ hybridization (FISH) and microelectrodes

    Microsoft Academic Search

    Kyu-Jung Chae; T. Rameshwar; Am Jang; Sung H. Kim; In S. Kim

    2008-01-01

    There is growing interest in the development of more cost-effective and retrofit technologies for the upgrade and expansion of existing wastewater treatment plants with extreme space constraints. A free-floating sponge media (BioCube) process, using a 24L lab scale reactor, was operated to study the nitrification profiles and microbial community. The COD removal efficiencies were maintained, at an average of 95%,

  2. Significance of cytogenetic and fluorescence in situ hybridization analysis in evaluating antichronic myeloid leukemia efficacy of different immune effector cells

    Microsoft Academic Search

    Chun-Rong Tong; Bo Hong; Jing-Ying Qiu; Zhong Chen; Dao-Pei Lu

    2002-01-01

    We report on the antileukemia effect of interleukin 2 (IL2) on different immune cells from 22 patients with chronic myeloid leukemia (CML). Bone marrow cells from these patients were first cultured in modified long-term bone marrow culture medium for several days, then separately cultured with lymphokine activated killer cells (LAK), cytokine-induced killer cells (CIK), and dendritic cell cocultured CIK (DC-CIK)

  3. Identification of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in Blood Cultures: a Multicenter Performance Evaluation of a Three-Color Peptide Nucleic Acid Fluorescence In Situ Hybridization Assay?

    PubMed Central

    Della-Latta, Phyllis; Salimnia, Hossein; Painter, Theresa; Wu, Fann; Procop, Gary W.; Wilson, Deborah A.; Gillespie, Wendy; Mender, Alayna; Crystal, Benjamin S.

    2011-01-01

    A multicenter evaluation was undertaken to evaluate the performance of a new three-color peptide nucleic acid fluorescence in situ hybridization assay that identifies isolates directly from blood cultures positive for Gram-negative bacilli (GNB). The assay correctly identified 100% (186/186) of the Escherichia coli isolates, 99.1% (109/110) of the Klebsiella pneumoniae isolates, and 95.8% (46/48) of the Pseudomonas aeruginosa isolates in this study. Negative assay results were correctly obtained for 162 of 165 other GNB (specificity, 98.2%). PMID:21490185

  4. Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection.

    PubMed

    Robertson, Kelly L; Vora, Gary J

    2012-01-01

    Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2). PMID:22258228

  5. One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection

    PubMed Central

    Tinawi-Aljundi, Rima; King, Lauren; Knuth, Shannon T; Gildea, Michael; Ng, Carrie; Kahl, Josh; Dion, Jacqueline; Young, Chris; Schervish, Edward W; Frontera, J Rene; Hafron, Jason; Kernen, Kenneth M; Di Loreto, Robert; Aurich-Costa, Joan

    2015-01-01

    Background Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH) probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance. Materials and methods In order to calculate clinical performance, we compared the OligoFISH results to the cytology and cystoscopy/pathology findings for 147 initial diagnoses and 399 recurrence monitorings. Finally, we compared clinical performance to published values for the UroVysion test, including both low- and high-grade tumors. Results Chromosomes 3, 6, 7, and 20 were highly involved in bladder carcinoma aneuploidy. At the initial diagnosis, we obtained 90.5% (95% confidence interval [CI]: 84.5%–94.7%) accuracy, 96.8% sensitivity (95% CI: 91.0%–99.3%), 79.2% specificity (95% CI: 65.9%–87.8%), 89.2% positive predictive value (PPV; 95% CI: 81.5%–94.5%), and 93.3% negative predictive value (NPV; 95% CI: 81.7%–97.3%). When monitoring for recurrence, we obtained 85.2% accuracy (95% CI: 81.3%–88.5%), 82.0% sensitivity (95% CI: 76.0%–87.1%), 88.4% specificity (95% CI: 83.2%–92.5%), 87.7% PPV (95% CI: 82.1%–92.0%), and 83.0% NPV (95% CI: 77.3%–87.8%). When looking at low- and high-grade tumors, the test showed 100% sensitivity for high-grade tumors (95% CI: 92.5%–100%) and 87.5% sensitivity (95% CI: 68.8%–95.5%) for low-grade tumors. All the clinical parameters for the OligoFISH panel were higher than the UroVysion test’s published performance. We found significantly higher clinical sensitivity and NPV at initial diagnosis and significantly higher specificity and PPV for recurrence. Conclusion The OligoFISH probe panel is a fast, easy, and reproducible test for bladder cancer diagnosis and monitoring, with excellent clinical performance and utility. PMID:25914883

  6. Fluorescence In Situ Hybridization for Rapid Identification of Achromobacter xylosoxidans and Alcaligenes faecalis Recovered from Cystic Fibrosis Patients

    Microsoft Academic Search

    Nele Wellinghausen; Beate Wirths; Sven Poppert

    2006-01-01

    Achromobacter xylosoxidans is a gram-negative, oxidase-pos- itive, non-glucose-fermenting rod that is widely distributed in the environment. The species is found in two subspecies, called A. xylosoxidans subsp. xylosoxidans and A. xylosoxidans subsp. denitrificans. Due to the close relation to Alcaligenes spp., the species has intermittently been named Alcaligenes (28); but in 1998, use of the initial name, Achromobacter xylosoxidans, was

  7. Cytomolecular characterization of rRNA gene sequences among Citrullus species and subspecies using fluorescent in situ hybridization (FISH) technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In previous studies, many DNA markers showed strong preferential (non-Mendelian) segregation in F2 and BC1 genetic populations derived from crosses between wild type watermelon [C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.] (CLC) and watermelon cultivar...

  8. Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

    SciTech Connect

    Korenberg, J.R.

    1997-12-31

    The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

  9. Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes

    Microsoft Academic Search

    KENNETH OLIVEIRA; GERHARD HAASE; CLETUS KURTZMAN; JENS JO; HENRIK STENDER; Boston Probes

    2001-01-01

    The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection

  10. Le diagnostic anténatal de la trisomie 21 par l'hybridation in situ en fluorescence (FISH): à propos des premiers tests réalisés au Maroc

    PubMed Central

    Lamzouri, Afaf; Natiq, Abdelhafid; Tajir, Mariam; Sendid, Mohamed; Sefiani, Abdelaziz

    2012-01-01

    Introduction Le but de cette étude était de présenter les premiers résultats de diagnostic anténatal de la trisomie 21 par la technique d'hybridation in situ en fluorescence (FISH) au Maroc et discuter son intérêt dans le diagnostic rapide de cette aneuploïdie. Méthodes Ce travail a été réalisé chez 23 femmes avec des grossesses à haut risque de trisomie 21. La moyenne d’âge des gestantes étaient de 37,43 ans avec des extrêmes de 21 et 43 ans. Toutes étaient musulmanes mariées, mariage légitimé par la Charia, dont trois mariages consanguins, sauf une originaire de la République Démocratique du Congo qui était chrétienne et concubine. La majorité des femmes étaient fonctionnaires et avaient un niveau de scolarisation moyen à élevé. Toutes les patientes ont bénéficié d'une consultation de génétique médicale au cours de laquelle il leur a été donné des informations sur la technique, son intérêt et ses limites. Il s'agit de femmes enceintes qui avaient soit un âge maternel élevé ou des signes d'appel échographiques et/ ou biochimiques. Une des patientes était porteuse d'une translocation robertsonienne t(14;21) équilibrée. Une amniocentèse a été réalisée chez toutes les gestantes et aucun avortement n'a était induit par ce geste invasif. L’âge gestationnel moyen à la première consultation était de 14 semaines d'aménorrhée (SA) et à l'amniocentèse était de 16 SA et 5 jours. L'analyse FISH a été réalisée, après consentement des couples, sur des cellules non cultivées à partir des échantillons de liquides amniotiques, en utilisant des sondes spécifiques du chromosome 21. Résultats Parmi les 23 patientes qui ont bénéficiées d'un diagnostic anténatal de la trisomie 21 par la technique FISH, nous avons pu rassurer 21 d'entre elles, et nous avons détecté deux cas de trisomie 21 fœtal. Conclusion La technique FISH permet un diagnostic anténatal rapide, en moins de 48h, de la trisomie 21 sur une faible quantité de liquide amniotique. Elle offre aux couples l'avantage de prendre, dans des délais raisonnables, la décision qui leur convient concernant la poursuite ou non de la grossesse. Elle permet souvent, avec un résultat normal, de rassurer rapidement les femmes enceintes trop angoissées. PMID:23330029

  11. Mapping of the gene encoding the [beta]-subunit of H[sup +], K[sup +]-ATPase to human chromosome 13q34 by fluorescence in situ hybridization

    SciTech Connect

    Song, I.; Brown, D.R.; Yamada, T.; Trent, J.M. (Univ. of Michigan Medical Center, Ann Arbor, MI (United States))

    1992-12-01

    To identify the chromosomal loci encoding this gene, 1 [mu]g of a clone of the human H[sup +], K[sup +]-ATPase subunit [beta] (designated HKB3-1-1a), which contains the coding region including exons 1 to 4 (approximately 17 kb), was labeled with biotin and hybridized to human metaphase chromosomes as previously described. A total of 32 metaphase cells were examined, and all cells examined had double fluorescent signals (i.e., one on each chromatid) on the terminal long arm of chromosome 13. In 17 cells double signals were observed on both homologs, and 15 cells had one homolog with a double signal on chromosome 13. Only chromosomes in which both chromatids displayed a signal were included for analyses, making the background hybridization essentially zero. The same cells hybridized for FISH had been previously G-banded (using trypsin Giemsa) and photographed to allow direct comparisons of the results. The results demonstrated that the sequences hybridizing to a DNA fragment that contains the coding region for the [beta]-subunit of H[sup +], K[sup +]-ATPase can be localized to 13q34. 16 refs., 1 fig.

  12. Detection of in-situ hybridization to human metaphase chromosomes by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Okamoto, Naoaki; Ishikawa, Mitsuru

    2000-04-01

    Detection of in situ hybridization to human metaphase chromosomes provides important information about gene mappings and about analysis of chromosomal disorders. We applied atomic force microscopy (AFM) to the detection of in situ hybridization to get better resolution as compared to light microscopy. Chromosomes were spread over a glass substrate and hybridized with DNA probes labeled with biotin or digoxigenin. The hybridized probes were reacted with streptavidin or anti-digoxigenin antibody, both of which were conjugated with 5-nm gold colloidal particles. We missed direct detection of the conjugated gold colloidal particles by micro-meter scale AFM scanning , but obtained clear topographic difference between the site of hybridization and the chromosome arm with the help of silver enhancement. We thus clearly detected the in situ hybridization using chromosome painting probes, alpha satellite probes, and locus specific gene probes by AFM. The in situ hybridization to DNA fiber was also detected by AFM. The detection of in situ hybridization by AFM has advantages over fluorescence in situ hybridization: no reduction of signal intensity under light irradiation. Application of AFM to the detection of in situ hybridization will be a useful method to analyze chromosomes.

  13. Standardised fluorescence in situ hybridisation in cytological and histological specimens

    Microsoft Academic Search

    Ludwig Wilkens; Heidrun Gerr; Dorothea Gadzicki; Hans Kreipe; Brigitte Schlegelberger

    2005-01-01

    Fluorescence in situ hybridisation (FISH) has gained major impact in the detection of chromosomal aberrations. The application of FISH, however, is hampered due to complicated protocols. We therefore designed a FISH protocol that allows the fast and reliable application on cytological and histological specimens. Cytological and histological specimens of bone marrow, lymph nodes, liver and breast were analysed with 14

  14. In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

    PubMed

    Yamaguchi, Tsuyoshi; Kawakami, Shuji; Hatamoto, Masashi; Imachi, Hiroyuki; Takahashi, Masanobu; Araki, Nobuo; Yamaguchi, Takashi; Kubota, Kengo

    2015-07-01

    In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ. PMID:25523128

  15. Assignment of the human skeletal muscle alpha actin gene (ACTA1) to 1q42 by fluorescence in situ hybridisation

    Microsoft Academic Search

    P. A. Akkari; H. J. Eyre; S. D. Wilton; D. F. Callen; C. Meredith; L. Kedes; N. G. Laing

    1994-01-01

    The human skeletal muscle alpha actin gene (ACTAl) has previously been localized to 1p21?qter using somatic cell hybrids and a specific probe from the 3’ untranslated region of the gene. Using fluorescence in situ hybridization the localization has been confirmed and the ACTAl gene precisely mapped to 1q42.Copyright © 1994 S. Karger AG, Basel

  16. A fluorescence in situ hybridization map of human chromosome 21 consisting of 30 genetic and physical markers on the chromosome: Localization of 137 additional YAC and cosmid clones with respect to this map

    SciTech Connect

    Gingrich, J.C.; Shadravan, F.; Lowry, S.R. (Lawrence Berkeley Lab., CA (United States))

    1993-07-01

    A fluorescence in situ hybridization (FISH) map of human chromosome 21 was compiled using yeast artificial chromosome (YAC) DNA probes that encode 28 markers physically and/or genetically mapped on the chromosome. Probes that recognize the centromere and rDNA repeat sequences in the p arm were also placed as reference markers on the FISH map. For each probe, the location of the fluorescence hybridization signal was measured on metaphase chromosomes with respect to fractional chromosome length (FL) from p-ter. The location of the markers was established with a standard error of [+-]1.9 Mb using from 9 to 63 FL measurements for each probe. The relative order and separation of the markers as determined by FISH are shown to correspond well to those of other maps of the chromosome. Fifty-one additional YAC and 86 cosmid clones were also localized by FISH with respect to the 30 markers on the chromosome. The cosmids, chosen at random from a flow-sorted chromosome 21 cosmid library, show some biases in chromosome distribution. 42 refs., 2 figs., 3 tabs.

  17. Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS? †

    PubMed Central

    Behrens, Sebastian; Lösekann, Tina; Pett-Ridge, Jennifer; Weber, Peter K.; Ng, Wing-On; Stevenson, Bradley S.; Hutcheon, Ian D.; Relman, David A.; Spormann, Alfred M.

    2008-01-01

    To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13C-carbon and 15N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities. PMID:18359832

  18. Edited by Oliver Hobert. Last revised May 24, 2012. Published December 13, 2012. This chapter should be cited as: Ji N. and van Oudenaarden A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos (December 13, 2012),

    E-print Network

    van Oudenaarden, Alexander

    FISH) of C. elegans worms and embryos (December 13, 2012), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.153.1, http://www.wormbook.org. Copyright: © 2012 Ni Ji and Alexander.vanoudenaarden@hubrecht.eu. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos* Ni Ji1

  19. Unconjugated estriol as an indication for prenatal diagnosis of steroid sulfatase deficiency by in situ hybridization

    Microsoft Academic Search

    Mohamed N. Ahmed; Allan Killam; Kathy H. Thompson; Mazin B. Qumsiyeh

    1998-01-01

    Background: Undetectable or very low unconjugated estriol (E3) levels in routine maternal serum screening are associated with steroid sulfatase deficiency, miscarriages, and anencephaly.Cases: Fluorescence in situ hybridization techniques were used in the diagnosis of steroid sulfatase deficiency prenatally in three cases with low or undetectable unconjugated E3 levels. Results showed a male fetus with a deleted steroid sulfatase region, but

  20. High-Resolution Mapping of Human Chromosome 11 by in Situ Hybridization with Cosmid Clones

    Microsoft Academic Search

    Peter Lichter; Chieh-Ju Chang Tang; Katherine Call; Gary Hermanson; Glen A. Evans; David Housman; David C. Ward

    1990-01-01

    Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by

  1. Whole Mount In Situ Hybridization On Mouse Embryos

    E-print Network

    De Robertis, Eddy M.

    Whole Mount In Situ Hybridization On Mouse Embryos Modified by Lise Zakin and Eddy De Robertis 2008 Henrique et al., 1995. Nature 375, 787-790. Note: This protocol works well for embryos up to 10.5 days post-coïtum (d.p.c.). For older embryos, use in situ on sections. Dissections -Dissect embryos in 1X PBS; remove

  2. Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

    Microsoft Academic Search

    H. M. Thomas; W. G. Morgan; M. R. Meredith; M. W. Humphreys; J. M. Leggett

    1994-01-01

    Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum ×

  3. In situ Measurements of Phytoplankton Fluorescence Using Low Cost Electronics

    PubMed Central

    Leeuw, Thomas; Boss, Emmanuel S.; Wright, Dana L.

    2013-01-01

    Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

  4. In situ measurements of phytoplankton fluorescence using low cost electronics.

    PubMed

    Leeuw, Thomas; Boss, Emmanuel S; Wright, Dana L

    2013-01-01

    Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

  5. Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

    2003-07-01

    Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

  6. Chromosomal abnormalities by conventional cytogenetics and interphase fluorescence in situ hybridization in chronic lymphocytic leukemia in Taiwan, an area with low incidence--clinical implication and comparison between the West and the East.

    PubMed

    Wu, Shang-Ju; Lin, Chien-Ting; Huang, Sheng-Yi; Lee, Fen-Yu; Liu, Ming-Chi; Hou, Hsin-An; Chen, Chien-Yuan; Ko, Bor-Sheng; Chou, Wen-Chien; Yao, Ming; Tang, Jih-Luh; Tsay, Woei; Tien, Hwei-Fang

    2013-06-01

    Chronic lymphocytic leukemia (CLL) is much less prevalent in Taiwan than in the West, but we have recently addressed the distinctly increasing incidence of CLL in Taiwan. We sought to find out whether there is any difference in cytogenetic abnormalities (CA) of CLL between the West and the East. We analyze the CA, by conventional cytogenetics (CG) and fluorescence in situ hybridization (FISH), and their clinical significance in 83 Taiwanese CLL patients and compared the data to those of Western countries. Thirty-five patients (42.2 %) possessed CG-CA and 58 (69.9 %) FISH-CA. By either CG or FISH, deletion of 17p or 11q was associated with poorer overall survival (OS) (P<0.001 and P=0.008, respectively), whereas isolated 13q deletion was associated with better OS (P=0.050). Trisomy 3 by CG was found in five patients; all of them were in Binet A stage but had strikingly poor OS (P<0.001). This prognostic impact was independent from the other CA and Binet stages. We conclude that, though the disease incidence is much different, the CA of CLL in Taiwan are similar to those in the West. The combined CG and FISH analysis is able to predict the patients' prognosis. The clinical significance of trisomy 3 warrants further validation. PMID:23417757

  7. Small RNA Detection by in Situ Hybridization Methods

    PubMed Central

    Urbanek, Martyna O.; Nawrocka, Anna U.; Krzyzosiak, Wlodzimierz J.

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  8. Interphase fluorescent in situ hybridization deletion analysis of the 9p21 region and prognosis in childhood acute lymphoblastic leukaemia (ALL): results from a prospective analysis of 519 Nordic patients treated according to the NOPHO-ALL 2000 protocol.

    PubMed

    Kuchinskaya, Ekaterina; Heyman, Mats; Nordgren, Ann; Söderhäll, Stefan; Forestier, Erik; Wehner, Peder; Vettenranta, Kim; Jonsson, Olafur; Wesenberg, Finn; Sahlén, Sigrid; Nordenskjöld, Magnus; Blennow, Elisabeth

    2011-03-01

    Interphase fluorescent in situ hybridization (FISH) was applied on diagnostic BM smears from 519 children with acute lymphoblastic leukaemia (ALL) in order to establish the frequency and prognostic importance of 9p21 deletion in children enrolled in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) - 2000 treatment protocol. Among the patients, 452 were diagnosed with B-cell precursor (BCP)-ALL and 66 with T-ALL. A higher incidence of 9p21 deletions was found in T-ALL (38%) compared to BCP-ALL (15·7%). Homozygous deletions were found in 19·7% of T-ALL and 4·0% of BCP-ALL; hemizygous deletions were found in 18·2% and 11·7% respectively. In our series, 9p21 deletions were detected in all age groups with a steady rise in the frequency with age. There was no significant difference in outcome between cases with or without 9p21 deletion or between cases with hemi- or homozygous deletions of 9p21. In conclusion, in this large series of childhood ALL deletion of 9p21 was not associated with worse prognosis. However, interphase FISH deletion analysis of 9p21 could be used as a first step to detect unfavourable subtle cytogenetic aberrations such as the dic(9;20) rearrangement. PMID:21241277

  9. Fluorescence in situ hybridization analysis using PAX8- and PPARG-specific probes reveals the presence of PAX8-PPARG translocation and 3p25 aneusomy in follicular thyroid neoplasms.

    PubMed

    Chia, Wai Kit; Sharifah, Noor Akmal; Reena, Rahayu Md Zin; Zubaidah, Zakaria; Clarence-Ko, Ching Huat; Rohaizak, Muhammad; Naqiyah, Ibrahim; Srijit, Das; Hisham, Abdullah Nor; Asmiati, Arbi; Rafie, Md Kaslan

    2010-01-01

    At the present time, the differentiation between follicular thyroid carcinoma (FTC) and adenoma can be made only postoperatively and is based on the presence of capsular or vascular invasion. The ability to differentiate preoperatively between the malignant and benign forms of follicular thyroid tumors assumes greater importance in any clinical setting. The PAX8-PPARG translocation has been reported to occur in the majority of FTC. In this study, a group of 60 follicular thyroid neoplasms [18 FTC, 1 Hurthle cell carcinoma (HCC), 24 follicular thyroid adenomas (FTA), 5 Hurthle cell adenomas (HCA), and 12 follicular variants of papillary thyroid carcinomas (FV-PTC)] were analyzed to determine the prevalence of the PAX8-PPARG translocation by fluorescence in situ hybridization. The PAX8-PPARG translocation was detected in 2/18 FTC (11.1%). In addition, 2/18 (11.1%) FTC and 1/5 (20%) HCA showed 3p25 aneusomy only. The frequency of the translocation detected in the study was lower compared to the earlier studies conducted in Western countries. This might be attributed to the ethnic background and geographic location. Detection of either the PAX8-PPARG translocation or the 3p25 aneusomy in FTC indicates that these are independent genetic events. It is hereby concluded that 3p25 aneusomy or PAX8-PPARG translocation may play an important role in the molecular pathogenesis of follicular thyroid tumors. PMID:19963130

  10. Fluorescence in situ hybridization identifies cryptic t(16;16)(p13;q22) masked by del(16)(q22) in a case of AML-M4 Eo.

    PubMed

    Merchant, Shakil H; Haines, Skip; Hall, Bryan; Hozier, John; Viswanatha, David S

    2004-08-01

    We report a patient presenting with acute myeloid leukemia (AML)-M4 Eo, in whom conventional cytogenetic analysis revealed a 46, XY, del(16)(q22) karyotype. Molecular analysis of the bone marrow cells using reverse transcriptase polymerase chain reaction (RT-PCR) identified a CBFbeta-MYH11, "type A" fusion transcript. However, despite a thorough reevaluation, a balanced chromosome 16 abnormality could not be definitively identified by cytogenetics. Since there exists a small possibility of obtaining a false-positive PCR result, fluorescence in situ hybridization (FISH) analysis using dual-color, break-apart probes for CBFbeta was performed to elucidate the mechanism of fusion gene formation and thus confirm the RT-PCR results. FISH analysis clearly revealed a cryptic t(16;16), which was probably masked by the del(16)(q22). FISH is the preferred diagnostic procedure to elucidate the CBFbeta-MYH11 fusion in this situation, and resolves the possibility of both false-positive and false-negative results with RT-PCR technique. Due to the improved prognosis of AML associated with the CBFbeta-MYH11 fusion compared to AML generally, we recommend the use of FISH for detection of inv(16)/t(16;16)/CBFbeta-MYH11 in patients with failed, complex, or apparently normal cytogenetics, and in whom the cell morphology indicates the strong possibility of this gene fusion. PMID:15269306

  11. Fluorescence In Situ Hybridization Identifies Cryptic t(16;16)(p13;q22) Masked By del(16)(q22) in a Case of AML-M4 Eo

    PubMed Central

    Merchant, Shakil H.; Haines, Skip; Hall, Bryan; Hozier, John; Viswanatha, David S.

    2004-01-01

    We report a patient presenting with acute myeloid leukemia (AML)-M4 Eo, in whom conventional cytogenetic analysis revealed a 46, XY, del(16)(q22) karyotype. Molecular analysis of the bone marrow cells using reverse transcriptase polymerase chain reaction (RT-PCR) identified a CBF?-MYH11, “type A” fusion transcript. However, despite a thorough reevaluation, a balanced chromosome 16 abnormality could not be definitively identified by cytogenetics. Since there exists a small possibility of obtaining a false-positive PCR result, fluorescence in situ hybridization (FISH) analysis using dual-color, break-apart probes for CBF? was performed to elucidate the mechanism of fusion gene formation and thus confirm the RT-PCR results. FISH analysis clearly revealed a cryptic t(16;16), which was probably masked by the del(16)(q22). FISH is the preferred diagnostic procedure to elucidate the CBF?-MYH11 fusion in this situation, and resolves the possibility of both false-positive and false-negative results with RT-PCR technique. Due to the improved prognosis of AML associated with the CBF?-MYH11 fusion compared to AML generally, we recommend the use of FISH for detection of inv(16)/t(16;16)/CBF?-MYH11 in patients with failed, complex, or apparently normal cytogenetics, and in whom the cell morphology indicates the strong possibility of this gene fusion. PMID:15269306

  12. Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

    PubMed Central

    Dedysh, Svetlana N.; Derakshani, Manigee; Liesack, Werner

    2001-01-01

    Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 106 cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 104 type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 106 type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 106 cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog. PMID:11571193

  13. Fluorescence in situ hybridization (FISH) mapping of human chromosome 1: Cytogenetic band localization of 71 NotI linking clones on chromosome 1q25

    Microsoft Academic Search

    J. Sumegi; C. G. Talmadge; D. K. Zhen

    1994-01-01

    Seventy-one human chromosome 1q25-qter-specific lambda clones have been isolated from NotI-linking libraries which were constructed using DNA from MCH206.1 somatic cell hybrid cells. These cells contain chromosome 1q25 translocated to chromosome Xp22 as the only human chromosomes in a mouse background. The NotI-linking clones have been mapped to cytogenetic bands. The relative order of ten NotI clones in 1q32 and

  14. Scanning force microscopical examination of metaphase chromosomes after in-situ hybridization

    NASA Astrophysics Data System (ADS)

    Papastavrou, Georg; Rinke, Bernd; Hausmann, Michael; Cremer, Christoph G.

    1996-12-01

    Chromosomes play a fundamental role in heredity. This reasons the interest in new highly resolving microscopical techniques for their analysis. New preparation techniques have offered a direct approach to detect specific nucleic acid sequences by in situ hybridization. Labelled DNA probes detected by fluorochrome conjugates make it possible to visualize regions down to single genes by light microscopy. Scanning force microscopy (SFM) provides the possibility to image surfaces of biological objects with a resolution one to two orders of magnitude better than a classical fluorescence light microscope. Here, air dried human metaphase chromosomes were examined by SFM before and after in situ hybridization. Different hybridization protocols were compared in their influence on chromosomal morphology. An immunogold technique was introduced for topographic labeling detection by SFM. By propidium iodide staining, identically the same chromosomes which were already examined by SFM were visualized by high resolution confocal light microscopy. The SFM results suggest that the hybridization procedure induced alterations in the overall chromosomal morphology which were not directly detectable by the fluorescence image in light microscopy. Using the immunogold labelling technique and silver enhancement, it was possible to study hybridization features and chromosomal morphology at high resolution simultaneously by SFM. The application of this approach may offer possibilities to investigate the hybridization mechanisms and to develop new hybridization protocols inducing minimal ultrastructural effects on the chromosomes.

  15. In situ hybridization to somatic metaphase chromosomes of potato

    Microsoft Academic Search

    R. G. F. Visser; R. Hoekstra; F. R. Leij; L. P. Pijnacker; B. Witholt; W. J. Feenstra

    1988-01-01

    An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a “ladderpattern” in genomic Southern's of Solanum tuberosum and Solanum phureja, but

  16. Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: Comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis

    SciTech Connect

    Visscher, D.W.; Wallis, T.; Ritchie, C.A. [Wayne State Univ., Detroit, MI (United States)

    1995-09-01

    We compared flow-cytometric DNA histogram pattern to counts of 4 fluorescent-labelled centromeric probes (chromosomes 1, 7, 8, and 17) in whole nuclei (WN) and in nuclei from formalin-fixed deparaffinized thin tissue section (TS) in 25 breast lesions. In benign lesions, signal gains (i.e., trisomic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (0-43%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chromosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal losses in benign TS, on average, were 50-150% greater than in matched WN preparations (chromosome 1: 21.7%, chromosome 7: 21.5%). Malignant lesions generally, but not always, displayed fewer monosomic nuclei and more trisomic nuclei in compared to TS, compatible with a slicing (i.e., nuclear truncation) artifact. Signal counts in carcinomas correlated well with flow cytometric DNA index; however, they were also characterized by evidence of genetic instability, manifest as signal gains in a subset of nuclei (10-25%) with individual probes in diploid range cases, as well as intratumoral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples. We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The minimum signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability. 10 refs., 1 fig., 3 tabs.

  17. Fixing Embryos for In Situ Hybridization Leslie Vosshall

    E-print Network

    1 Fixing Embryos for In Situ Hybridization Leslie Vosshall 2/7/2001 1. A few days in advance, set old plates in the fly waste container in the fly room. 3. The evening before the first embryo the embryos on the old plate as described below. First, use a spatula or kimwipe to remove the large blob

  18. Comparative Cytogenetics Analysis of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians with C0t-1 DNA by Fluorescence In Situ Hybridization

    PubMed Central

    Hu, Li-Ping; Shang, Wen-Cong; Sun, Yan; Wang, Shan; Ren, Xiao-Liang; Huang, Xiao-Ting; Bao, Zhen-Min

    2011-01-01

    The chromosomes of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians were studied by FISH using C. farreri C0t-1 DNA probes. The results showed that C0t-1 DNA signals spread on all chromosomes in the three scallops, whereas signal density and intensity were different strikingly. Clustering brighter signals presented in the centromeric and telomeric regions of most C. farreri chromosomes, and in the centromeric or pericentromeric regions of several P. yessoensis chromosomes. Comparative analysis of the mapping indicated a relatively higher homology in the repetitive DNA sequences of the genome between C. farreri and P. yessoensis than that between C. farreri and A. irradians. In addition, FISH showed that the distribution of C0t-1 DNA clustering signals in C. farreri displayed completely similar signal bands between homologous chromosomes. Based on the C0t-1 DNA fluorescent bands, a more exact karyotype of C. farreri has been obtained. In this study, the comparative analysis based on C0t-1 DNA provides a new insight into the chromosomal reconstructions during the evolution process, and it is helpful for understanding an important source of genomic diversity, species relationships, and genome evolution. PMID:21845202

  19. The fate of Helicobacter pylori phagocytized by Acanthamoeba polyphaga demonstrated by fluorescent in situ hybridization and quantitative polymerization chain reaction tests

    EPA Science Inventory

    Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pyl...

  20. Frequency of aneuploidy in in vitro-matured MII oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds as determined by dual-color fluorescent in situ hybridization.

    PubMed

    Nicodemo, D; Pauciullo, A; Cosenza, G; Peretti, V; Perucatti, A; Di Meo, G P; Ramunno, L; Iannuzzi, L; Rubes, J; Di Berardino, D

    2010-03-01

    The current study was undertaken to investigate the aneuploidy rates in in vitro-matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro-matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to the standard methods and analyzed by FISH using "Xcen" and "5" chromosome-specific painting probes, produced by chromosome microdissection and Degenerate Oligonucleotide Primer- Polymerase Chain Reaction (DOP-PCR). Oocytes with unreduced chromosome number were 10.1% and 16.7% in the two breeds, respectively. To avoid bias due to possible artifacts, the aneuploidy rates were determined by analyzing only oocytes with the corresponding polar bodies. In the Italian Friesian, 100 of 143 (69.9%) secondary MII oocytes showed clear MII plates with corresponding first polar bodies and were scored for aneuploidy detection; one oocyte was "nullisomic" for chromosome X (1.0%) and one "disomic" for chromosome 5 (1.0%). In the Italian Brown, 100 of 120 (83.3%) MII oocytes with corresponding first polar bodies were analyzed; one oocyte was nullisomic (1.0%) and one was disomic (1.0%), both for chromosome 5. Totally, 303 oocytes were analyzed, 40 of which showed an unreduced chromosome complement (13.2%); of 200 MII oocytes with the corresponding first polar bodies, the aneuploidy rate (nullisomy+disomy) for the two chromosomes scored was 2%. Assuming that each chromosome is equally involved in aneuploidy, it results that in cattle oocytes matured in vitro, at least 30% of the oocytes (1x30 haploid chromosomes) should be aneuploid. Premature separation of sister chromatids (PSSC) was also observed in 2% of the oocytes in the Italian Friesian breed involving chromosome 5 and in 1% of the Italian Brown breed involving the X chromosome. Estimation of the "baseline" level of aneuploidy in the in vitro-matured oocytes of the various domestic animal species and breeds is, to our opinion, a useful reference for improving the in vitro production of embryos as well as for monitoring future trends of the reproductive health of the species/breeds engaged in zootechnical productions, especially in relation to management errors and environmental hazards. PMID:20022097

  1. Determination of cutoff values to detect small aneuploid clones by interphase fluorescence in situ hybridization: the Poisson model is a more appropriate approach. Should single-cell trisomy 8 be considered a clonal defect?

    PubMed

    Castagné, Chantal; Mühlematter, Dominique; Beyer, Valérie; Parlier, Valérie; van Melle, Guy; Jotterand, Martine

    2003-12-01

    We applied a dual-color interphase in situ fluorescence hybridization (I-FISH) technique using centromeric probes specific to chromosomes 7 and 8 on 20 control samples in order to define the statistical model best suited to determine cutoff values for detection of small abnormal clones. We found that the Poisson model is a more appropriate approach than a Gaussian model. Then, based on the analysis of 91 samples from 80 patients with myelocytic malignant hemopathies and either clonal or nonclonal -7 or +8 as determined with conventional cytogenetics (CC), we compared the respective power of I-FISH and CC for detection of aneuploidy, with special emphasis on the potential contribution of I-FISH as a complement to CC in the case of small abnormal clones. The I-FISH results were positive in samples with clonal -7 or +8 according to CC analysis. Whereas I-FISH was negative in samples with nonclonal -7 according to CC, thus confirming the reliability of the criteria used to define the clonality of -7; the situation was different with nonclonal +8. I-FISH revealed the clonality of +8 in most samples with single-cell +8. In several cases, however, the unquestionable clonal nature of +8, as evidenced during follow-up, could not be established with either CC or I-FISH according to accepted criteria. Our data suggest that, in case of a single metaphase with +8, the general rule should be amended and the single-cell +8 should be considered and reported as potentially clonal. PMID:14623458

  2. Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: a study of the GIMEMA CML WP.

    PubMed

    Testoni, Nicoletta; Marzocchi, Giulia; Luatti, Simona; Amabile, Marilina; Baldazzi, Carmen; Stacchini, Monica; Nanni, Mauro; Rege-Cambrin, Giovanna; Giugliano, Emilia; Giussani, Ursula; Abruzzese, Elisabetta; Kerim, Simonetta; Grimoldi, Maria Grazia; Gozzetti, Alessandro; Crescenzi, Barbara; Carcassi, Carlo; Bernasconi, Paolo; Cuneo, Antonio; Albano, Francesco; Fugazza, Giuseppina; Zaccaria, Alfonso; Martinelli, Giovanni; Pane, Fabrizio; Rosti, Gianantonio; Baccarani, Michele

    2009-12-01

    In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926. PMID:19797518

  3. Evaluation of HER2/neu Status by Immunohistochemistry Using Computer-Based Image Analysis and Correlation With Gene Amplification by Fluorescence In Situ Hybridization Assay: A 10-Year Experience and Impact of Test Standardization on Concordance Rate.

    PubMed

    Sarode, Venetia R; Xiang, Qun Diane; Christie, Alana; Collins, Rebecca; Rao, Roshni; Leitch, A Marilyn; Euhus, David; Haley, Barbara

    2015-07-01

    Context .- The American Society of Clinical Oncology/College of American Pathologists proposed several recommendations for human epidermal growth factor receptor 2 (HER2) test standardization. One suggestion was that image analysis (IA) could be useful for scoring of HER2/neu immunohistochemistry. The utilization of IA in a real-world practice in a large cohort of cases has not been previously reported. Objectives .- To compare HER2/neu quantification by IA with gene amplification by fluorescence in situ hybridization (FISH); to determine sensitivity, specificity, and concordance rates with the FISH assay; and to determine association between HER2 status with estrogen receptor (ER), progesterone receptor (PR), and Ki-67 expression. Design .- We evaluated HER2 results performed by immunohistochemistry and FISH in conjunction with ER, PR, and Ki-67 in 3093 invasive breast cancer cases from 2002 to 2011. Results .- The overall concordance between immunohistochemistry and FISH was 87.3% (1768 of 2026). When analyzed by year, there was an improvement in the positive concordance rate from 49.4% (44 of 89) to 95.0% (57 of 60) (P < .001). The negative concordance rate was at least 95% with a median false-negative rate of 1.5%. In the FISH+ group, amplification ratio showed significant correlation with IA scores (P < .001). Positive versus negative HER2 status was associated with lower ER and PR levels (P < .001) and higher Ki-67 expression (P < .001). Conclusion .- Scoring of HER2/neu by IA was associated with high false-positive rates before 2008. Improvement in concordance rate after 2008 may be due to proper tissue handling, improved HER2/neu scoring by IA, and assay standardization. PMID:26125432

  4. In situ hybridization to detect spatial gene expression in medaka

    Microsoft Academic Search

    A. R. Tompsett; J. W. Park; X. Zhang; P. D. Jones; J. L. Newsted; D. W. T. Au; E. X. H. Chen; R. Yu; R. S. S. Wu; R. Y. C. Kong; J. P. Giesy; M. Hecker

    2009-01-01

    A whole-animal tissue section in situ hybridization (ISH) system with radio-labeled probes was developed to detect differential gene expression among tissues of the small, oviparous teleost fish, Japanese medaka (Oryzias latipes). Because of its tissue- and gender-specific expression, gonadal aromatase (CYP19a) was selected as a model gene to demonstrate the potential of the system. The ISH system was validated with

  5. The detection of HIV by in situ hybridization

    SciTech Connect

    Shapshak, P.; Sun, N.C.; Resnick, L.; Hsu, M.Y.; Tourtellotte, W.W.; Schmid, P.; Conrad, A.; Fiala, M.; Imagawa, D.T. (Mount Sinai Medical Center, Miami Beach, FL (USA))

    1990-03-01

    A simplified method of in situ hybridization is described for the detection of HIV targets. This standardized method can be applied to sections of formalin-fixed paraffin-embedded tissues, cell blocks, and smears of cultured cells using {sup 3}H- or {sup 35}S-labeled DNA and {sup 35}S-labeled RNA probes. In order to use elevated stringencies in the hybridization and wash steps, tissue sections and cells are covalently bonded to silanated glass slides without their subsequent loss from the slides. Routine hematoxylin and eosin or Romanovsky's stains enable the identification of the cells detected by in situ hybridization. HIV-infected neuroblastoma and lymphoid cell lines, lymph nodes, bone marrow, kidney, as well as brain tissue from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex are used to demonstrate the generality of the procedure. The standardized method described widens the ease and applicability of in situ hybridization in the investigation of pathologic tissues with the use of diverse specimens and probes.

  6. Neuroanatomical tracing combined with in situ hybridization: Analysis of gene expression patterns within brain circuits of interest

    Microsoft Academic Search

    Lorena Conte-Perales; Pedro Barroso-Chinea; Alberto J. Rico; Virginia Gómez-Bautista; Iciar P. López; Elvira Roda; Floris G. Wouterlood; José L. Lanciego

    2009-01-01

    Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols. The subsequent detection at the

  7. DETECTION OF HYPERDIPLOIDY IN RAT INTERPHASE HEPATOCYTES FOLLOWING TREATMENT IN VIVO WITH VINBLASTINE SULFATE USING FLUORESCENCE IN SITU HYBRIDIZATION (FISH). (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  8. Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers.

    PubMed

    Zhang, Luoping; Lan, Qing; Guo, Weihong; Li, Guilan; Yang, Wei; Hubbard, Alan E; Vermeulen, Roel; Rappaport, Stephen M; Yin, Songnian; Rothman, Nathaniel; Smith, Martyn T

    2005-05-30

    Benzene is an established human leukemogen. The mechanism of benzene-induced leukemogenesis, however, remains unclear, but chromosomal damage is thought to play a critical role. We previously reported that the loss of chromosomes 5 and 7 (monosomy 5 and 7) and the gain of chromosomes 8 and 21 (trisomy 8 and 21) are significantly increased in benzene-exposed workers in comparison to matched controls. To determine if selective effects of benzene can occur, we employed three-color painting on an 8-square slide to screen numerical changes in all 24 human chromosomes (OctoChrome FISH) in a pilot study of 11 subjects (6 exposed to >5 ppm benzene and 5 age- and sex-matched controls). The effects of benzene on each chromosome were assessed as the incidence rate ratio (IRR) from a Poisson regression model with the strongest effects being reflected by the highest IRR values. Monosomy of chromosomes 5, 6, 7 and 10 had the highest IRRs and statistical significance in this preliminary study (IRR>2.5, p<0.01). On the other hand, the monosomy levels of six other chromosomes (1, 4, 9, 11, 22 and Y) were unchanged in the exposed workers with IRRs close to 1.0. Similarly, selective effects were also observed on trisomy induction with chromosomes 8, 9, 17, 21 and 22 (IRR>2.5, p<0.01). These results suggest that benzene has the capability of producing selective effects on certain chromosomes, which is supported by our in vitro findings showing that chromosomes 5 and 7 are more sensitive to loss than other chromosomes following exposure to benzene metabolites. We are currently investigating potential mechanisms for this induction of selective aneuploidy. PMID:15935807

  9. Interspecific hybridisation in rhinoceroses: Confirmation of a Black × White rhinoceros hybrid by karyotype, fluorescence in situ hybridisation (FISH) and microsatellite analysis

    Microsoft Academic Search

    T. J. Robinson; V. Trifonov; I. Espie; E. H. Harley

    2005-01-01

    Black and white rhinoceroses are among the most charismatic megaherbivores and have become flagship species for international conservation. They are often subject to intense management that includes being compressed unnaturally in space and density. We present chromosomal and microsatellite evidence to substantiate the first recorded instance of interspecific hybridisation between them. The data suggest that the genetic integrity of the

  10. Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Udo Friedrich; Jan Lenke

    2006-01-01

    Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target

  11. Apparent mosaicism for del(17)(p11.2) ruled out by fluorescence in situ hybridization in a Smith-Magenis syndrome patient

    SciTech Connect

    Juyal, R.C.; Shaffer, L.G.; Lupski, J.R.; Greenberg, F.; Baldini, A.; Patel, P.I. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-11-20

    Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome typically associated with a deletion of band p11.2 of human chromosome 17. Finucane et al. reported a 14-year-old boy with mild physical and behavior manifestations of SMS. No evidence for deletion was initially evident in 20 peripheral blood lymphocytes examined at 850 band level of resolution. Examination of metaphase chromosomes of skin fibroblasts showed a deletion of 17p11.2 in 25/25 cells examined which was consistent with the patient`s clinical manifestations of SMS. Subsequent examination of 25 cells from peripheral blood cultures indicated that 11% of cells harbored a deletion at 17p11.2, thus suggesting a mosaicism for the deletion. A third study of 20 peripheral blood lymphocytes examined at 550-850 band length resolution in a different laboratory, indicated that 13 cells had no apparent deletion, 4 cells had an apparent deletion and 3 cells were questionable. 7 refs.

  12. International, collaborative assessment of limitations of chromosome-specific probes (CSP) and fluorescent in situ hybridization (FISH): Analysis of expected detections in 73,000 prenatal cases

    SciTech Connect

    Evans, M.I.; Henry, G.P.; Miller, W.A. [Wayne State Univ., Detroit, MI (United States)] [and others

    1994-09-01

    FISH and CSP have been proposed to reduce karyotyping need. The purpose of this study was to assess the potential efficacy of CSP-FISH using currently available probes (13, 18, 21, X, & Y) in large, prenatal diagnostic centers. Results (1990-1993) from 7 centers in 4 countries were divided by those expected to be detectable by currently available probes, and those which would be missed assuming 10% probe efficacy. 72,994 karyotypes included 699 trisomy 21`s, 352 trisomy 18`s, 136 trisomy 13`s, 358 sex chromosome aneuploidies, 70 triploidies, and 855 others (translocations, inversions, deletions, markers). Of 2,613 abnormalities, 1,745 would be detectable (66.8%). [Detroit 55.7%, Stockholm 68.3%, Boston 52.6%, Denver 61.3%, Muenster 77.0%, London 84.5%, Philadelphia 69.4%]. Centers with high proportions of referrals for ultrasound anomalies had the highest CSP-FISH positives secondary to increased T 18 & 13. We conclude: (1) 73,000 karyotypes show relatively consistent incidences of the common trisomies, sex chromosome abnormalities, and other chromosome abnormalities among the centers. (2) The proportion expected detectable by FISH-CSP technology varies from 52.6% to 84.5%, averaging 66.8%. (3) 1/3 of the karyotypic abnormalities would be missed, and therefore, replacement of complete karyotyping with FISH would have unacceptably high false-negative rates for routine evaluation. (4) FISH-CSP, while useful when positive for anomalies, is not sufficient when negative to obviate the need for a complete karyotype.

  13. Assignment of the human neuropeptide Y gene to chromosome 7p15.1 by nonisotopic in situ hybridization

    SciTech Connect

    Baker, E.; Sutherland, G.R. [Women`s and Children`s Hospital, Adelaide (Australia)] [Women`s and Children`s Hospital, Adelaide (Australia); Hort, Y.J. [Garvan Inst. of Medical Research, Sydney (Australia)] [and others] [Garvan Inst. of Medical Research, Sydney (Australia); and others

    1995-03-01

    A 15-kb genomic fragment containing the whole human NPY gene in the vector {lambda}GEM-11 was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 5 ng/{mu}l to metaphases from two males. The fluorescence in situ hybridization method was modified from that previously described in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a CCD camera and computer enhanced.

  14. Physical mapping of the Nile tilapia ( Oreochromis niloticus) genome by fluorescent in situ hybridization of repetitive DNAs to metaphase chromosomes—a review

    Microsoft Academic Search

    Cesar Martins; Claudio Oliveira; Adriane P. Wasko; Jonathan M. Wright

    2004-01-01

    The Nile tilapia (Oreochromis niloticus) has received increasing scientific interest over the past few decades for two reasons: first, tilapia is an enormously important species in aquaculture worldwide, especially in regions where there is a chronic shortage of animal protein; and second, this teleost fish belongs to the fascinating group of cichlid fishes that have undergone a rapid and extensive

  15. Localization of the human prostate transglutaminase (Type IV) gene (TGM4) to chromosome 3p21.33-p22 by fluorescence in situ hybridization

    SciTech Connect

    Gentile, V.; Grant, F.J.; Porta, R. [Univ. of Naples, (Italy)] [and others

    1995-05-01

    Transglutaminases (EC 2.3.2.13) catalyze the post-translational modifications of proteins by the formation of{var_epsilon}({gamma}-glutamyl)lysine insopeptide bonds. The number of transglutaminases recently described in eukaryotic cells includes at least five distinct enzymes that have been localized both intracellularly (tissue transglutaminase, keratinocyte transglutaminase, hair follicle transglutaminase) and extracellularly (factor XIIIa and prostate transglutaminase). Although these enzymes share some common features, such as the amino acid sequence in the active site and a strict calcium dependence for their catalytic activity, many differences in the biochemical and immunological properties have been described among the different members of this family. These findings suggest that each molecular form of transglutaminase, catalyzing the cross-linking of specific substrate proteins in specific biological districts, plays a different role in various physiological processes (i.e., blood coagulation, keratinocyte terminal differentiation, cell differentiation, apoptosis, seminal fluid coagulation, and sperm immunogenicity suppression). 10 refs., 1 fig.

  16. Human 60-kDa SS-A/Ro ribonucleoprotein autoantigen gene (SSA2) localized to 1q31 by fluorescence in situ hybridization

    SciTech Connect

    Chan, E.K.L.; Tan, E.M. [Scripps Research Institute, La Jolla, CA (United States)] [Scripps Research Institute, La Jolla, CA (United States); Ward, D.C. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others] [Yale Univ. School of Medicine, New Haven, CT (United States); and others

    1994-09-01

    Human autoantibodies to intracellular antigens are often found in patients with systemic rheumatic diseases. Our laboratory and others have reported the cloning of the cDNAs for two protein autoantigens. The 60-kDa protein is a member of the RNA binding protein family containing an RNA binding motif and has been shown to be primarily responsible for direct interaction with hY-RNAs. The assignment of the 60-kDa autoantigen gene (SSA2) to 1q31 may allow further investigation of the genetic basis of human autoimmune diseases in which ss-A/Ro antibodies are observed. 14 refs., 1 fig.

  17. Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

    PubMed Central

    Conde, Esther; Suárez-Gauthier, Ana; Benito, Amparo; Garrido, Pilar; García-Campelo, Rosario; Biscuola, Michele; Paz-Ares, Luis; Hardisson, David; de Castro, Javier; Camacho, M. Carmen; Rodriguez-Abreu, Delvys; Abdulkader, Ihab; Ramirez, Josep; Reguart, Noemí; Salido, Marta; Pijuán, Lara; Arriola, Edurne; Sanz, Julián; Folgueras, Victoria; Villanueva, Noemí; Gómez-Román, Javier; Hidalgo, Manuel; López-Ríos, Fernando

    2014-01-01

    Background Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. Results All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations. PMID:25248157

  18. Localization of the gene encoding the secretin receptor, SCTR, on human chromosome 2q14.1 by fluorescence in situ hybridization and chromosome morphometry

    SciTech Connect

    Mark, Hon Fong Louie; Chow, Billy K.C. [Univ. of Hong Kong (Hong Kong)] [Univ. of Hong Kong (Hong Kong)

    1995-10-10

    It has been over 90 years since the discovery of secretin, a duodenal mucosal hormone that stimulates the secretion of bicarbonate, enzymes, and potassium ions by the pancreas. It is now known that secretin is one of the most potent physiological regulators of bicarbonate, water, potassium ion, and enzyme secretion from the pancreas. Other pharmacological actions of secretin have also been reported, with effector tissues throughout the body, including the brain, heart, stomach, intestine, and kidney. The rat secretin is a 27-amino-acid peptide that is synthesized as a precursor protein of 134 amino acids in specific endocrine cells. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor is a new type of the G protein-coupled receptor with seven transmembrane segments. 9 refs., 1 fig.

  19. In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence

    E-print Network

    Walter, M.Todd

    In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence David] The physics regarding the retention and mobilization of colloids in saturated and unsaturated conditions remains poorly understood, partially because of the inability to measure colloid concentrations in situ

  20. Novel, in-situ Raman and fluorescence measurement techniques: Imaging using optical waveguides

    Microsoft Academic Search

    Jerry Chance Carter

    2000-01-01

    The following dissertation describes the development of methods for performing standoff and in- situ Raman and fluorescence spectroscopy for chemical imaging and non-imaging analytical applications. The use of Raman spectroscopy for the in- situ identification of crack cocaine and cocaine.HCl using a fiberoptic Raman probe and a portable Raman spectrograph has been demonstrated. We show that the Raman spectra of

  1. Root genomics: towards digital in situ hybridization

    PubMed Central

    Scheres, Ben; van den Toorn, Henk; Heidstra, Renze

    2004-01-01

    Separation of cell types and developmental stages in the Arabidopsis root and subsequent expression profiling have yielded a valuable dataset that can be used to select candidate genes for detailed study and to start probing the complexities of gene regulation in plant development. PMID:15186485

  2. Establishment of the genomic in situ hybridization (GISH) technique for analysis in interspecific hybrids of Passiflora.

    PubMed

    Melo, C A F; Silva, G S; Souza, M M

    2015-01-01

    The genomic in situ hybridization (GISH) technique was applied to Passiflora interspecific F1 HD13-133 hybrids (Passiflora sublanceolata x Passiflora foetida) and HD15-101 (Passiflora gardineri x Passiflora gibertii), and the backcrossed hybrids (BC1) HD18-106 and HD18-113 (Passiflora sublanceolata x HD13-133). GISH was performed using genomic probes prepared with the DNA from the paternal genitor, whereas the maternal DNA was used as blocking DNA and employed at various concentrations (20X, 40X, 60X, and 100X) in relation to the probe concentration. At the same time, GISH was applied with the use of simultaneous probes from both genomes, paternal and maternal, that were detected with avidin-FITC and anti-digoxigenin-rhodamine, respectively. Both methodologies allowed the distinguishing of the maternal and paternal genomes, thus confirming the hybrid nature of all the analyzed genotypes. Furthermore, the presence of recombinant chromosomes in BC1 hybrids revealed the occurrence of meiotic recombination in HD13 hybrids. This application of the GISH technique is an important step towards genomic analyses of Passiflora hybrids: it can broaden the phylogenetic and evolutionary studies of the genus and, at the same time, contribute to breeding programs. PMID:25867365

  3. In situ hybridization of plant meiotic and mitotic chromosomes: differences in signal detection.

    PubMed

    Clark, M S; Parker, J S

    1992-09-01

    The technique of in situ hybridization to both meiotic and mitotic chromosomes of Rumex acetosa is described. Differences in the efficiency of signal detection were observed between the two types of material. The implications of these results for in situ hybridization to other plant species are explored. PMID:1300148

  4. Bicolor detection of two target DNAs by non-radioactive in situ hybridization

    Microsoft Academic Search

    A. H. N. Hopman; J. Wiegant; A. K. Raap; J. E. Landegent; M. Ploeg; P. Duijn

    1986-01-01

    A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different

  5. Trigenomic origin of the hexaploid Psammopyrum athericum (Triticeae: Poaceae) revealed by in-situ hybridization

    Microsoft Academic Search

    Pernilla Ellneskog-Staam; Björn Salomon; Roland von Bothmer; Kesara Anamthawat-Jònsson

    2001-01-01

    The genomic constitution of the hexaploid Psammopyrum athericum was studied with in-situ DNA hybridization using both genomic DNA and isolated cloned sequences as probes. A genomic probe from Thinopyrum bessarabicum (E genome) hybridized successfully to 14 chromosomes of Ps. athericum and a probe from Festucopsis serpentinii (L genome) hybridized to another 14 chromosomes. The remaining chromosomes did not hybridize, apart

  6. The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation

    ERIC Educational Resources Information Center

    Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

    2014-01-01

    "In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

  7. Demonstration of tissue-specific gene expression by in situ hybridization

    Microsoft Academic Search

    L. M. Angerer; K. H. Cox; R. C. Angerer

    1987-01-01

    In situ hybridization has emerged as a valuable tool for the identification of individual cells expressing specific genes. Recently, methods have become sufficiently sensitive to detect mRNAs present at the level of only a few molecules per cell. When mRNAs are expressed in only a small fraction of the cells in a mixed population, in situ hybridization may be the

  8. In Situ Surface Plasmon Resonance Imaging Detection of DNA Hybridization to Oligonucleotide

    E-print Network

    In Situ Surface Plasmon Resonance Imaging Detection of DNA Hybridization to Oligonucleotide Arrays, and these arrays have been used in DNA hybridization experiments with in situ surface plasmon resonance (SPR on Gold Surfaces Andrew J. Thiel, Anthony G. Frutos, Claire E. Jordan, Robert M. Corn, and Lloyd M. Smith

  9. Solar cells Improved Hybrid Solar Cells via in situ UV Polymerization

    E-print Network

    Sibener, Steven

    Solar cells Improved Hybrid Solar Cells via in situ UV Polymerization Sanja Tepavcevic, Seth B-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples mobility of the photoactive layer can be enhanced. 1. Introduction Hybrid solar cells have been developed

  10. Evaluation of the impact of bioaugmentation and biostimulation by in situ hybridization and microelectrode.

    PubMed

    Satoh, Hisashi; Okabe, Satoshi; Yamaguchi, Yuki; Watanabe, Yoshimasa

    2003-05-01

    Three rotating disk biofilm reactors were operated to evaluate whether bioaugmentation and biostimulation can be used to improve the start-up of microbial nitrification. The first reactor was bioaugmented during start-up period with an enrichment culture of nitrifying bacteria, the second reactor received a synthetic medium containing NH(4)(+) and NO(2)(-) to facilitate concomitant proliferation of ammonia- and nitrite-oxidizing bacteria, and the third reactor was used as a control. To evaluate the effectiveness of bioaugmentation and biostimulation approaches, time-dependent developments of nitrifying bacterial community and in situ nitrifying activity in biofilms were monitored by fluorescence in situ hybridization (FISH) technique and microelectrode measurements of NH(4)(+), NO(2)(-), NO(3)(-), and O(2). In situ hybridization results revealed that addition of the enrichment culture of nitrifying bacteria significantly facilitated development of dense nitrifying bacterial populations in the biofilm shortly after, which led to a rapid start-up and enhancement of in situ nitrification activity. The inoculated bacteria could proliferate and/or survive in the biofilm. In addition, the addition of nitrifying bacteria increased the abundance of nitrifying bacteria in the surface of the biofilm, resulting in the higher nitrification rate. On the other hand, the addition of 2.1mM NO(2)(-) did not stimulate the growth of nitrite-oxidizing bacteria and did inhibit the proliferation of ammonia-oxidizing bacteria instead. Thus, the start-up of NO(2)(-) oxidation was unchanged, and the start-up of NH(4)(+) oxidation was delayed. In all the three biofilm reactors, data sets of time series analyses on population dynamics of nitrifying bacteria determined by FISH, in situ nitrifying activities determined by microelectrode measurements, and the reactor performances revealed an approximate agreement between the appearance of nitrifying bacteria and the initiation of nitrification activity, suggesting that the combination of these techniques was a very powerful monitoring tool to evaluate the effectiveness of bioaugmentation and biostimulation strategies. PMID:12691906

  11. Fetal t(5p;21q) misdiagnosed as monosomy 21: A plea for in situ hybridization studies

    SciTech Connect

    Gill, P.; Uhrich, S.; Cheng, E.; Disteche, C. [Univ. of Washington Medical Center, Seattle, WA (United States)

    1994-10-01

    We report a case of 45,XY,-5,-21,+der (5)t(5;21) (p13 or p14;q11.2 or q21) that was prenatally misdiagnosed as complete monosomy 21 and terminated at 24 weeks of gestation. Subsequent fluorescence in situ hybridization studies with a chromosome 21 painting probe documented the cryptic unbalanced translocation. 17 refs., 2 figs., 1 tab.

  12. [Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].

    PubMed

    Chen, Jian-Min; Hong, Yi-Huan; Wang, You-Ping; Bowley, Steve; Wan, Jian-Min

    2006-02-01

    Physical localization of ribosomal genes in diploid and tetraploid alfalfa (Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It was revealed that 45s gene was only located at nucleolus organizer region (NOR) with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3 loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as probe to hybridize with tetraploid species in alfalfa, both diploid species were successfully hybridized with tetraploid chromosomes, only showing the difference in hybridization signals in different numbers of chromosomes. Chromosomes of alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of distribution of DAPI banding were consistent with C-banding for M. coerulea. The possible origination of tetraploid alfalfa was discussed based on DAPI banding patterns and FISH analysis for ribosomal genes . PMID:16520314

  13. Improved hybrid solar cells via in situ UV-polymerization.

    SciTech Connect

    Tepavcevic, S.; Darling, S. B.; Dimitrijevic, N. M.; Rajh, T.; Sibener, S. J.; Univ. of Chicago

    2009-08-03

    One approach for making inexpensive inorganic-organic hybrid photovoltaic (PV) cells is to fill highly ordered TiO{sub 2} nanotube (NT) arrays with solid organic hole conductors such as conjugated polymers. Here, a new in situ UV polymerization method for growing polythiophene (UV-PT) inside TiO{sub 2} NTs is presented and compared to the conventional approach of infiltrating NTs with pre-synthesized polymer. A nanotubular TiO{sub 2} substrate is immersed in a 2,5-diiodothiophene (DIT) monomer precursor solution and then irradiated with UV light. The selective UV photodissociation of the C-I bond produces monomer radicals with intact {pi}-ring structure that further produce longer oligothiophene/PT molecules. Complete photoluminescence quenching upon UV irradiation suggests coupling between radicals created from DIT and at the TiO{sub 2} surface via a charge transfer complex. Coupling with the TiO{sub 2} surface improves UV-PT crystallinity and {pi}-{pi} stacking; flat photocurrent values show that charge recombination during hole transport through the polymer is negligible. A non-ideal, backside-illuminated setup under illumination of 620-nm light yields a photocurrent density of {approx} 5 {micro}A cm{sup -2} - surprisingly much stronger than with comparable devices fabricated with polymer synthesized ex situ. Since in this backside architecture setup we illuminate the cell through the Ag top electrode, there is a possibility for Ag plasmon-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples the conjugated polymer to the TiO{sub 2} surface, the absorption of sunlight can be improved and the charge carrier mobility of the photoactive layer can be enhanced.

  14. A new method for the accurate in situ monitoring of chlorophyll a fluorescence in lichens and bryophytes

    Microsoft Academic Search

    Mark Schlensog; Burkhard Schroeter

    2001-01-01

    Abstract:A new method has been developed to measure chlorophyll a fluorescence of bryophytes and lichens in situ in the field. Specially designed aluminium stands fixed to rocks or other suitable substrata allow several cryptogam thalli to be measured sequentially with only one pulse amplitude modulated (PAM) chlorophylla fluorescence analyser. The stands guarantee a fixed position for the fluorescence probe in

  15. [Characterization of chromosomal rearrangements by in situ hybridization in glioblastoma].

    PubMed

    Bourgeois, C A; Kokalj-Vokac, N; Dutrillaux, A M; Poisson, M; Delattre, J Y; Vega, F; Malfoy, B; Dutrillaux, B

    1994-05-01

    In a case of glioblastoma, the following karyotype was determined: 47, X, - Y, + der(1) t(1;9)(p21;p23), t(1;9)(p21;p23), + 3, + 7, der(9) t(Y;9)(q11;p21), - 13, t(13;16)(p13,p11), del(14)(q11q22). Classical satellite DNAs are mainly located in chromosomes 1, 9, 15, 16 and Y. Because, most of these chromosomes were implicated in the rearrangements, a detailed cytogenetic study was undertaken. This study included in situ hybridization of the satellite and alphoid DNAs of chromosomes 1, 9, 16 and Y combined with various chromosome banding methods (DA-DAPI, quinacrine mustard and R-banding). The data obtained, demonstrated that the breakpoints were always located outside the areas containing the satellite and alphoid DNAs. The situation observed here differs from that reported in breast cancers for which a high proportion of the breakpoints occur within these areas. These findings suggest that in glioblastoma, chromosome rearrangements result from different mechanisms than those implicated in breast cancers. Thus, in cancers, chromosomal instabilities may result from several mechanisms. PMID:7538360

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

    Microsoft Academic Search

    HJ Tanke; J Wiegant; RPM van Gijlswijk; V Bezrookove; H Pattenier; RJ Heetebrij; EG Talman; AK Raap; J Vrolijk

    1999-01-01

    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

  17. A capillary-based probe for in situ detection of enhanced fluorescence signals

    NASA Astrophysics Data System (ADS)

    Long, F.; Xiao, R.; Zhu, A. N.; Shi, H. C.; Wang, S. Q.

    2013-07-01

    A simple, compact, and high sensitivity capillary-based probe for the in situ detection of fluorescence signals with high sensitivity is demonstrated. A home-made single–multi-mode fiber coupler that is coaxially aligned with the capillary-based probe provides for the transmission of excitation light and the collection and transmission of fluorescence. We propose a conceptually straightforward theoretical model to optimize the factors affecting the fluorescence-capture capability of the capillary-based probe. The fluorescence signal detected by fiber-optic spectroscopy non-linearly increases with the length of the capillary-based probe. In addition, the thicker the capillary tube wall is, the less the fluorescence signals determined are. The performance of the proposed probe is evaluated experimentally by measuring the fluorescence spectra of Cy5.5 dye and blue-green algae. The experimental results show that the proposed probe provides more than a ten-fold increase in fluorescence signal compared with direct measurements by a flat-tipped multi-mode fiber probe. The advantages of the capillary-based probe, which include its simple and compact structure, excellent light collection efficiency, requirement of small sample volume, and recoverability of samples, allow its wide application to in situ detection in the medical, forensic, biological, geological, and environmental fields with high sensitivity.

  18. Analysis of chromosome segregation during mammalian meiosis using combined immunofluorescence and fluorescence in situ hubridization

    SciTech Connect

    Hunt, P.A.; Embury, P.B.; Mroz, K.M. [Case Western Univ., Cleveland, OH (United States)] [and others

    1994-09-01

    Meiotic non-disjunction is thought to occur in 10-20% of all human oocytes, making this the most common genetic abnormality in our species. Aberrant recombination has been implicated in the genesis of these errors; however, direct studies of the meiotic process have been hampered by the lack of material and appropriate technology. We have developed a technique for the evaluation of meiosis in intact mammalian oocytes that combines immunofluorescence and fluorescence in situ hybridization (FISH). This allows for simultaneous, 3-dimensional visualization of the meiotic spindle, the alignment of the chromosomes on the spindle, and the placement of specific chromosomes. We have used this technology to follow meiotic progression in oocytes from XO female mice to evaluate the behavior of an unsynapsed chromosome during mammalian meiosis. Perturbations in chromosome behavior are evident early in meiosis: during the formation of the first meiotic spindle, the univalent X chromosome is properly positioned. With the onset of anaphase, the single X chromosome most commonly segregates as an intact chromosome, although equational segregation of the X chromatids is seen in a significant minority (approximately 20%) of oocytes. These observations demonstrate that failure of pairing/recombination can result in segregation of sister chromatids at meiosis I. This has obvious implications for human non-disjunction, much of which is thought to be due to recombination deficiencies; accordingly, we are now extending our studies to include analyses of human oocytes.

  19. Fluorescent in situ sequencing on polymerase colonies Robi D. Mitra,a,1

    E-print Network

    Church, George M.

    of enzyme during sequencing cycles. Next, we present two novel types of reversibly dye-labeled nucleotideFluorescent in situ sequencing on polymerase colonies Robi D. Mitra,a,1 Jay Shendure,a Jerzy isolation, amplification, and sequencing can be achieved by the use of polymerase colonies (polonies

  20. Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

    PubMed Central

    Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

    2012-01-01

    A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

  1. A method for cellular localization of gene expression via quantitative in situ hybridization in plants.

    PubMed

    Küpper, Hendrik; Seib, Laura Ort; Sivaguru, Mayandi; Hoekenga, Owen A; Kochian, Leon V

    2007-04-01

    A quantitative in situ hybridization technique (quantitative whole-mount in situ hybridization, QISH) for plants is described. It employs direct hybridization of fluorescently labelled gene-specific oligonucleotides in large tissue pieces combined with optical sectioning. It dramatically increases the throughput compared with conventional antibody- and microtome-based in situ mRNA hybridization methods, while simultaneously eliminating artefact-prone preparation steps that prevent reliable quantification in conventional methods. The key feature of this technique is the quantification of gene expression using housekeeping genes (cytosolic GAPDH and 18S RNA) as internal standards. This feature enables a correction of varying cytoplasm/vacuole ratios in different cell types, as well as tissue optical effects and non-specific signals. The quantitative nature of the technique allows for analysis of gene expression in response to different environmental conditions, as well as tissue- and age-dependent differences in gene expression patterns. In addition to testing tissue permeabilization, structural preservation, specificity, linearity and tissue optical effects, we verified the reliability of the technique with three Arabidopsis thaliana genes of known function and distribution. These were the rbcL gene for ribulose 1,5-bisphosphate carboxylase, the developmentally related gene SCARECROW (AtSCR) and PHOT-1, a photoreceptor kinase. As expected, rbcL mRNA was found in all photosynthetic cells, while SCR mRNA was detected mainly in bundle sheath cells and PHOT-1 was found predominantly in epidermal and cortical cells of the apical hook of light-grown seedlings. As an application example, QISH was used to measure transcript abundance for a zinc transporter from the ZIP family of transporters in the Zn/Cd hyperaccumulator model plant, Thlaspi caerulescens, and the related non-accumulator Thlaspi arvense. This showed that QISH can be used to compare differences in mRNA levels between cell types, plant growth conditions and plant species. Messenger RNA for the zinc transporter gene ZNT1 was abundant in photosynthetic cells, but not in the epidermal storage cells where metal hyperaccumulation in T. caerulescens occurs. This indicates that ZNT1 does not directly participate in metal hyperaccumulation within the leaf. Growing T. caerulescens with high zinc levels strongly reduced ZNT1 transcript abundance in the spongy mesophyll cells, but less in the other cell types. In T. arvense, ZNT1 mRNA levels were generally much lower, and were furthermore drastically reduced by growth at increased zinc levels, confirming earlier reports regarding ZNT1 regulation in these two Thlaspi species. PMID:17397510

  2. Orientation of the Friedreich`s ataxia linkage group by fluorescent in situ hybridisation

    SciTech Connect

    Hillermann, R.; Wilkes, D.; Pook, M. [Imperial College, London (United Kingdom)] [and others

    1994-09-01

    The tight linkage between the Friedreich`s ataxia (FRDA) locus and the DNA marker loci originally used to assign the mutation to chromosome 9q13-21.1 has caused great difficulty in determining the precise order of the disease locus with respect to the markers. Investigation of a single-recombination event identified by a collaborative study indicated that the most probable order for the linkage group is cen-FRDA-D9S5-D9S15-qter. To confirm this order, essential for the final cloning strategy, we have undertaken fluorescent in situ hybridization studies on pro-metaphase chromosomes. Cosmid clones isolated from the proximal and distal ends of a YAC contig spanning a 1.2 Mb interval containing the gene were labelled with biotin and digoxigenin, respectively, and analyzed utilising dual color detection methods. An orientation of cen-D9S5-D9S15-qter was demonstrated on normal chromosomes, which confirms and strengthens the genetic data. These markers were also orientated on a polymorphic inverted chromosome 9 and a corresponding order demonstrated, suggesting that the inversion does not disrupt or interfere with the region containing the FRDA gene.

  3. Understanding aquatic microbial processes using EEM's and in-situ fluorescence sensors

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Attridge, John; Rushworth, Cathy; Cox, Tim; Anesio, Alexandre; Reynolds, Darren

    2015-04-01

    The diverse origin of dissolved organic matter (DOM) in aquatic systems is well documented within the literature. Previous literature indicates that coloured dissolved organic matter (CDOM) is, in part, transformed by aquatic microbial processes, and that dissolved organic material derived from a microbial origin exhibits tryptophan-like fluorescence. However, this phenomenon is not fully understood and very little data is available within the current literature. The overall aim of our work is to reveal the microbial-CDOM interactions that give rise to the observed tryptophan-like fluorescence. The work reported here investigates the microbial processes that occur within freshwater aquatic samples, as defined by the biochemical oxygen demand (BOD) test, as a function of the T1 peak (?ex/em 280/330-370 nm). A series of standard water samples were prepared using glucose, glutamic acid, BOD dilution water and a bacterial seed (Cole-Parmer BOD microbe capsules). Samples were spiked with CDOM (derived from an environmental water body) and subjected to time resolved BOD analysis and as excitation-emission fluorescence spectroscopy. All EEM spectral data was interrogated using parallel factor analysis (PARAFAC) in an attempt to determine the presence and dominance (relative intensities) of the CDOM-related and T1-related fluorophores within the samples. In-situ fluorescence sensors (Chelsea Technologies Group Ltd.) were also used to monitor the T1 fluorescence peak (UviLux Tryptophan) and the CDOM fluorescence peak (UviLux CDOM) during experiments. Tryptophan-like fluorescence was observed (albeit transient) in both spiked and un-spiked standard water samples. By furthering our understanding of aquatic organic matter fluorescence, its origin, transformation, fate and interaction with aquatic microbiological processes, we aim to inform the design of a new generation in-situ fluorescence sensor for the monitoring of aquatic ecosystem health.

  4. Quantifying the photothermal efficiency of gold nanoparticles using tryptophan as an in situ fluorescent thermometer.

    PubMed

    Chiu, Ming-Jui; Chu, Li-Kang

    2015-06-24

    The photothermal efficiencies, denoting the efficiency of transducing incident light to heat, of gold nanoparticles of different diameters (? = 22-86 nm) were quantified upon exposure at 532 nm. The fluorescence of tryptophan at 300-450 nm upon 280 nm excitation serves as an in situ fluorescent thermometer to illustrate the evolution of the average temperature change in the heating volume of the nanoparticle solution. The fluorescence intensity decreases as the temperature increases, having a linear gradient of 2.05% fluorescence decrease per degree Celsius increment from 20 to 45 °C. The presence of gold nanoparticles at the nM level does not perturb the temperature-dependent fluorescence of tryptophan in terms of fluorescence contour and temperature response. The heating volume was defined by overlapping the collimated 532 nm laser (? = 0.83 mm) for exciting the nanoparticles and the 280 nm continuous-wave beam (? = 0.81 mm) for exciting tryptophan in a 2 mm × 2 mm square tube, and the fluorescence was collected perpendicularly to the collinear alignment. This method has satisfactory reproducibility and a sufficient temperature detectivity of 0.2 °C. The profiles of the average temperature evolution of the mixtures containing nanoparticles and tryptophan were derived from the evolution of fluorescence and analyzed using collective energy balancing. The relative photothermal efficiencies for different sizes of gold nanoparticles with respect to the 22 nm nanoparticle agree with those predicted using Mie theory. The employment of tryptophan as a fluorescent thermometer not only provides an in situ tool to monitor the photothermal effect of nanostructures but is also applicable to thermal imaging in biological applications. PMID:26068797

  5. Genomic in situ hybridization analysis of a trigenomic hybrid involving Solanum and Lycopersicon species.

    PubMed

    Ali, S N; Huigen, D J; Ramanna; Jacobsen, E; Visser, R G

    2001-04-01

    A 4x potato (+) tomato fusion hybrid (2n = 4x = 48) was successfully backcrossed with a diploid Lycopersicon pennellii (2n = 2x = 24). Genomic in situ hybridization (GISH) on somatic and meiotic chromosomes confirmed that the progenies were triploids (2n = 3x = 36) and possessed three different genomes: potato, tomato, and L. pennellii. Therefore, they have been called trigenomic hybrids. Total genomic probes of both Lycopersicon species were found to hybridize mutually, whereas the potato genome was clearly differentiated. During metaphase I, bivalents were formed predominantly between tomato and L. pennellii chromosomes and the univalents of potato chromosomes were most common. Trivalents in all cases included homoeologous chromosomes of potato, tomato, and L. pennellii. However, the triploids were totally sterile as determined from extensive crossing. On chromosome doubling of triploids by shoot regeneration from callus, hexaploids (2n = 6x = 72) were obtained. Despite exhibiting clear allohexaploid behaviour by forming 36 bivalents at meiosis, these were also completely sterile like their triploid counterparts. In spite of this drawback, the prospects of chromosome pairing between potato L. pennellii and Solanum genomes does open the possibilities for bringing the two genera close. PMID:11341741

  6. Regulatory pathway analysis by high-throughput in situ hybridization

    SciTech Connect

    Visel, Axel; Carson, James P.; Oldekamp, Judit; Warnecke, Marei; Jakubcakova, Vladimira; Zhou, Xunlei; Shaw, Chad; Alvarez-Bolado, Gonzalo; Eichele, Gregor

    2007-10-01

    Automated in situ hybridization (ISH) permits construction of comprehensive atlases of gene expression patterns in mammals. When web-accessible, such atlases become searchable digital expression maps of individual genes and offer an entryway to elucidate genetic interactions and signaling pathways. An atlas housing ~1,000 spatial gene expression patterns of the mid-gestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising 90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This ordered hundreds of complex expression patterns into a matrix that reflected the embryonic architecture and the relatedness of patterns of expression. Clustering yielded twelve distinct groups of expression pattern. Because of similarity of expression patterns within a group, members of this group may be components of regulatory cascades. We focused on one group, which is composed of 83 genes, including Pax6, an evolutionary conserved transcriptional master mediator of the development. Using functional studies, ISH on Pax6-deficient embryos and Pax6 binding site identification and validation by means of electromobility shift assays, we identified numerous genes that are transcriptionally regulated by Pax6. Hence cluster analysis of annotated gene expression patterns obtained by robotic ISH is an entryway for identification of components of signaling cascades in mammals.

  7. Morphological investigation of hybrid materials composed of phenolic resin and silica prepared by in situ polymerization

    Microsoft Academic Search

    K Haraguchi; Y Usami; K Yamamura; S Matsumoto

    1998-01-01

    Morphological investigations for hybrid materials composed of phenolic resin and silica, prepared by in situ polymerization of silicon alkoxide, were performed using TEM. Irregularly shaped silica particles a few tens of nanometres in size, composed of hyper-fine particles, are homogeneously dispersed in the transparent hybrid materials forming a partial or uniform silica network depending on the silica content. In the

  8. Elucidating Structure and Function In Vivo With Hybrid Fluorescence and Magnetic Resonance Imaging

    Microsoft Academic Search

    Mark Niedre; Vasilis Ntziachristos

    2008-01-01

    While the mathematics, physics, and technology behind magnetic resonance (MR) and fluorescence image formation are distinctively different, the two modalities have significant complementary features to impart strong preclinical and clinical application synergies. Traditionally, hybrid MR and fluorescence imaging implied the use of a system where optical and MR signals can be concurrently acquired. In this case, the common geometry allows

  9. Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation

    Microsoft Academic Search

    Ki-Byung Lim; Jannie Wennekes; Jong de J. H. S. G. M; Evert Jacobsen; Tuyl van J. M

    2001-01-01

    Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the

  10. The fabrication of rare earth covalent luminescent hybrid materials with potential molecular bridge by in situ sol-gel process

    SciTech Connect

    Zhao Limin [Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092 (China); Yan Bing [Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092 (China)]. E-mail: byan@tongji.edu.cn

    2006-01-05

    1,2,4-Benzenetricar boxylic acid (abbreviated as TMA) was modified to achieve a functional molecular bridge (TMA-APMES) with double reactivity by the reaction with a cross-linking molecule (3-aminopropyl-methyl-diethoxylsiliane, APMES). The modified functional ligand further behaves as a bridge both coordinates to Ln{sup 3+} through oxygen atom and occurs in situ sol-gel process with matrix precursor (tetraethoxysilane, TEOS) through co-hydrolysis and co-polycondensation reaction. Then a novel molecular hybrid material (named as hybrid Ln{sup 3+}) with double chemical bond (Tb-O coordination bond and Si-O covalent bond) resulted. Ultraviolet absorption, phosphorescence, and fluorescence spectra were applied to characterize the photophysical properties of the obtained hybrid material. The strong luminescence of Tb{sup 3+} substantiates optimum energy couple and effective intramolecular energy transfer between the triplet state energy of modified ligand bridge and emissive energy level of Tb{sup 3+}.

  11. Examining Hedgehog pathway genes GLI3, SHH, and PTCH1 and the p53 target GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster using fluorescence in situ hybridization uncovers GLIPR1/GLIPR1L1/GLIPR1L2 deletion in 9% of patients with multiple myeloma.

    PubMed

    Tam, Michael; Lin, Pei; Hu, Peter; Lennon, Patrick A

    2010-01-01

    Mutations in genes regulating cell cycle and apoptosis are considered major culprits for the malignant transformation of cancer cells. Aberrant activation of the Hedgehog (HH) signaling pathway which primarily regulates genes involved in cell growth, proliferation, survival and apoptosis has been demonstrated in multiple myeloma. Mutations resulting in defective components of the p53 pathway, which serves a critical role in mediating cellular stress response by triggering DNA repair, cell cycle arrest, senescence and apoptosis, have also been identified. This study focuses on detecting copy number variations for the GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster of the p53 pathway and three elements of the HH pathway, SHH, PTCH1 and GLI3 in multiple myeloma (MM) using fluorescence in situ hybridization (FISH). In eighteen samples, there was no evidence of abnormal copy number for PTCH1, GLI3 or SHH. Thus, it is unlikely that copy number variations of these genes are linked to multiple myeloma. However, a deletion of the GLIPR1/GLIPR1L1/ GLIPR1L2 gene cluster, all p53 targets, was found in three of 32 samples (9.4%) indicating that these deleted genes may have significant implications in MM. Further studies should be performed to determine the role of the GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster in the pathogenesis of multiple myeloma. PMID:20978342

  12. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. PMID:24514660

  13. A Hybrid Approach of Using Wavelets and Fuzzy Clustering for Classifying Multispectral Florescence In Situ Hybridization Images

    PubMed Central

    Dandpat, Ashok Kumar

    2006-01-01

    Multicolor or multiplex fluorescence in situ hybridization (M-FISH) imaging is a recently developed molecular cytogenetic diagnosis technique for rapid visualization of genomic aberrations at the chromosomal level. By the simultaneous use of all 24 human chromosome painting probes, M-FISH imaging facilitates precise identification of complex chromosomal rearrangements that are responsible for cancers and genetic diseases. The current approaches, however, cannot have the precision sufficient for clinical use. The reliability of the technique depends primarily on the accurate pixel-wise classification, that is, assigning each pixel into one of the 24 classes of chromosomes based on its six-channel spectral representations. In the paper we introduce a novel approach to improve the accuracy of pixel-wise classification. The approach is based on the combination of fuzzy clustering and wavelet normalization. Two wavelet-based algorithms are used to reduce redundancies and to correct misalignments between multichannel FISH images. In comparison with conventional algorithms, the wavelet-based approaches offer more advantages such as the adaptive feature selection and accurate image registration. The algorithms have been tested on images from normal cells, showing the improvement in classification accuracy. The increased accuracy of pixel-wise classification will improve the reliability of the M-FISH imaging technique in identifying subtle and cryptic chromosomal abnormalities for cancer diagnosis and genetic disorder research. PMID:23165039

  14. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    SciTech Connect

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-03-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of /sup 35/S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

  15. Localization of messenger RNAs encoding three GABA transporters in rat brain: an in situ hybridization study

    Microsoft Academic Search

    Margaret M. Durkin; Kelli E. Smith; Laurence A. Borden; Richard L. Weinshank; Theresa A. Branchek; Eric L. Gustafson

    1995-01-01

    Localization of the messenger RNAs encoding three ?-aminobutyric acid (GABA) transporters, termed GAT-1, GAT-2, and GAT-3, has been carried out in rat brain using radiolabeled oligonucleotide probes and in situ hybridization histochemistry. Hybridization signals for GAT-1 mRNA were observed over many regions of the rat brain, including the retina, olfactory bulb, neocortex, ventral pallidum, hippocampus, and cerebellum. At the microscopic

  16. Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector

    PubMed Central

    Seblova, Veronika; Lewis, Michael D.; Mauricio, Isabel; Volf, Petr; Miles, Michael A.

    2011-01-01

    Background The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described. Methodology/Principal Findings Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro. Conclusions/Significance For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits. PMID:21637755

  17. Retrospective study of myocardial canine parvovirus infection by in situ hybridization.

    PubMed

    Waldvogel, A S; Hassam, S; Weilenmann, R; Tratschin, J D; Siegl, G; Hänichen, T; Briner, J; Pospischil, A

    1991-07-01

    The diagnosis of myocardial canine parvovirus (CPV) infection used to depend on the presence of pathognomonic intranuclear inclusion bodies. The in situ hybridization technique, however, allowed to detect CPV specific nucleic acid in myocardial tissue where no inclusion bodies were found. Hence, we applied this technique to check formalin-fixed paraffin-embedded myocardial tissue from puppies with heart lesions for the presence of CPV. The tissues had been collected between 1977 and 1989. A biotinylated probe was used for in situ hybridization. This way CPV specific nucleic acid was detected in 3 dogs where CPV myocarditis had not been diagnosed on routinely stained slides because of the lack of intranuclear inclusion bodies. However, in spite of the application of the in situ hybridization technique no further myocardial CPV infection was detected in puppies with heart lesions from after 1979, confirming that the number of puppies with myocardial CPV infection declined after that year. PMID:1656656

  18. Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product

    SciTech Connect

    Boschman, G.; Rens, W.; Slater, R.; Aten, J. (Univ. of Amsterdam (Netherlands)); Buys, C.; Veen, A. van der; Osinga, J. (State Univ. of Groningen (Netherlands))

    1993-01-01

    A method combining flow sorting and molecular cytogenetic techniques was used to identify an unknown marker chromosome in the bladder tumor cell line J82. The marker chromosome was isolated by dual parameter sorting after staining with Hoechst 33258 and chromomycin [Lambda]3. DNA amplification of 300 isolated chromosomes by polymerase chain reaction using the Alu-primer Bk33 and the Lines-primer LH5 was carried out. The DNA was labelled using Bio-11-dUTP and applied to human lymphocyte metaphase cells in a suppressive in situ hybridization procedure. Fluorescence was visible over chromosome 20 and over the distal one-half of 6p. Together the fluorescent regions accounted for only 60% of the marker length, indicating a possible duplication of chromosome 20 material. This was subsequently confirmed by applying bicolor in-situ hybridization using chromosome 6- and 20-specific DNA libraries to metaphase cells of the J82 culture.

  19. In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

    Microsoft Academic Search

    T. Kindaichi; I. Tsushima; Y. Ogasawara; M. Shimokawa; Noriatsu Ozaki; Hisashi Satoh; Satoshi Okabe

    2007-01-01

    We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organi- zation, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that \\

  20. Genetic mapping of the Adh locus in the repleta group of Drosophila by in situ hybridization.

    PubMed

    Labrador, M; Naveira, H; Fontdevila, A

    1990-01-01

    A biotinylated probe of the Adh (alcohol dehydrogenase) gene of Drosophila melanogaster was used for in situ hybridization on polytene chromosomes of D. mojavensis and D. buzzatii, two species of the repleta group of the genus Drosophila. Hybridization showed that the Adh gene maps at the G1a band of the third chromosome. This is in accordance with a previous result obtained through the use of interspecific hybrid asynapsis as a cytological marker and establishes the limits of the precision of this method. PMID:2185305

  1. In Situ Microscopic Observation of the Crystallization Process of Molecular Microparticles by Fluorescence Switching.

    PubMed

    Ye, Xin; Liu, Yang; Lv, Yun; Liu, Guangfeng; Zheng, Xiaoxin; Han, Quanxiang; Jackson, Kenneth A; Tao, Xutang

    2015-06-26

    To clearly understand the solid-state amorphous-to-crystalline transformation is a long-standing challenge because such crystallization occuring in confined environments is difficult to observe directly. We developed an in situ and real-time imaging procedure to record the interface evolution in a solid-state crystallization of molecular amorphous particles. The method, by employing a tetra-substituted ethene with novel morphology-dependent fluorescence, which can distinguish the interfaces between the crystalline and amorphous phase by fluorescence color, is a simple and practical method to probe the inner process of a molecular microparticle. The crystallization of amorphous microparticles in different cases was clearly recorded, where the perfect microparticles and those with defects demonstrate diverse destinies. The details disclosed in this observation will deepen the understanding for a series of solid-state crystallization that we know little about before. PMID:25974083

  2. Nano-SiO 2 in-situ hybrid polyurethane leather coating with enhanced breathability

    Microsoft Academic Search

    Yi Chen; Haojun Fan; Ruowang Liu; Jixin Yuan

    2010-01-01

    A novel in-situ nano hybrid technique combined with industrialized wet phase inversion coating-forming process was developed for the modification\\u000a of polyurethane (PU) leather coating with nano-SiO2. During the wet phase inversion process, nano-SiO2 particles were in-situ generated synchronously as polyurethane resin coagulated. Scanning electron microscope analysis indicated that when the SiO2 concentration was limited within 1.5 wt%, the size scale

  3. Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes.

    PubMed

    Wu, Wei; Zhang, Lei; Yao, Lun; Tan, Xiaoming; Liu, Xufeng; Lu, Xuefeng

    2015-01-01

    Construction of highly efficient microbial cell factories producing drop-in biofuel alkanes is severely limited due to the lack of a fast detection method against alkanes. Here we first developed a sensitive fluorescent biosensor for rapid and in situ monitoring of intracellular alkane synthesis. Using GFP as reporter, the biosensor could actively respond to the intracellular alkane products, especially for the mid- and long-chain alkanes synthesized in the recombinant Escherichia coli and give a concentration-dependent fluorescence response. Our results also suggested the feasibility of developing high-throughput strategies basing on the alkane biosensor device in E. coli, and thus will greatly facilitate the application of directed evolution strategies to further improve the alkane-producing microbial cell factories. PMID:26039923

  4. Fluorescence spectroscopy of collagen crosslinking: non-invasive and in situ evaluation of corneal stiffness

    NASA Astrophysics Data System (ADS)

    Franco, Walfre; Ortega-Martinez, Antonio; Zhu, Hong; Wang, Ruisheng; Kochevar, Irene E.

    2015-03-01

    Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

  5. Characterization of Chemoautotrophic Bacterial Symbionts in a Gutless Marine Worm (Oligochaeta, Annelida) by Phylogenetic 16S rRNA Sequence Analysis and In Situ Hybridization

    Microsoft Academic Search

    NICOLE DUBILIER; OLAV GIERE; DANIEL L. DISTEL; ANDCOLLEEN M. CAVANAUGH

    1995-01-01

    ThephylogeneticrelationshipsofchemoautotrophicendosymbiontsinthegutlessmarineoligochaeteInanid- rilus leukodermatus to chemoautotrophic ecto- and endosymbionts from other host phyla and to free-living bacteria were determined by comparative 16S rRNA sequence analysis. Fluorescent in situ hybridizations confirmed that the 16S rRNA sequence obtained from these worms originated from the symbionts. The symbiontsequenceisuniquetoI.leukodermatus.Inphylogenetictreesinferredbybothdistanceandparsimony methods, the oligochaete symbiont is peripherally associated with one of two clusters of chemoautotrophic symbionts that

  6. Effectiveness of fluorescence-based methods to detect in situ demineralization and remineralization on smooth surfaces.

    PubMed

    Moriyama, C M; Rodrigues, J A; Lussi, A; Diniz, M B

    2014-01-01

    This study aimed to evaluate the effectiveness of fluorescence-based methods (DIAGNOdent, LF; DIAGNOdent pen, LFpen, and VistaProof fluorescence camera, FC) in detecting demineralization and remineralization on smooth surfaces in situ. Ten volunteers wore acrylic palatal appliances, each containing 6 enamel blocks that were demineralized for 14 days by exposure to a 20% sucrose solution and 3 of them were remineralized for 7 days with fluoride dentifrice. Sixty enamel blocks were evaluated at baseline, after demineralization and 30 blocks after remineralization by two examiners using LF, LFpen and FC. They were submitted to surface microhardness (SMH) and cross-sectional microhardness analysis. The integrated loss of surface hardness (?KHN) was calculated. The intraclass correlation coefficient for interexaminer reproducibility ranged from 0.21 (FC) to 0.86 (LFpen). SMH, LF and LFpen values presented significant differences among the three phases. However, FC fluorescence values showed no significant differences between the demineralization and remineralization phases. Fluorescence values for baseline, demineralized and remineralized enamel were, respectively, 5.4 ± 1.0, 9.2 ± 2.2 and 7.0 ± 1.5 for LF; 10.5 ± 2.0, 15.0 ± 3.2 and 12.5 ± 2.9 for LFpen, and 1.0 ± 0.0, 1.0 ± 0.1 and 1.0 ± 0.1 for FC. SMH and ?KHN showed significant differences between demineralization and remineralization phases. There was a negative and significant correlation between SMH and LF and LFpen in the remineralization phase. In conclusion, LF and LFpen devices were effective in detecting demineralization and remineralization on smooth surfaces provoked in situ. PMID:24902775

  7. Classifying Fruit Fly Early Embryonic Developmental Stage Based on Embryo In situ Hybridization Images

    Microsoft Academic Search

    Hua Zhong; Wei-bang Chen; Chengcui Zhang

    2009-01-01

    In this paper, we present a supervised classification system for sorting Drosophila embryonic in situ hybridization (ISH) images according to their developmental stages. The proposed system first segments the embryo from an image and registers it for subsequent texture feature extraction. In order to extract the most distinguishing features for classifying developmental stages, we identify several areas of interest in

  8. RNA in situ hybridization makes sperm doesn't make sperm

    E-print Network

    RNA in situ hybridization makes sperm doesn't make sperm #12;spe-47 promoter spe-47 3' UTR structures in developing sperm: the Fibrous Body (FB) Membranous Organelle (MO) complexes. A mutation in spe regions of the protein. #12;Structural alignment of SPE-8 with crystal structures from human c-Abl protein

  9. Coexistence of t(15;17) and t(15;16;17) detected by fluorescence in situ hybridization in a patient with acute promyelocytic leukemia: A case report and literature review

    PubMed Central

    ZHANG, RUI; KIM, YOUNG-MI; WANG, XIANFU; LI, YAN; PANG, HUI; LEE, JI-YUN; LI, SHIBO

    2014-01-01

    Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid ?-receptor (RARA) gene at 17q21. The current study presents the case of a 54-year-old female with APL carrying the atypical PML/RARA fusion signal due to a novel complex variant translocation t(15;16;17)(q22;q24;q21), as well as the classical PML/RARA fusion signal. Subsequent array comparative genomic hybridization revealed somatic, cryptic deletions on 3p25.3, 8q23.1 and 12p13.2-p13.1, and a duplication on 8q11.2; however, no genetic material loss or gain was observed in the breakpoint regions of chromosomes 15, 16 or 17. To the best of our knowledge, this is the first report of the coexistence of two abnormal clones, one classical and one variant, presenting simultaneously in addition to cryptic chromosome segmental imbalances in an adult APL patient. PMID:25120648

  10. Cochlear whole mount in situ hybridization: identification of longitudinal and radial gradients

    Microsoft Academic Search

    Tiffany N Judice; Nathan C Nelson; Chase L Beisel; Duane C Delimont; Bernd Fritzsch; Kirk W Beisel

    2002-01-01

    The morphology of the organ of Corti has a radial asymmetry and also changes longitudinally from base to apex. Cellular localization of transcripts within the inner ear has relied primarily on the use of sectioned tissue with in situ hybridization. However, radial and longitudinal gradients of expression are not readily recognized using sectioned tissue owing to problems in visualization of

  11. Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes Leslie Vosshall

    E-print Network

    1 Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall DAY to the top with wash solution and place embryos on a nutator during the washing period. 1. Prepare fixed embryos according to attached protocol until 70% ethanol step. 2. Remove an aliquot of embryos sufficient

  12. ELF{trademark}: A new fluorogenic alkaline phosphatase substrate for nonisotopic mRNA in situ hybridization

    SciTech Connect

    Singer, V.; Paragas, V.; Zhang, Y.Z. [Molecular Probes, Inc., Eugene, OR (United States)

    1994-09-01

    Molecular Probes` researchers have developed a novel alkaline phosphatase substrate that gives rise to a bright yellow-green fluorescent precipitate at the site of enzymatic activity. We refer to this process as ELF, for Enzyme-Labeled Fluorescence. We found that the ELF substrate is an ideal tool for mRNA in situ hybridization. Not only are ELF signals much brighter than those generated using conventional fluorophores, resulting in film exposure times approximately one-fortieth as long as those needed for fluorescein-labeled probes or secondary detection reagents, but the ELF signal is also many-fold more photostable. The ELF signal develops in seconds to minutes, in contrast to radioactive detection, which requires days to weeks for adequate signal development. In addition, because the ELF precipitate has a Stokes shift of greater than 100 nm, we found that ELF signals can be easily distinguished from sample autofluorescence and from signals arising from other fluorescent probes or counterstrains. Also, unlike signals from chromogenic enzyme substrates, the fluorescent ELF signals are easily distinguished from pigmented tissues, even in zebrafish whole-mount embryos.

  13. Development of a fluorescent in situ method for visualization of enteric viruses.

    PubMed

    Rawsthorne, Helen; Phister, Trevor G; Jaykus, Lee-Ann

    2009-12-01

    Studying the interactions between enteric pathogens and their environment is important to improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate, and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV); the process was confirmed by using a sandwich-type approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots with an emission range of 635 to 675 nm (Q-Dots 655). The assay demonstrated a relative fluorescence of 528 +/- 48.1 and 112 +/- 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity, nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surfaces of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ. PMID:19854924

  14. Single-cell electroendocytosis on a micro chip using in situ fluorescence microscopy.

    PubMed

    Lin, Ran; Chang, Donald C; Lee, Yi-Kuen

    2011-12-01

    Electroendocytosis (EED), i.e. electric field-induced endocytosis, is a technique for bio-molecule and drug delivery to cells using a pulsed electric field lower than that applied in electroporation (EP). Different from EP in which nanometer-sized electropores appear on the plasma membrane lipid bilayer, EED induces cell membrane internalization and fission via endocytotic vesicles. In this study, we conduct comprehensive experimental study on the EED of HeLa cells using a micro chip and the corresponding endocytotic vesicles were visualized and investigated by using FM4-64 fluorescent dye and in situ fluorescence microscopy. The uptake of molecules by the EED of cells was characterized by average intracellular fluorescent intensity from a large number (>2,000) of single cells. The EED efficiency was determined as a function of three electric parameters (electric field strength, pulse duration, total electric treatment time). The EED efficiency as a function of electric field strength clearly shows biphasic characteristics at different experimental conditions. The EED experiments using cytoskeleton inhibitors illustrate unique mechanisms distinct from EP. This study provides a foundation for further on-chip study of the time-dependent mechanism of EED at the single-cell level. PMID:21800146

  15. A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization

    PubMed Central

    Dierschke, Tom; Mandáková, Terezie; Lysak, Martin A.; Mummenhoff, Klaus

    2009-01-01

    Background and Aims Incongruence between chloroplast and nuclear DNA phylogenies, and single additive nucleotide positions in internal transcribed spacer (ITS) sequences of polyploid Australian/New Zealand (NZ) Lepidium species have been used to suggest a bicontinental hybrid origin. This pattern was explained by two trans-oceanic dispersals of Lepidium species from California and Africa and subsequent hybridization followed by homogenization of the ribosomal DNA sequence either to the Californian (C-clade) or to the African ITS-type (A-clade) in two different ITS-lineages of Australian/NZ Lepidium polyploids. Methods Genomic in situ hybridization (GISH) was used to unravel the genomic origin of polyploid Australian/NZ Lepidium species. Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) probes was applied to test the purported ITS evolution, and to facilitate chromosome counting in high-numbered polyploids. Key Results In Australian/NZ A-clade Lepidium polyploids, GISH identified African and Australian/NZ C-clade species as putative ancestral genomes. Neither the African nor the Californian genome were detected in Australian/NZ C-clade species and the Californian genome was not detected in Australian/NZ A-clade species. Five of the eight polyploid species (from 7x to 11x) displayed a diploid-like set of rDNA loci. Even the undecaploid species Lepidium muelleriferdinandi (2n = 11x = 88) showed only one pair of each rDNA repeat. In A-clade allopolyploids, in situ rDNA localization combined with GISH corroborated the presence of the African ITS-type. Conclusions The nuclear genomes of African and Australian/NZ C-clade species were detected by GISH in allopolyploid Australian/NZ Lepidium species of the A-clade, supporting their hybrid origin. The presumed hybrid origin of Australian/NZ C-clade taxa could not be confirmed. Hence, it is assumed that Californian ancestral taxa experienced rapid radiation in Australia/NZ into extant C-clade polyploid taxa followed by hybridization with African species. As a result, A-clade allopolyploid Lepidium species share the Californian chloroplast type and the African ITS-type with the C-clade Australian/NZ polyploid and African diploid species, respectively. PMID:19589857

  16. Detection of canine parvovirus in naturally infected dogs with enteritis and myocarditis by in situ hybridization.

    PubMed

    Nho, W G; Sur, J H; Doster, A R; Kim, S B

    1997-07-01

    An improved method for the diagnosis of canine parvovirus using in situ hybridization in standard formalin-fixed, paraffin-embedded tissue sections was developed. A digoxigenin-labeled probe complementary to DNA sequences that code for the entire sequence of the capsid protein VP-1 and the middle part of the sequence of the capsid protein VP-2 was designed. Specific histologic localization of canine parvovirus-infected cells was demonstrated in small intestine, tonsil, lymph node, thymus, spleen, heart, liver, and kidney from dogs diagnosed at necropsy with canine parvovirus infection. The in situ hybridization accurately pinpointed the specific sites of viral infection. The detection of canine parvovirus in liver, kidney, and heart tissues together in the same pups could represent an enhanced virulence of this strain of canine parvovirus and suggests a broadened tissue tropism not seen before in Korean strains of canine parvovirus. PMID:9249164

  17. In situ synthesis of photocatalytically active hybrids consisting of bacterial nanocellulose and anatase nanoparticles.

    PubMed

    Wesarg, Falko; Schlott, Franziska; Grabow, Janet; Kurland, Heinz-Dieter; Heßler, Nadine; Kralisch, Dana; Müller, Frank A

    2012-09-18

    Bacterial nanocellulose (BNC) is an extraordinary biopolymer with a wide range of potential technical applications. The high specific surface area and the interconnected pore system of the nanofibrillar BNC network suggest applications as a carrier of catalysts. The present paper describes an in situ modification route for the preparation of a hybrid material consisting of BNC and photocatalytically active anatase (TiO(2)) nanoparticles (NPs). The influence of different NP concentrations on the BNC biosynthesis and the resulting supramolecular structure of the hybrids was investigated. It was found that the number of colony forming units (CFUs) and the consumption of glucose during biosynthesis remained unaffected compared to unmodified BNC. During the formation of the BNC network, the NPs were incorporated in the whole volume of the accruing hybrid. Their distribution within the hybrid material is affected by the anisotropic structure of BNC. The photocatalytic activity (PCA) of the BNC-TiO(2) hybrids was determined by methanol conversion (MC) under UV irradiation. These tests demonstrated that the NPs retained their PCA after incorporation into the BNC carrier structure. The PCA of the hybrid material depends on the amount of incorporated NPs. No alteration of the photocatalyst's efficiency was found during repeated PCA tests. In conclusion, the in situ integration of photocatalytically active NPs into BNC represents an attractive possibility to extend its fields of application to porous filtering media for drinking water purification and air cleaning. PMID:22925063

  18. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    PubMed Central

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  19. Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization

    Microsoft Academic Search

    M. I. Lima; M. E. N. Fonseca; R. Flores; E. W. Kitajima

    1994-01-01

    Summary In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigeninlabelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while

  20. Development of a combined portable x-ray fluorescence and Raman spectrometer for in situ analysis.

    PubMed

    Guerra, M; Longelin, S; Pessanha, S; Manso, M; Carvalho, M L

    2014-06-01

    In this work, we have built a portable X-ray fluorescence (XRF) spectrometer in a planar configuration coupled to a Raman head and a digital optical microscope, for in situ analysis. Several geometries for the XRF apparatus and digital microscope are possible in order to overcome spatial constraints and provide better measurement conditions. With this combined spectrometer, we are now able to perform XRF and Raman measurements in the same point without the need for sample collection, which can be crucial when dealing with cultural heritage objects, as well as forensic analysis. We show the capabilities of the spectrometer by measuring several standard reference materials, as well as other samples usually encountered in cultural heritage, geological, as well as biomedical studies. PMID:24985805

  1. Detection of Hepatitis B Virus DNA in Hepatocytes, Bile Duct Epithelium, and Vascular Elements by in situ Hybridization

    NASA Astrophysics Data System (ADS)

    Blum, Hubert E.; Stowring, Linda; Figus, Annalena; Montgomery, Carolyn K.; Haase, Ashley T.; Vyas, Girish N.

    1983-11-01

    A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefulness of in situ hybridization in the study of pathogenesis of HBV infection.

  2. Fig. 1a-d. In situ hybridization of Allizirn metaphase chromosomes. Yellow indicates areas hybridized with rDNA probe. Orange chromosomal material is hybridized and visualized with propidium iodide. Arrows with a number show the labelled chromosomes

    E-print Network

    Baker, Robert J.

    #12;#12;Fig. 1a-d. In situ hybridization of Allizirn metaphase chromosomes. Yellow indicates areas, hybrid 81215,ll metaphase cells each of six different individuals were evaluated. Of the 16 chromosomes hybridized with rDNA probe. Orange chromosomal material is hybridized and visualized with propidium iodide

  3. Assignment of the genes encoding the human chloride channels, CLCNKA and CLCNKB, to 1p36 and of CLCN3 to 4q32-q33 by in situ hybridization

    SciTech Connect

    Saito-Ohara, Fumiko; Uchida, Shinichi; Takeuchi, Yasuo [Tokyo Medical and Dental Univ. (Japan)] [and others] [Tokyo Medical and Dental Univ. (Japan); and others

    1996-09-01

    This report describes the localization of the genes encoding the human chloride channels, CLCNKA and CLCNKB, to human chromosome 1p36 and of CLCN3 to human chromosome 4q32-33 using fluorescence in situ hybridization. Mutations in these voltage-gated chloride channel genes have been implicated in various hereditary diseases. 18 refs., 1 fig.

  4. In situ measurement of UV fluence rate distribution by use of a micro fluorescent silica detector.

    PubMed

    Li, Mengkai; Qiang, Zhimin; Li, Tinggang; Bolton, James R; Liu, Changlong

    2011-04-01

    The fluence rate (FR) distribution in an ultraviolet (UV) reactor was determined experimentally in situ by use of a novel 360° micro fluorescent silica detector (MFSD). The UV response of the MFSD was systematically characterized, and the results indicated that this detector responded only to UV in the range from 210 to 280 nm. The nonlinearity was found to be less than 1% as the FR varied from 0.083 to 2110 ?W/cm2. The luminescent signal increased by 0.11% for every degree increase in temperature in the studied range of 0-55 °C. FR distribution tests were performed in different media (air or water) with the water transmittance at either 95% or 85% determined in a 1-cm path length. The FR distribution of the near-lamp region (e.g., radius<50 mm) was well determined with the nearest distance to the sleeve being less than 3 mm. Comparisons were made between the experimental data and the calculations by use of the UVCalc model. This work demonstrates that the MFSD is a novel technique that can provide in situ and real-time measurements of the FR distribution in a UV reactor. PMID:21388180

  5. Novel, in-situ Raman and fluorescence measurement techniques: Imaging using optical waveguides

    NASA Astrophysics Data System (ADS)

    Carter, Jerry Chance

    The following dissertation describes the development of methods for performing standoff and in- situ Raman and fluorescence spectroscopy for chemical imaging and non-imaging analytical applications. The use of Raman spectroscopy for the in- situ identification of crack cocaine and cocaine.HCl using a fiberoptic Raman probe and a portable Raman spectrograph has been demonstrated. We show that the Raman spectra of both forms of cocaine are easily distinguishable from common cutting agents and impurities such as benzocaine and lidocaine. We have also demonstrated the use of Raman spectroscopy for in-situ identification of drugs separated by thin layer chromatography. We have investigated the use of small, transportable, Raman systems for standoff Raman spectroscopy (e.g. <20 m). For this work, acousto-optical (AOTF) and liquid crystal tunable filters (LCTF) are being used both with, and in place of dispersive spectrographs and fixed filtering devices. In addition, we improved the flexibility of the system by the use of a modified holographic fiber-optic probe for light and image collection. A comparison of tunable filter technologies for standoff Raman imaging is discussed along with the merits of image transfer devices using small diameter image guides. A standoff Raman imaging system has been developed that utilizes a unique polymer collection mirror. The techniques used to produce these mirrors make it easy to design low f/# polymer mirrors. The performance of a low f/# polymer mirror system for standoff Raman chemical imaging has been demonstrated and evaluated. We have also demonstrated remote Raman hyperspectral imaging using a dimension-reduction, 2-dimensional (2-D) to 1-dimensional (1-D), fiber optic array. In these studies, a modified holographic fiber-optic probe was combined with the dimension-reduction fiber array for remote Raman imaging. The utility of this setup for standoff Raman imaging is demonstrated by monitoring the polymerization of dibromostyrene. To further demonstrate the utility of in- situ spectral imaging, we have shown that small diameter (350 ?m) image guides can be used for in-situ measurements of analyte transport in thin membranes. This has been applied to the measurement of H2O diffusion in Nafion™ membranes using the luminescent compound, [Ru(phen)2dppz] 2+, which is a H2O indicator.

  6. [Demonstration of the angiogenic cytokine vascular endothelial growth factor (VEGF) by non radioactive in situ hybridization].

    PubMed

    Strik, M W; Laus, C; Duchrow, M; Broll, R; Bruch, H P

    1997-12-01

    In tumor angiogenesis Vascular Endothelial Growth Factor (VEGF) has an important role due to its target cell specificity. It is expressed by the tumor and effects on a paracrine pathway. To increase the understanding of its regulation, it is necessary to identify those cells releasing VEGF. This can be done by in situ hybridization (ISH). In this paper we present a protocol for non-radioactive ISH for VEGF colonic tissue. With this protocol it is possible to perform hybridization within one day. PMID:9480624

  7. An in-situ fluorescence-based optical extensometry system for imaging mechanically loaded bone.

    PubMed

    Price, Christopher; Li, Wen; Novotny, John E; Wang, Liyun

    2010-06-01

    The application and quantification of well-controlled tissue strains is required for investigations into mechanisms of tissue adaptation within the musculoskeletal system. Although many commercial and custom extensometry systems exist for large biological samples, integrated loading/strain measurement for small samples is not as readily available. Advanced imaging modules such as laser scanning microscopy provide in situ, minimally invasive tools to probe cellular and molecular processes with high spatiotemporal resolution. Currently, a need exists to devise loading/strain measurement systems that can be integrated with such advanced imaging modules. We describe the development and validation of a fluorescence-based, optical extensometry system directly integrated within a confocal microscopy platform. This system allows in situ measurement of surface strain and is compatible with the direct imaging of cellular processes within small bone samples. This optical extensometry system can accurately and reproducibly measure physiologically relevant surface strains (200 to 3000 microstrain) in beams machined from various well-characterized materials, including bovine femoral cortex, and in intact murine tibia. This simple system provides a powerful tool to further our investigation of the relationships between mechanical loading, fluid and solute transport, and mechanosensation within the musculoskeletal system. PMID:20041487

  8. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    PubMed

    Müller-Lucks, Annika; Bock, Sinja; Wu, Binghua; Beitz, Eric

    2012-01-01

    Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality. PMID:22848743

  9. Site Specific Synthesis and in-situ Immobilization of Fluorescent Silver Nanoclusters on DNA Nanoscaffolds Using Tollens Reaction

    SciTech Connect

    Pal, Suchetan; Varghese, R.; Yan, Hao; Liu, Yan

    2011-01-01

    DNA strands with specific sequences and covalently attached sugar moieties were used for the site-specific incorporation of the sugar units on a DNA origami scaffold. This approach enabled the subsequent site-specific synthesis and in situ immobilization of fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment with the Tollens reagent.

  10. Chromosome pairing in allotetraploid hybrids of Festuca pratensis x Lolium perenne revealed by genomic in situ hybridization (GISH).

    PubMed

    Zwierzykowski, Zbigniew; Zwierzykowska, Elzbieta; Taciak, Magdalena; Jones, Neil; Kosmala, Arkadiusz; Krajewski, Pawe?

    2008-01-01

    Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift. PMID:18409011

  11. In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in very low numbers.

    PubMed

    Cho, Il-Hoon; Mauer, Lisa; Irudayaraj, Joseph

    2014-07-15

    Consumption of foods contaminated with pathogenic bacteria is a major public health concern. Foods contain microorganisms, the overwhelming majority of which are nonpathogenic, some are responsible for food spoilage, and some cause serious illness leading to death or a variety of diseases in humans. The key challenge in food safety is to rapidly screen foods to determine the presence of pathogens so that appropriate intervention protocols can be pursued. A simple fluorometric immunological method in combination with a magnetic concentration step was developed for rapid detection of target bacteria with high sensitivity and specificity in less than 2h without enumeration. The method constitutes performing an in-situ immunoassay on a magnetic bead through the formation of a sandwich complex of the target bacteria and the probe (detection antibody-denatured BSA labelled with fluorophores) followed by the release of fluorophores by means of enzymatic digestion with proteinase K. The limit of detection (LOD) was <5 CFU/mL of the tested pathogens (Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes) in buffer. When the pathogens were inoculated in foods (spinach, chicken, and milk), the LOD was under 5 CFU/mL for E. coli O157:H7, S. typhimurium and L. monocytogenes. Furthermore, the method was highly specific in detecting the target pathogens in a multiplex format. The developed in-situ fluorescent immunomagnetic sensor approach offers distinct advantages because it is rapid, highly sensitive, and easy to use and could therefore be potentially used as a pathogen screening tool. PMID:24583684

  12. Preparative in situ hybridization: selection of chromosome region-specific libraries on mitotic chromosomes.

    PubMed

    Hozier, J; Graham, R; Westfall, T; Siebert, P; Davis, L

    1994-02-01

    We have developed preparative in situ hybridization (Prep-ISH) of complex DNA populations to mitotic chromosomes as a means of generating chromosome region-specific DNA subpopulations. Prep-ISH is a combination of two cytogenetic techniques: in situ hybridization of DNA molecules to mitotic chromosomes and chromosome microdissection. Here, we present test cases demonstrating the feasibility of this approach on mouse and human genomes, using single nuclei, single chromosomes, or single chromosomal subregions to assess sensitivity, specificity, and representation of the Prep-ISH technique. Prep-ISH has a number of applications in studies of gene expression and genome organization, including efficient cytogenetic sorting of tissue-specific cDNAs and genomic DNA libraries. In addition, Prep-ISH is likely to dramatically reduce the number of candidate genes to aid in gene discovery efforts and to improve efficiency of developing transcription maps and YAC and cosmid contigs through defined cytogenetic regions. PMID:8188286

  13. Quantification of biotinylated RNA probes for in situ hybridization using chemiluminescence

    Microsoft Academic Search

    J. J. Bijl; E. Rieger; J. W. Oostveen; C. J. L. M. Meijer; J. M. M. Walboomers; C. B. M. Oudejans

    1994-01-01

    For reliable detection of mRNA by non-radioactive in situ hybridization, calibration and standardization of the individual steps involved are essential. We describe a method that allows determination of the size and integrity as well as quantification of biotinylated RNA probes in a single experiment. Serial dilutions of biotinylated RNA probes generated by promotor-mediated in vitro transcription were size-separated by gel

  14. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

    Microsoft Academic Search

    Mary E. Harper; Grady F. Saunders

    1981-01-01

    Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the 3H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on

  15. In situ and in vitro comparison of laser fluorescence with visual inspection in detecting occlusal caries lesions

    Microsoft Academic Search

    Andréia Bolzan de Paula; Juliana Álvares Duarte Bonini Campos; Michele Baffi Diniz; Josimeri Hebling; Jonas Almeida Rodrigues

    2011-01-01

    The aim of this study was to compare the in situ and in vitro performances of a laser fluorescence (LF) device (DIAGNOdent\\u000a 2095) with visual inspection for the detection of occlusal caries in permanent teeth. Sixty-four sites were selected, and\\u000a visual inspection and LF assessments were carried out, in vitro, three times by two independent examiners, with a 1-week interval

  16. Determination of gene expression patterns using in situ hybridization to Drosophila testes

    Microsoft Academic Search

    Ceri A Morris; Elizabeth Benson; Helen White-Cooper

    2009-01-01

    We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult

  17. L'utilisation des techniques d'hybridation in situ et de clonage de squences d'ADN pour

    E-print Network

    Paris-Sud XI, Université de

    L'utilisation des techniques d'hybridation in situ et de clonage de séquences d'ADN pour l des méthodes utilisées pour le clonage de séquences d'ADN dans des vecteurs comme des plasmides'hybridation in situ est basée sur le principe de la formation de duplexes ARN-ADN ou (1) Adresse actuelle :Department

  18. RNA in situ hybridization in whole mount embryos and cell histology adapted for marine elasmobranchs.

    PubMed

    Theodosiou, Nicole A

    2013-01-01

    Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs. Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development. While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression. Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries. PMID:23603660

  19. High-throughput physical mapping of chromosomes using automated in situ hybridization.

    PubMed

    George, Phillip; Sharakhova, Maria V; Sharakhov, Igor V

    2012-01-01

    Projects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila, allows the user to visualize more details on chromosomes than the regular squashing technique. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol. PMID:22782181

  20. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans.

    PubMed Central

    Jurtshuk, R J; Blick, M; Bresser, J; Fox, G E; Jurtshuk, P

    1992-01-01

    A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism. Images PMID:1381173

  1. Assignment of the phosducin (PDC) gene to human chromosome 1q25-1q32. 1 by somatic cell hybridization and in situ hybridization

    SciTech Connect

    Sparkes, R.S.; Kojis, T.; Klisak, I.; Heinzmann, C.; Bateman, J.B. (Jules Stein Eye Institute, Los Angeles, CA (United States)); Lee, R.H. (Univ. of California Developmental Neurology Lab., Sepulveda, CA (United States)); Shinohara, T. (National Eye Institute, Bethesda, MD (United States)); Craft, C.M. (Univ. of Texas Southwestern Medical Center, Dallas, TX (United States))

    1993-11-01

    Phosducin is a soluble photoreceptor phosphoprotein that probably modulates phototransduction in the retina and thus qualifies as a potential candidate gene for retinitis pigmentosa. Using both human/mouse somatic cell hybrids and in situ hybridization to human metaphase chromosomes, the authors have mapped this gene to chromosome 1q25-1q32.1. 18 refs., 2 figs.

  2. [Application of in situ micro energy dispersive X-ray fluorescence analysis in mineralogy].

    PubMed

    Yang, Hai; Ge, Liang-Quan; Gu, Yi; Zhang, Qing-Xian; Xiong, Sheng-Qing

    2013-11-01

    Thirteen rock samples were collected for studying the variation of element content in the mineral during the alteration process from Xinjiang, China. The IED-6000 in situ micro energy dispersive X-ray fluorescence developed by CDUT was applied to get chemical and physical data from minerals. The non-destructive spectrometer is based on a low-power Mo-anode X-ray tube and a Si-PIN peltier cooled X-ray detector. The unique design of the tube's probe allows very close coupling of polycapillary and makes the use of micro-area measurement feasible and efficient. The spectrometer can be integrated into any microscope for analysis. The long axis diameter of beam spot is about 110 microm. According to micro-EDXRF measurement, the tetrahedrite was corrected to pyrite, improving the efficiency and accuracy of the mineral identification. The feldspar of mineralized rock sample is rich in Cu and Zn which can be used as prospecting indicator elements. Element content of Cr, Mn and Co shows negative correlation with the degree of mineralization. PMID:24555398

  3. Sensitivity to DNA-damage induction and chromosomal alterations in mucosa cells from patients with and without cancer of the oropharynx detected by a combination of Comet assay and fluorescence in situ hybridization

    Microsoft Academic Search

    Ulrich A. Harréus; Norbert H. Kleinsasser; Sabina Zieger; Barbara Wallner; Max Reiter; Paul Schuller; Alexander Berghaus

    2004-01-01

    In addition to exogenous risk factors, the development of head and neck cancer is based on genetic alterations and individual sensitivity to mutagens. The DNA-damaging effect of xenobiotics and the location of chromosomal changes warrant further investigation. The aim of this study was to evaluate variance in structural genetic changes in human epithelia as target cells for head and neck

  4. The Fate of the Nucleolus during Mitosis: Comparative Analysis of Localization of Some Forms of Pre-rRNA by Fluorescent in Situ Hybridization in NIH/3T3 Mouse Fibroblasts

    PubMed Central

    Shishova, K.V.; Zharskaya, ?.?.; Zatsepina, ?.V.

    2011-01-01

    Nucleolus is the major structural domain of the cell nucleus, which in addition to proteins contains ribosomal RNA (rRNA) at different stages of maturation (or pre-rRNA). In mammals, the onset of mitosis is accompanied by the inhibition of rRNA synthesis, nucleolus disassembly, and the migration of pre-rRNA to the cytoplasm. However, the precise role of cytoplasmic pre-rRNA in mitosis remains unclear, and no comparative analysis of its different forms at consequent mitotic stages has thus far been performed. The focus of this research was the study of the localization of pre-rRNA in mitotic NIH/3T3 mouse fibroblasts by fluorescentin situhybridization (FISH) with probes to several regions of mouse primary 47S pre-rRNA transcripts and by confocal laser microscopy. The results reveal that all types of pre-rRNA appear in the cytoplasm at the beginning of mitosis, following the breakdown of the nucleolus and nuclear envelope. However, not all pre-rRNA are transported by chromosomes from maternal cells into daughter cells. At the end of mitosis, all types of pre-rRNA and 28S rRNA can be visualized in nucleolus-derived foci (NDF), structures containing many proteins of mature nucleoli the appearance of which indicates the commencement of nucleologenesis. However, early NDF are enriched in less processed pre-RNA, whereas late NDF contain predominantly 28S rRNA. Altogether, the results of this study strengthen the hypotheses that postulate that different forms of pre-rRNA may play various roles in mitosis, and that NDF can be involved in the maturation of pre-rRNA, remaining preserved in the cytoplasm of dividing cells. PMID:22649709

  5. Characterization of wheat-Thinopyrum partial amphiploids by meiotic analysis and genomic in situ hybridization.

    PubMed

    Fedak, G; Chen, Q; Conner, R L; Laroche, A; Petroski, R; Armstrong, K W

    2000-08-01

    A combination of genomic in situ hybridization (GISH) and meiotic pairing analysis of wheat-Thinopyrum partial amphiploids was employed to identify the genomic constitution and relationships between partial amphiploids derived from wheat and wheatgrass crosses. On the basis of similarities in the meiotic behavior and GISH patterns, the alien chromosomes of two of eight partial amphiploids, TAF46 and 'Otrastayuskaya 38', were judged to originate from Th. intermedium, whereas Th. ponticum was one of the parents of the other six partial amphiploids; PWM706, PWM206, PWM209, PWMIII, OK7211542, and Ag-wheat hybrid. Each of these partial amphiploids was found to contain a synthetic alien genome composed of different combinations of St-, J-, or Js-genome chromosomes. For relatedness of partial amphiploid lines, meiotic analysis of F1 hybrids and GISH results were generally complementary, but the latter offered greater precision in identifying constituent genomes. PMID:10984185

  6. Micromechanics of fracture in a ceramic\\/metal composite studied by in situ fluorescence spectroscopy I: Foundations and stress analysis

    Microsoft Academic Search

    Giuseppe Pezzotti; Wolfgang H. Müller

    2002-01-01

    in situ   microstress fields that develop during fracture in an exemplary ceramic\\/metal composite. Stress maps are obtained over a\\u000a relatively large area in the neighborhood of a propagating crack at both zero and critical loading conditions. Theoretical\\u000a and experimental issues of concern for measurement of local complex stress fields are dealt with. In particular, a review\\u000a of the fluorescence spectroscopy

  7. Detection of prolactin messenger RNA in rat anterior pituitary by in situ hybridization.

    PubMed Central

    Lloyd, R. V.; Landefeld, T. D.

    1986-01-01

    The effects of chronic diethylstilbestrol treatment on rat prolactin mRNA was analyzed by in situ hybridization histochemistry. Forty-day-old female rats were treated with 10 mg diethylstilbestrol in Silastic tubes for 3, 6, and 9 weeks. Estrogen treatment for 9 weeks increased pituitary wet weight (51.6 +/- 2.4 versus 7.9 +/- 0.31 mg for controls), serum prolactin (4155 +/- 571 versus 47.1 +/- 8.9 ng/ml for controls), and the percentage of immunoreactive prolactin cells (69% +/- 3% versus 34% +/- 2% for controls). In situ hybridization studies showed an increase in rat prolactin mRNA with increasing duration of estrogen treatment. After 9 weeks of estrogen treatment, there was a 2.3-fold increase in rat prolactin mRNA. 3H-cDNA was distributed diffusely throughout the anterior pituitary in both normal and hyperplastic pituitaries. There were no separate foci of adenomatous pituitary with increased labeling or with increased immunoreactive PRL cells. Although transplantable pituitary MtT/W15 tumors secreted very large amounts of PRL, compared with pituitaries from DES-treated rats, rat prolactin mRNA as evaluated by mean grain counts was considerably less in the MtT/W15 tumor than in DES-treated pituitary cells. These results show that in situ hybridization histochemistry can be used to detect changes in rat prolactin mRNA in tissue sections from the anterior pituitary with chronic estrogen treatment and that these pituitaries show a diffuse increase in immunoreactive prolactin cells and cellular prolactin mRNA, rather than distinct adenomatous areas within the glands. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3777138

  8. Using in situ hybridization and PFGE Southern hybridization to detect translocation breakpoints in a BOR/TRPS patient cell line

    SciTech Connect

    Gu, J.Z.; Sapru, M.; Smith, D. [Univ. of Houston, TX (United States)] [and others

    1994-09-01

    Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by ear malformations, cervical fistulae, hearing loss and renal abnormalities. We have integrated the Genethon YAC contig maps with additional markers in the chromosome 8q region genetically linked by a unique patient cell line. This cell line is from a patient who has both the branchio-oto-renal syndrome and tricho-rhino-phalangeal syndrome (TRPS). High resolution cytogenetics demonstrated a direct insertion of materials from 8q13.3q21.13 to 8q24.11. TRPS has been previously linked to deletions involving 8q24.11-q24.13. The rearrangement in this patient suggests that TRPS results from loss of gene function due to insertion at the 8q24.11 breakpoint and the possible location for the BOR gene is at either of the two breakpoints of 8q13.3 and 8q21.13. We have constructed cosmid contigs in 8q24.11. In situ hybridization with cosmids mapped to these locations as probes has helped to narrow down the breakpoints. Combinations of cosmids on either side or overlapping the 8q24.11 breakpoint show split signals on one chromosome 8q arm due to insertion of the materials from the proximal region. Cosmids mapped to the TRPS deletion region have been used to hybridize to pulsed field gel genomic blots of DNA from the patient cell line and detected rearranged genomic fragments. Both in situ hybridization and genomic PFGE Southern blot will be used to precisely locate the breakpoints.

  9. HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity.

    PubMed

    Hanna, Wedad M; Rüschoff, Josef; Bilous, Michael; Coudry, Renata A; Dowsett, Mitch; Osamura, Robert Y; Penault-Llorca, Frédérique; van de Vijver, Marc; Viale, Giuseppe

    2014-01-01

    Trastuzumab-containing therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely assigned using in situ hybridization to assess HER2 gene amplification, but interpretation of in situ hybridization results may be challenging in tumors with chromosome 17 polysomy or intratumoral genetic heterogeneity. Apparent chromosome 17 polysomy, defined by increased chromosome enumeration probe 17 (CEP17) signal number, is a common genetic aberration in breast cancer and represents an alternative mechanism for increasing HER2 copy number. Some studies have linked elevated CEP17 count ('polysomy') with adverse clinicopathologic features and HER2 overexpression, although there are numerous discrepancies in the literature. There is evidence that elevated CEP17 ('polysomy') count might account for trastuzumab response in tumors with normal HER2:CEP17 ratios. Nonetheless, recent studies establish that apparent 'polysomy' (CEP17 increase) is usually related to focal pericentromeric gains rather than true polysomy. Assigning HER2 status may also be complex where multiple cell subclones with distinct HER2 amplification characteristics coexist within the same tumor. Such genetic heterogeneity affects up to 40% of breast cancers when assessed according to a College of American Pathologists guideline, although other definitions have been proposed. Recent data have associated heterogeneity with unfavorable clinicopathologic variables and poor prognosis. Genetically heterogeneous tumors harboring HER2-amplified subclones have the potential to benefit from trastuzumab, but this has yet to be evaluated in clinical studies. In this review, we discuss the implications of apparent polysomy 17 and genetic heterogeneity for assigning HER2 status in clinical practice. Among our recommendations, we support the use of mean HER2 copy number rather than HER2:CEP17 ratio to define HER2 positivity in cases where coamplification of the centromere might mask HER2 amplification. We also highlight a need to harmonize in situ hybridization scoring methodology to support accurate HER2 status determination, particularly where there is evidence of heterogeneity. PMID:23807776

  10. Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization.

    PubMed

    Lima, M I; Fonseca, M E; Flores, R; Kitajima, E W

    1994-01-01

    In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigenin-labelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. This is in contrast with the subcellular localization of potato spindle tuber viroid and some other related viroids, which are mainly found in the nucleus. PMID:7998844

  11. Dual Labeling of Pseudomonas putida with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid

    PubMed Central

    Nancharaiah, Y. Venkata; Wattiau, Pierre; Wuertz, Stefan; Bathe, Stephan; Mohan, S. Venkata; Wilderer, Peter A.; Hausner, Martina

    2003-01-01

    We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies. PMID:12902279

  12. In situ fluorescence spectroscopy correlates ionomer degradation to reactive oxygen species generation in an operating fuel cell.

    PubMed

    Prabhakaran, Venkateshkumar; Arges, Christopher G; Ramani, Vijay

    2013-11-21

    The rate of generation of reactive oxygen species (ROS) within the polymer electrolyte membrane (PEM) of an operating proton exchange member fuel cell (PEMFC) was monitored using in situ fluorescence spectroscopy. A modified barrier layer was introduced between the PEM and the electrocatalyst layer to eliminate metal-dye interactions and fluorescence resonance energy transfer (FRET) effects during measurements. Standard fuel cell operating parameters (temperature, relative humidity, and electrode potential) were systematically varied to evaluate their influence on the rate of ROS generation during PEMFC operation. Independently, the macroscopic rate of PEM degradation was measured by monitoring the fluoride ion emission rate (FER) in the effluent stream at each operating condition. The ROS generation reaction rate constant (estimated from the in situ fluorescence experiments) correlated perfectly with the measured FER across all conditions, demonstrating unequivocally for the first time that a direct correlation exists between in situ ROS generation and PEM macroscopic degradation. The activation energy for ROS generation within the PEM was estimated to be 12.5 kJ mol(-1). PMID:24092495

  13. In situ IO Measurements in the Marine Boundary Layer Using Laser-Induced Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Heard, D. E.; Bale, C. S.; Bloss, W. J.; Commane, R.; Furneaux, K. L.; Ingham, T.; Whalley, L. K.

    2007-12-01

    The iodine monoxide (IO) radical plays an important role in the chemistry of the marine boundary layer (MBL). It is formed by the reaction of ozone with iodine atoms generated by the photolysis of I2 and photo-labile iodocarbons and at night from the reaction of NO3 with I2 and O3. IO is implicated in ozone destruction, DMS oxidation and new particle formation. IO has been measured previously using LP-DOAS, with absorption paths of several kilometres, and MAX-DOAS both of which are associated with significant spatial averaging over the halogen source regions. An in situ laser-induced fluorescence (LIF) technique has been developed to detect {IO} radicals in the MBL (Whalley et. al. (2007), J. Atmos. Chem. 58: 19 - 39) and has been employed in two separate instruments. In both field instruments, an all solid-state pulsed laser operating at 445 nm is used to excite IO in the A 2 ?3/2 \\ (?'=2) \\ ? \\ X 2 ?3/2 \\ (?"=0) electronic transition, with off-resonant fluorescence detected at 520.3 nm in the (2,5) band. The sensitivity of each instrument is determined by the generation of known concentrations of IO (between 5 and 100 pptv) from the photolysis of N2O at 185 nm followed by the subsequent reaction of oxygen atoms with CF3I. The first instrument was deployed in Roscoff, North-western France during August/September 2006 as part of the RHaMBLe (Reactive Halogens in the Marine Boundary Layer) campaign. The instrument limit of detection was 0.4 pptv for a 5 min integration time, with an uncertainty of 23%. Located on a small jetty, the instrument measured significant levels of IO on 11 days, with up to 29 pptv observed (10 second average). IO displayed a clear diurnal profile with a maximum at low tide, and lower concentrations observed on some nights. This is compared with LP-DOAS (University of Leeds) measurements also made at the site. The second instrument was deployed twice in 2007. The instrumental limit of detection was also found to be 0.4 pptv for a 5 minute integration time, with an overall uncertainty of 23%. The first deployment was aboard the RSS Discovery to measure open ocean IO over the Mauritanian upwelling during May/June 2007. The second deployment was to Mace Head, Western Ireland during August 2007. At Mace Head, IO was observed on all 9 measurement days, with a maximum of 28 pptv observed (1 min average) coinciding with low (spring) tide.

  14. Assignment of the human RT6 gene to 11q13 by PCR screening of somatic cell hybrids and in situ hybridization

    SciTech Connect

    Koch-Nolte, F.; Haag, F.; Kuehl, M.; Thiele, H.G.; Singh, S. (Univ. Hospital, Hamburg (Germany)); Van Heyningen, V. (Medical Research Council, Edinburgh (United Kingdom)); Hoovers, J. (Univ. of Amsterdam (Netherlands)); Grzeschik, K.H. (Univ. of Marburg (Germany))

    1993-11-01

    RT6 is a T cell membrane protein that has attracted interest because a defect in RT6 expression is associated with susceptibility to autoimmune type I diabetes in DP-BB rats and NOD mice. Using PCR screening of human/rodent somatic cell hybrids and fluorescence in situ hybridization, the authors have determined that the gene for the human RT6 homologue is located at 11q13, centromeric to the gene for tyrosinase (TYR, albino locus) and telomeric to that for fibroblast growth factor 4 (FGF4). The data suggest that the human RT6 gene constitutes a new linkage group with TYR and the gene for olfactory marker protein (OMP) on 11q, which has a counterpart in mouse chromosome 7. Thus, in the human, the RT6 locus is dissociated from the hemoglobin [beta] chain locus (HBB) and its neighboring conserved linkage group at 11q15, in contrast to the mouse, in which RT6 shows a tighter linkage to Hbb than to Tyr. The results support the conclusion that there has been considerable intrachromosomal reshuffling of linked genes since the divergence of primates and rodents. 9 refs., 1 fig.

  15. A New In Situ Method of Determining Relative Abundances and Charge States of Implanted Transition Metals in Individual Grains Using Synchrotron X-Ray Fluorescence

    SciTech Connect

    Kitts, K.; Sutton, S.; Newville, M. (NIU); (UofC)

    2007-03-06

    We report on a new in situ method of determining relative abundances and charge states of implanted transition metals in individual grains using synchrotron X-ray fluorescence. In order to determine in situ the relative abundances and charge states of the transition metals in implanted solar wind in individual lunar plagioclase grains, we have developed a new microbeam x-ray fluorescence method using the synchrotron x-ray microprobe at the Advanced Photon Source (GSECARS sector 13) at Argonne National Laboratory.

  16. Atmospheric NO2: in situ laser-induced fluorescence detection atparts per trillion mixing ratios

    SciTech Connect

    Thornton, Joel A.; Wooldridge, Paul J.; Cohen, Ronald C.

    1999-08-04

    NO2 affects the abundance of O3 and OH throughout theatmosphere, exerting influence over stratospheric ozone, on the oxidativecapacity of the atmosphere, on the greenhouse effect, on production oforganic and inorganic aerosol, and on production of photochemical smog.In the troposphere, NO2 mixing ratios vary from 100s of parts per billion(ppb) in cities to below 10 parts per trillion (ppt) in remote locations.We describe a laser-induced fluorescence instrument designed for accurate(+-5 percent, 1s), continuous, autonomous, in situ observations of NO2with the sensitivity (15 ppt/10 secat S/N=2) and portability necessary tostudy NO2 anywhere in the troposphere. The technique is advantageousbecause it is spectroscopically specific and direct in that it does notrequire conversion of NO2 into another species (e.g., NO) prior todetection eliminating a class of potential interferences. Performance ofthe instrument is illustrated with 15 weeks (July-Oct 1998) ofobservations at the University of California, Blodgett Forest fieldstation located in the foothills of the Sierra Nevada and 4 weeks (June15-July 15, 1999) in Nashville, TN during the Southern Oxidants Study.Ambient concentrations of NO2 at Blodgett Forest varied from below 50 pptto 4000 ppt and NO2 ranged from 5 to 50 percent of total reactivenitrogen, while in Nashville, TN concentrations ranged from 1 ppb to 75ppb.

  17. Mapping of the calcium-sensing receptor gene (CASR) to human Chromosome 3q13.3-21 by fluorescence in situ hybridization, and localization to rat Chromosome 11 and mouse Chromosome 16

    Microsoft Academic Search

    N. Janicic; E. Soliman; Z. Pausova; M. F. Seldin; M. Rivière; J. Szpirer; C. Szpirer; G. N. Hendy

    1995-01-01

    The calcium-sensing receptor (CASR), a member of the G-protein coupled receptor family, is expressed in both parathyroid and kidney, and aids these organs in sensing extracellular calcium levels. Inactivating mutations in the CASR gene have been described in familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the CASR gene have been described in autosomal dominant hypoparathyroidism

  18. Tracking the composition and dominant components of the microbial community via polymerase chain reaction-denaturing gradient gel electrophoresis and fluorescence in situ hybridization during vermiconversion for liquid-state excess sludge stabilization.

    PubMed

    Xu, Ting; Xing, Meiyan; Yang, Jian; Lv, Baoyi; Duan, Ting; Nie, Jing

    2014-09-01

    To quantitatively explore the microbial community modified by earthworms, a vermifilter (VF, with earthworms) and a conventional biofilter (BF, without earthworms) were continuously operated to stabilize excess sludge. The results demonstrated a positive role imposed by earthworms on compositions and dominant components of microbial community in the VF. For one thing, the phyla Actinobacteria and Acidobacteria were only detected in the VF, which might explain for the higher Shannon index of bacteria in the VF (H = 2.58) than that in the BF (H = 1.99). For another, the total proportion of dominant bacteria in the VF increased by 23% compared to the BF. Moreover, quantification analysis explicitly noted that the dominant bacteria in VF were ?-proteobacteria (27 ± 2%) and ?-proteobacteria (24 ± 1%) while that in BF was Bacteroidetes (21 ± 1%). In conclusion, stimulated by earthworms, a unique microbial community developed in the VF, thus improving the stabilization of excess sludge. PMID:24971951

  19. Direct detection of cytomegalovirus from bronchoalveolar lavage samples by using a rapid in situ DNA hybridization assay.

    PubMed

    Gleaves, C A; Myerson, D; Bowden, R A; Hackman, R C; Meyers, J D

    1989-11-01

    An in situ DNA hybridization assay was compared with centrifugation culture for rapid detection of cytomegalovirus (CMV) from bronchoalveolar lavage (BAL) samples. Eighty BAL samples were inoculated into both centrifugation culture and standard culture. Cytospin preparations of the BAL samples were studied in a 75-min in situ DNA hybridization assay using the PathoGene CMV kit (Enzo Biochem, Inc., New York, N.Y.). Of the 80 samples, 39 (49%) were positive for CMV; 37 of 39 (95%) were positive by centrifugation culture, 34 of 39 (87%) were positive in standard culture, 24 of 39 (62%) were positive by in situ hybridization, and 20 of 39 (56%) were positive by histologic and/or immunofluorescence techniques. The in situ hybridization assay detected 23 of the 37 samples positive in centrifugation culture, for a sensitivity of 62% and a specificity of 98%. We conclude that the in situ hybridization assay is a specific and more rapid test than centrifugation culture and standard culture for diagnosis of CMV pulmonary infection. For the clinical laboratory, however, current hybridization methods are not sufficiently sensitive to replace centrifugation culture for detection of CMV in BAL specimens. PMID:2553766

  20. Mercury Vapor Release from Broken Compact Fluorescent Lamps and In Situ Capture by New Nanomaterial Sorbents

    PubMed Central

    2008-01-01

    The projected increase in the use of compact fluorescent lamps (CFLs) motivates the development of methods to manage consumer exposure to mercury and its environmental release at the end of lamp life. This work characterizes the time-resolved release of mercury vapor from broken CFLs and from underlying substrates after removal of glass fragments to simulate cleanup. In new lamps, mercury vapor is released gradually in amounts that reach 1.3 mg or 30% of the total lamp inventory after four days. Similar time profiles but smaller amounts are released from spent lamps or from underlying substrates. Nanoscale formulations of S, Se, Cu, Ni, Zn, Ag, and WS2 are evaluated for capture of Hg vapor under these conditions and compared to conventional microscale formulations. Adsorption capacities range over 7 orders of magnitude, from 0.005 (Zn micropowder) to 188?000 ?g/g (unstabilized nano-Se), depending on sorbent chemistry and particle size. Nanosynthesis offers clear advantages for most sorbent chemistries. Unstabilized nano-selenium in two forms (dry powder and impregnated cloth) was successfully used in a proof-of-principle test for the in situ, real-time suppression of Hg vapor escape following CFL fracture. PMID:18754507

  1. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

    SciTech Connect

    Edelhoff, S.; Disteche, C.M. [Univ. of Washington School of Medicine, Seattle, WA (United States)] [Univ. of Washington School of Medicine, Seattle, WA (United States); Lai, C. [Scripps Research Inst., LaJolla, CA (United States)] [Scripps Research Inst., LaJolla, CA (United States)

    1995-01-01

    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  2. Large-scale expression screening by automated whole-mount in situ hybridization.

    PubMed

    Quiring, Rebecca; Wittbrodt, Beate; Henrich, Thorsten; Ramialison, Mirana; Burgtorf, Carola; Lehrach, Hans; Wittbrodt, Joachim

    2004-07-01

    Gene expression profiling is an important component of functional genomics. We present a time and cost efficient high-throughput whole-mount in situ technique to perform a large-scale gene expression analysis in medaka fish (Oryzias latipes) embryos. Medaka is a model system ideally suited for the study of molecular genetics of vertebrate development. Random cDNA clones from an arrayed stage 20 medaka plasmid library were analyzed by whole-mount in situ hybridization on embryos of three representative stages of medaka development. cDNA inserts were colony PCR amplified in a 384-format. The PCR products were used to generate over 2000 antisense RNA digoxigenin probes in a high-throughput process. Whole-mount in situ hybridization was carried out in a robot and a broad range of expression patterns was observed. Partial cDNA sequences and expression patterns were documented with BLAST results, cluster analysis, images and descriptions, respectively; collectively this information was entered into a web-based database, "MEPD" (http://www.embl-heidelberg.de/mepd/), that is publicly accessible. PMID:15210201

  3. Assignment of the GLG1 gene for MG-160, a fibroblast growth factor and E-selectin binding membrane sialoglycoprotein of the Golgi apparatus, to chromosome 16q22-q23 by fluorescence in situ hybridization

    SciTech Connect

    Mourelatos, Z.; Gonatas, J.O.; Gonatas, N.K. [Univ. of Pennsylvania, Pittsburgh, PA (United States)] [and others] [Univ. of Pennsylvania, Pittsburgh, PA (United States); and others

    1995-07-20

    MG-160, originally isolated from rat brain, is a conserved membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of virtually all cells. MG-160 appears in the developing chicken embryo as early as 12 h and persists in most cells throughout embryonic development and in the adult animal. The early appearance and ubiquitous presence of MG-160 suggest that the protein plays an important role in the biogenesis and function of the Golgi apparatus. The rat cDNA of MG-160 encodes a polypeptide of 1171 amino acids (AA) with a short cytoplasmic carboxy-terminus of 13 aa, a single transmembrane domain, and a large cysteine-rich intraluminal domain. MG-160 binds basic fibroblast growth factor (bFGF) and displays 90% identity of amino acids with CFR, a cystein-rich receptor for several fibroblast growth factors (FGFs), that was isolated form chicken embryos. However, unlike the fibroblast growth factor receptors (FGFRs) involved in signal transduction, MG-160 and its chicken homologue CFR do not display the extracellular IG-like repeats and the intracytoplasmic tyrosin kinase domains. In addition to CFR, MG-160 is highly homologous to ESL-1, a ligand for E-selectin. 16 refs., 1 fig.

  4. Chromosomal localization of the human ECT2 proto-oncogene to 3q26.1 {yields} q26.2 by somatic cell analyses and fluorescence in situ hybridization

    SciTech Connect

    Takai, Setsuo; Yamada, Kiyomi [International Medical Center of Japan, Tokyo (Japan)] [International Medical Center of Japan, Tokyo (Japan); Long, J.E. [National Cancer Institute, Bethesda, MD (United States)] [and others] [National Cancer Institute, Bethesda, MD (United States); and others

    1995-05-01

    The mouse ect2 oncogene was identified in a search of mitogenic signal transducers using a novel expression cloning strategy. In this system, the introduction of an appropriate expression-cDNA library prepared from the mRNA of one cell type into another can result in the morphological transformation of the recipient cells. The predicted Ect2 protein has sequence similarity within a central core of 255 amino acids to the products of the breakpoint-cluster gene, bcr, the yeast cell cycle gene, CDC24, the dbl and vav oncogenes, and the RasGrf gene. Baculovirus-expressed Ect2 protein shows specific binding to Rho and Rac proteins, indicating that Ect2 is a new member of transforming proteins that can interact with Rho-like proteins of the Ras superfamily. The ect2 foci exhibit an unusual stellate morphology and are capable of forming tumors when subcutaneously inoculated in nude mice. These results indicate that the ect2 gene acts as an oncogene. To investigate the involvement of the ect2 in human tumors, the authors isolated the human homolog, ECT2, of mouse ect2 and determined its chromosomal localization. 13 refs., 1 fig.

  5. Oligomeric interface modifiers in hybrid polymer solar cell prototypes investigated by fluorescence voltage spectroscopy.

    PubMed

    Reeja-Jayan, B; Koen, Katherine A; Ono, Robert J; Vanden Bout, David A; Bielawski, Christopher W; Manthiram, Arumugam

    2015-04-28

    Carboxylated oligothiophenes were evaluated as interfacial modifiers between the organic poly(3-hexylthiophene) (P3HT) and inorganic TiO2 layers in bilayer hybrid polymer solar cells. Carboxylated oligothiophenes can be isolated using conventional purification techniques resulting in pure, monodisperse molecules with 100% carboxylation. Device prototypes using carboxylated oligothiophenes as interfacial modifiers showed improved performance in the open-circuit voltage and fill factor over devices using unmodified oligothiophenes as interfacial modifiers. X-ray photoelectron spectroscopy (XPS) studies supported the idea that interface layer adhesion was improved by functionalizing oligothiophenes with a carboxyl moiety. Wide-field fluorescence images revealed that devices made using carboxylated oligothiophenes had fewer aggregates in the P3HT layers atop the modified TiO2 surface. Hysteresis seen in the fluorescence intensity as a function of applied bias, obtained from In-Device Fluorescence Voltage Spectroscopy (ID-FVS), was found to be a diagnostic criterion of the quality of the hybrid interface modification. The best interfaces were found using oligothiophenes functionalized with carboxylates, which created smooth layers on TiO2, and showed no hysteresis, suggesting elimination of interfacial charge traps. However, this hysteresis could be re-introduced by increasing the scan rate of the applied bias, suggesting that smooth P3HT layers created by carboxylated oligothiophene interface modifiers were necessary but not sufficient for sustaining improved photovoltaic properties especially during long-term device operation. PMID:25804286

  6. A submicroscopic unbalanced subtelomeric translocation t(2p;10q) identified by fluorescence in situ hybridization: fetus with increased nuchal translucency and normal standard karyotype with later growth and developmental delay, rhombencephalosynapsis (RES).

    PubMed

    Lespinasse, J; Testard, H; Nugues, F; Till, M; Cordier, M P; Althuser, M; Amblard, F; Fert-Ferrer, S; Durand, C; Dalmon, F; Pourcel, C; Jouk, P S

    2004-01-01

    Reaching an accurate diagnosis in children with mental retardation associated or not with dysmorphic signs is important to make precise diagnosis of a syndrome and for genetic counseling. A female case with severe growth and development delay, dysmorphic features and feeding disorder is presented. Antenataly, the fetus was observed to have increased nuchal translucency and a slight hypoplastic cerebellum. A standard karyotype was normal. RES and a submicroscopic unbalanced subtelomeric translocation t(2p; 10q) were demonstrated after birth. We show that within the framework of a collaborative approach, a concerted research of submicroscopic subtelomeric rearrangements should be performed in case of mental retardation associated with facial dysmorphic features, and when other etiologies or non-genetic factors (iatrogenic, toxic, infectious, metabolic...) have been ruled out. PMID:15581840

  7. Localization of the genes encoding the melanocortin-2 (Adrenocorticotropic hormone) and melanocortin-3 receptors to chromosomes 18p11. 2 and 20q13. 2-q13. 3 by fluorescence in situ hybridization

    SciTech Connect

    Gantz, I.; Tashiro, Takao; Konda, Yoshitaka; Shimoto, Yoshimasa; Miwa, Hiroto; Munzert, G.; Barcroft, C.; Glover, T.; Yamada, Tadataka (Univ. of Michigan Medical Center, Ann Arbor, MI (United States))

    1993-10-01

    Adrenocorticotropic hormone (ACTH) and [alpha]-, [beta]-, and [gamma]-melanocyte-stimulating hormone (MSH) are products of propiomelanocortin post-translational processing. These compounds are collectively labeled as melanocortins (MC). Aside from their established effects on the regulation of the adrenal cortex (ACTH) and melanocytes ([alpha]-MSH), the melanocortins have been implicated in a broad array of physiological events. Melanocortins mediate their effects through cell membrane receptors belonging to the superfamily of seven transmembrane G-protein-linked receptors. Using the technique of polymerase chain reaction with primers based on conserved areas of the seven transmembrane G-protein-linked receptor family, the authors recently isolated an [open quotes]orphan[close quotes] subfamily of this receptor group. Within the past year, two of these receptors were identified as specific for [alpha]-MSH (MC1) and ACTH (MC2). They have recently described a third melanocortin receptor (MC3) that appears to recognize the core heptapeptide sequence of melanocortins with equal potency and efficacy and identified its presence in the brain, placenta, and gut. Using the FISH technique, they localized the ACTH and the melanocortin-3 receptors to chromosome loci 18p11.2 and 20q12.3-q13.2, respectively. 19 refs., 1 fig.

  8. Turn-on fluorescent dopamine sensing based on in situ formation of visible light emitting polydopamine nanoparticles.

    PubMed

    Yildirim, Adem; Bayindir, Mehmet

    2014-06-01

    Dopamine is the principle biomarker for diseases such as schizophrenia, Huntington's, and Parkinson's, and the need is urgent for rapid and sensitive detection methods for diagnosis and monitoring of such diseases. In this Article, we report a turn-on fluorescent method for rapid dopamine sensing which is based on monitoring the intrinsic fluorescence of in situ synthesized polydopamine nanoparticles. The assay uses only a common base and an acid, NaOH and HCl to initiate and stop the polymerization reaction, respectively, which makes the assay extremely simple and low cost. First, we studied the in situ optical properties of polydopamine nanoparticles, for the first time, which formed under different alkaline conditions in order to determine optimum experimental parameters. Then, under optimized conditions we demonstrated high sensitivity (40 nM) and excellent selectivity of the assay. With its good analytical figures of merit, the described method is very promising for detection of dopamine related diseases. PMID:24803112

  9. In situ amplification signaling-based autonomous aptameric machine for the sensitive fluorescence detection of cocaine.

    PubMed

    Xie, Su-Jin; Zhou, Hui; Liu, Dengyou; Shen, Guo-Li; Yu, Ruqin; Wu, Zai-Sheng

    2013-06-15

    The development of autonomous DNA machines and their use for specific sensing purpose have recently attracted considerable research attention. In existing autonomous machines, the target recognition process and signal transduction are separated from each other. This results in misunderstanding of the operation behavior, and the assay capability is compromised when serving as a sensing tool. In this communication, the integrated signal transduction-based autonomous aptameric machine, in which the recognition element and signal reporters are integrated into a DNA strand, is developed. This new machine can execute the in situ amplification of target binding-induced signal. The authentic operation behavior of autonomous DNA machine is discovered: the machine's products directly hybridize to the "track" rather than to the signaling probes. Along this line, the machine is employed to detect the cocaine in a more straightforward fashion, and improved assay characteristics (for example, the dynamic response range is widened by more than 500-fold) are achieved. Our efforts not only clarify the concept described in traditional autonomous DNA machines but also have made technological advancements that are expected to be especially valuable in designing nucleic acid-based machines employed in basic research and medical diagnosis. PMID:23399471

  10. Site-selective in situ growth of fluorescent polymer-antibody conjugates with enhanced antigen detection by signal amplification.

    PubMed

    Zhang, Libin; Zhao, Wenguo; Liu, Xinyu; Wang, Guilin; Wang, Yang; Li, Dong; Xie, Liangzhi; Gao, Yan; Deng, Haiteng; Gao, Weiping

    2015-09-01

    This paper reports a new and general in situ methodology to grow fluorescent polymer conjugates from the interchain disulfide bridging sites of a monoclonal antibody. Atom transfer radical polymerization (ATRP) initiators were attached to a monoclonal antibody at its interchain disulfide bridging sites by disulfide re-bridging to yield a macroinitiator. Subsequent in situ ATRP of PEG-like monomers with dye-functionalized monomers from the macroinitiator formed antibody-polymer-dye conjugates with site-selectivity and tunable dye-to-antibody ratios. Notably, these conjugates can amplify antigen detection signal by reducing label-density dependent fluorescence quenching and by increasing dye-to-antibody ratios. The method developed may be applicable to a variety of antibodies, dyes and drugs to create a number of antibody-polymer-dye/drug conjugates for advanced diagnosis and therapy of diseases. PMID:26102329

  11. Making a Hybrid Microfluidic Platform Compatible for In Situ Imaging by Vacuum-Based Techniques

    SciTech Connect

    Yang, Li; Yu, Xiao-Ying; Zhu, Zihua; Thevuthasan, Suntharampillai; Cowin, James P.

    2011-10-26

    A self-contained microfluidic-based device was designed and fabricated for in situ imaging of aqueous surfaces using vacuum techniques. The device is a hybrid between a microfluidic PDMS block and external accessories, all portable on a small platform (10 cm-8 cm). The key feature is that a small aperture with a diameter of 2-3 micrometers is opened to the vacuum, which serves as a detection window for in situ imaging of aqueous surfaces. Vacuum compatibility and temperature drop due to water vaporization are the two most important challenges in this invention. Theoretical calculations and fabrication strategies are presented from multiple design aspects. In addition, results from the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of aqueous surfaces are presented.

  12. Detection of white spot syndrome virus (WSSV) of Penaeus chinensis by in situ hybridization

    NASA Astrophysics Data System (ADS)

    Zhan, Wen-Bin; Wang, Yuan-Hong; Zhang, Zhi-Dong; Hideo, Fukuda

    2000-09-01

    White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV-infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

  13. Limitations of in situ hybridization with total genomic DNA in routine screening for alien introgressions in wheat.

    PubMed

    Lukaszewski, A J; Lapinski, B; Rybka, K

    2005-01-01

    In situ hybridization with total genomic DNA (GISH) has become a powerful tool in characterization of alien introgressions in wheat. With recent simplification it can now be used in large scale screening for new chromosome constructs. Its level of resolution in routine applications was tested on sets of recombined wheat-rye chromosomes with genetically determined positions of the translocation breakpoints. The resolution level of GISH visualized by an enzymatic color reaction was much lower than that of GISH with fluorescent probes but both techniques failed to reveal the presence of some distally located breakpoints. The limits of resolution for the two methods were at least 9.8 and 3.5 cM of the relative genetic lengths of chromosome arms, respectively, in configurations with proximal rye and terminal wheat segments when rye DNA was used as a probe. When wheat DNA was used as a probe, a terminal wheat segment estimated to be ca. 1.6 cM in length could not be visualized. An example of induced recombination between a chromosome of Agropyron elongatum and wheat illustrates that these resolution limits of GISH may hamper isolation of critical translocation breakpoints in a chromosome engineering effort. PMID:15753599

  14. In Situ Airborne Measurement of Formaldehyde with a New Laser Induced Fluorescence Instrument

    NASA Astrophysics Data System (ADS)

    Arkinson, H.; Hanisco, T. F.; Cazorla, M.; Fried, A.; Walega, J.

    2012-12-01

    Formaldehyde (HCHO) is a highly reactive and ubiquitous compound in the atmosphere that originates from primary emissions and secondary formation by photochemical oxidation of volatile organic compounds. HCHO is an important precursor to the formation of ozone and an ideal tracer for the transport of boundary layer pollutants to higher altitudes. In situ measurements of HCHO are needed to improve understanding of convective transport mechanisms and the effects of lofted pollutants on ozone production and cloud microphysics in the upper troposphere. The Deep Convective Clouds and Chemistry Project (DC3) field campaign addressed the effects of deep, midlatitude continental convective clouds on the upper troposphere by examining vertical transport of fresh emissions and water aloft and by characterizing subsequent changes in composition and chemistry. Observations targeting convective storms were conducted over Colorado, Alabama, and Texas and Oklahoma. We present measurements of the In Situ Airborne Formaldehyde instrument (ISAF), which uses laser induced fluorescence to achieve the high sensitivity and fast time response required to detect low concentrations in the upper troposphere and capture the fine structure characteristic of convective storm outflow. Preliminary results from DC3 indicate that the ISAF is able to resolve concentrations ranging from under 35 ppt to over 35 ppb, spanning three orders of magnitude, in less than a few minutes. Frequent, abrupt changes in HCHO captured by the ISAF are corroborated by similar patterns observed by simultaneous trace gas and aerosol measurements. Primary HCHO emissions are apparent in cases when the DC-8 flew over combustion sources or biomass burning, and secondary HCHO formation is suggested by observations of enhanced HCHO concurrent with other elevated hydrocarbons. Vertical transport of HCHO is indicated by measurements of over 6 ppb from outflow in the upper troposphere. The DC-8 payload also included the Difference Frequency Generation Absorption Spectrometer instrument (DFGAS), which detects HCHO via infrared absorption. Preliminary data suggests the ISAF and DFGAS HCHO measurements typically agree to within 15%. Initial results from DC3 illustrate the efficacy of the novel ISAF instrument for detection of HCHO to elucidate convective transport of boundary layer pollutants and subsequent effects in the upper troposphere.

  15. Bovine Adenovirus Type 10 Identified in Fatal Cases of Adenovirus-Associated Enteric Disease in Cattle by In Situ Hybridization

    Microsoft Academic Search

    JOAN A. SMYTH; MARIA BENKO; DEBORAH A. MOFFETT; ANDBALAZS HARRACH

    1996-01-01

    A severe, naturally occurring enteric disease of cattle in which adenovirus inclusions are present in the intestinal vascular endothelium has been recognized in several countries; three different adenovirus serotypes have been isolated from affected animals. An in situ hybridization technique for the detection of bovine adenoviral DNA was developed and applied to tissue from 13 cattle in Northern Ireland diagnosed

  16. X-Ray Diffraction and Fluorescence Measurements for In-situ Planetary Instruments

    NASA Astrophysics Data System (ADS)

    Hansford, G.; Hill, K. S.; Vernon, D.; Ambrosi, R. M.; Bridges, J.; Hutchinson, I.

    2010-12-01

    The X-Ray Diffraction (XRD) instruments are core components of the forthcoming NASA Mars Science Laboratory (MSL) and ESA/NASA EXOMARS missions and will provide the first demonstrations of an XRF/XRD instrument’s capabilities in-situ on an extraterrestrial planetary surface. The University of Leicester team is part of the Italy-UK collaboration that is responsible for building the ExoMars X-Ray Diffraction instrument, Mars XRD. The ExoMars X-ray diffraction instrument incorporates an 55-Fe radioisotope source and three fixed-position CCDs to simultaneously acquire an X-Ray fluorescence spectrum and a diffraction pattern providing a measurement of both elemental and mineralogical composition [1]. The CCDs cover an angular range from 6 to 65-deg enabling the analysis of silicates, from clays, or other phyllosilicates characterised by varying d-spacings, to oxides, and carbonates or evaporites. The identification of hydrous minerals may help identify past Martian hydrothermal systems capable of preserving traces of life. Here we present some initial findings from XRF and XRD tests carried out at the University of Leicester using an 55-Fe source and X-ray sensitive CCD [1]. The XRD/XRD test system consists of a single CCD on a motorised arm, an 55-Fe X-ray source, source collimator and a sample table which approximately replicate the reflection geometry of the XRD instrument. It was used to test geological reference standard materials and Martian analogues. Incidence angle and CCD angles on both the diffraction and fluorescence results were evaluated. A key area of interest is the effect of sample roughness on the XRD/XRF results. We present results from testing pressed powder pellet samples of varying surface roughness, and a comparison with model results [2]. So far we have found that increased roughness causes a reduced intensity at lower take-off angles. Several methods for measuring surface roughness of the samples have been used including an Alicona Infinite Focus microscope. [1] Marinangeli et al. (2007) LPSC #1322 [2] Hansford et al. (2010) EGU General Assembly 2010

  17. Evaluation of Bacterivory of Rotifera Based on Measurements of in-Situ Ingestion of Fluorescent Particles, Including Some Comparisons with Cladocera

    Microsoft Academic Search

    A. L. Ooms-Wilms; G. Postema; R. D. Gulati

    1995-01-01

    Bacterivory of pelagic rotifers and cladocerans in eutrophic Lake Loosdrecht (The Netherlands) was determined by microscopic observation of in situ tracer particle uptake. Ingestion rates of rotifer species using 0.51 mu m microspheres or fluorescently labelled bacteria as tracers differed, with one exception. The ingestion rates depended on both the species and the tracer type. For cladocerans, fluorescently labelled bacteria

  18. X-Ray Diffraction and Fluorescence Measurements for In Situ Planetary Instruments

    NASA Astrophysics Data System (ADS)

    Hansford, G.; Hill, K. S.; Talboys, D.; Vernon, D.; Ambrosi, R.; Bridges, J.; Hutchinson, I.; Marinangeli, L.

    2011-12-01

    The ESA/NASA ExoMars mission, due for launch in 2018, has a combined X-ray fluorescence/diffraction instrument, Mars-XRD, as part of the onboard analytical laboratory. The results of some XRF (X-ray fluorescence) and XRD (X-ray diffraction) tests using a laboratory chamber with representative performance are reported. A range of standard geological reference materials and analogues were used in these tests. The XRD instruments are core components of the forthcoming NASA Mars Science Laboratory (MSL) and ESA/NASA ExoMars missions and will provide the first demonstrations of the capabilities of combined XRD/XRF instrumentation in situ on an extraterrestrial planetary surface. The University of Leicester team is part of the Italy-UK collaboration that is responsible for building the ExoMars X-ray diffraction instrument, Mars-XRD [1,2]. Mars-XRD incorporates an Fe-55 radioisotope source and three fixed-position charge-coupled devices (CCDs) to simultaneously acquire an X-ray fluorescence spectrum and a diffraction pattern providing a measurement of both elemental and mineralogical composition. The CCDs cover an angular range of 2? = 6° to 73° enabling the analysis of a wide range of geologically important minerals including phyllosilicates, feldspars, oxides, carbonates and evaporites. The identification of hydrous minerals may help identify past Martian hydrothermal systems capable of preserving traces of life. Here we present some initial findings from XRF and XRD tests carried out at the University of Leicester using an Fe-55 source and X-ray sensitive CCD. The XRF/XRD test system consists of a single CCD on a motorised arm, an Fe-55 X-ray source, a collimator and a sample table which approximately replicate the reflection geometry of the Mars-XRD instrument. It was used to test geological reference standard materials and Martian analogues. This work was funded by the Science and Technology Facilities Council, UK. References [1] Marinangeli, L., Hutchinson, I., Baliva, A., Stevoli, A., Ambrosi, R., Critani, F., Delhez, R., Scandelli, L., Holland, A., Nelms, N. & the Mars-XRD Team, Proceedings of the 38th Lunar and Planetary Science Conference, 12 - 16 March 2007, League City, Texas, USA. [2] L. Marinangeli, I. B. Hutchinson, A. Stevoli, G. Adami, R. Ambrosi, R. Amils, V. Assis Fernandes, A. Baliva, A. T. Basilevsky, G. Benedix, P. Bland, A. J. Böttger, J. Bridges, G. Caprarelli, G. Cressey, F. Critani, N. d'Alessandro, R. Delhez, C. Domeneghetti, D. Fernandez-Remolar, R. Filippone, A. M. Fioretti, J. M. Garcia Ruiz, M. Gilmore, G. M. Hansford, G. Iezzi, R. Ingley, M. Ivanov, G. Marseguerra, L. Moroz, C. Pelliciari, P. Petrinca, E. Piluso, L. Pompilio, J. Sykes, F. Westall and the MARS-XRD Team, EPSC-DPS Joint Meeting 2011, 3 - 7 October 2011, La Cité Internationale des Congrès Nantes Métropole, Nantes, France.

  19. Transferrin Gene Expression Visualized in Oligodendrocytes of the Rat Brain by Using in situ Hybridization and Immunohistochemistry

    Microsoft Academic Search

    Bertrand Bloch; Theodora Popovici; Mariano J. Levin; David Tuil; Axel Kahn

    1985-01-01

    The presence and production of transferrin in the adult rat brain have been investigated using both immunohistochemistry and in situ hybridization in tissue sections. Indirect immunofluorescence with four distinct antisera against rat and human transferrin and one monoclonal antibody against human transferrin demonstrated labeling of the cytoplasm of oligodendrocytes (a category of glial cells) in most parts of the brain,

  20. Detection of neurotrophic factors in taste buds by laser capture microdissection, immunohistochemistry, and in situ hybridization.

    PubMed

    Suzuki, Yuko; Mizoguchi, Itaru; Uchida, Nobuhiko

    2007-07-01

    Neurotrophic factors are thought to function in the survival and maintenance of the taste buds and nerve fibers innervating them. Laser capture microdissection (LCM) coupled with the reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA of neurotrophic factors and their receptors in the taste buds of adult mouse circumvallate papillae. Results showed mRNAs of the ciliary neurotrophic factor (CNTF), its receptor (CNTFR), glial cell line-derived neurotrophic factor (GDNF), GDNF family receptors alpha-1 (GFRalpha-1), GFRalpha-2, and RET tyrosine kinase receptor (RET), neurotrophin (NT)3, NT4/5, tyrosine kinase (Trk) C, nerve growth factor (NGF), and TrkA were expressed in the isolated taste buds. Among these neurotrophic factors, GDNF, GFRalpha-1, GFRalpha-2, NT3, NT4/5, NGF, and TrkA were previously found in the taste buds immunohistochemically and were detected at the mRNA level in the present study. The present immunohistochemical study revealed that CNTF, CNTFR, and the RET tyrosine kinase receptor, which binds GDNF family/ receptor complexes, were also expressed in the taste buds. However, by in situ hybridization, mRNAs of CNTF and RET were not detected in the taste buds from adult mice although they were found in those from early postnatal mice. CNTFR mRNA did not show any specific pattern in the taste buds. Moreover, mRNA expressions of NT4/5 and TrkC was re-examined by in situ hybridization; however no specific pattern was found for them in the taste buds. In summary, LCM is a useful tool for the detection of a relatively small amount of mRNA, such as that of neurotrophic factors and receptors in the taste buds. PMID:17827669

  1. SUPERSENSITIVE IN SITU HYBRIDIZATION BY TYRAMIDE SIGNAL AMPLIFICATION AND NANOGOLD SILVER STAINING: THE CONTRIBUTION OF AUTOMETALLOGRAPHY AND CATALYZED REPORTER DEPOSITION TO THE REJUVENATION OF IN SITU HYBRIDIZATION.

    SciTech Connect

    TUBBS,R.R.PETTAY,J.GROGAN,T.CHEUNG,A.L.M.POWELL,R.D.HAINFELD,J.HAUSER-KRONBERGER,C.HACKER,G.W.

    2002-04-17

    It is peculiar that in situ hybridization (ISH), a technique with many similarities to immunohistochemistry (IHC), has not enjoyed the phenomenal growth in both basic research and clinical applications as has its sister technique IHC. Since the late 1970s, when immunoperoxidase techniques began to be applied to routine diagnostic material and to numerous research applications, there has been a natural evolution of the IHC procedure. Namely, only a few primary antibodies were available commercially at the onset, and only one indirect and the peroxidase-antiperoxidase (PAP) technique detection systems were in place. With the advent of avidin-biotin detection systems and monoclonal antibodies, and a viable commercial market, extraordinary growth of the procedure's applications in clinical research and diagnostic pathology occurred during the subsequent two decades. Today, IHC is automated and widely used for research purposes and, to a large extent, has become a routine diagnostic ''special stain'' in most clinical laboratories. During the same period, ISH enjoyed very little growth in both research and diagnostic applications. What has accounted for this lack of maturation of the technique? The success of IHC is part of the reason measuring a gene's encoded protein routinely and inexpensively, particularly as automation evolved, rendered IHC a more viable choice in many instances. Inherent comparative sensitivity of the procedures has also clearly been a factor. Unfortunately, the chromogenic procedures in place are often insufficiently sensitive to detect the relatively low amounts of DNA and RNA levels at which the clinical utility is to be found.

  2. The detection of Jonah gene transcripts in Drosophila by in situ hybridization.

    PubMed

    Akam, M E; Carlson, J R

    1985-01-01

    The Jonah genes constitute a family of approximately 20 genes expressed at two periods during the development of Drosophila melanogaster. They are expressed only in the midgut, where they yield very abundant transcripts of approximately 900 bases. The function of their products is not known. We have used in situ hybridization to show that transcripts homologous to two members of the Jonah family have quite different distributions within the midgut. Transcripts closely homologous to Jon65Aiv are expressed throughout most of the anterior midgut and in the posterior section of the middle midgut. Transcripts closely homologous to Jon99C beta are expressed only in the anterior region of the posterior midgut. PMID:2410252

  3. Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

    NASA Astrophysics Data System (ADS)

    Meng, Qinglei; Wang, Shi; Zhang, Lingling; Huang, Xiaoting; Bao, Zhenmin

    2013-01-01

    In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop ( Chlamys farreri).

  4. In situ hybridization of ribosomal DNA labelled with 125 iodine to metaphase and lampbrush chromosomes from newts

    Microsoft Academic Search

    Sally Hennen; S. Mizuno; H. C. Macgregor

    1975-01-01

    Methods are described for in situ hybridization of ribosomal DNA from Xenopus laevis, labelled in vitro with 125iodine, to mitotic and lampbrush chromosomes from Triturus cristatus carnifex. The hybridization reaction was carried out in a mixture containing 50% formamide, 4 x SSC, 0.1 M KI, at 37° C, or in 2 x SSC, 0.1 M KI at 65° C. Autoradiographs

  5. Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

    PubMed Central

    Pirttilä, Anna Maria; Laukkanen, Hanna; Pospiech, Helmut; Myllylä, Raili; Hohtola, Anja

    2000-01-01

    Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified as Methylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts. PMID:10877808

  6. A novel genome of C and the first autotetraploid species in the Setaria genus identified by genomic in situ hybridization

    Microsoft Academic Search

    Yongqiang Wang; Hui Zhi; Wei Li; Haiquan Li; Yongfang Wang; Zhanjing Huang; Xianmin Diao

    2009-01-01

    Genomic in situ hybridization (GISH) was used to investigate the genomic relationships among some newly collected species of genus Setaria. Previous work identified that S. viridis and S. adhaerens carry genomes A and B, respectively. GISH patterns obtained in this report clearly distinguished the genome of S. grisebachii from the known genomes A and B, and indicated its new genomic

  7. In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Wang, Jianling; Zhang, Gen; Li, Qiwei; Jiang, Hui; Liu, Chongyang; Amatore, Christian; Wang, Xuemei

    2013-01-01

    Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors.

  8. Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.

    PubMed

    Speel, E J

    1999-08-01

    In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed. PMID:10460463

  9. Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus: In situ hybridization histochemistry and immunocytochemistry

    Microsoft Academic Search

    Ellen L. Sutin; David M. Jacobowitz

    1990-01-01

    1.In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem.2.Alternate serial sections were hybridized with a 48-base,35S-labeled synthetic oligonucleotide probe encoding SP usingin situ hybridization histochemistry

  10. Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

    PubMed Central

    Uttenthal, A; Larsen, S; Lund, E; Bloom, M E; Storgård, T; Alexandersen, S

    1990-01-01

    Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed. Images PMID:2159543

  11. Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

    PubMed Central

    Wei, Qiaozhi; Manley, Nancy R.; Condie, Brian G.

    2011-01-01

    Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments. PMID:22005971

  12. Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization

    Microsoft Academic Search

    Robert H. Singer; Gary L. Langevin; Jeanne Bentley Lawrence

    1989-01-01

    We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole- mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybrid- ized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point

  13. Detection of Intrahepatic Replication of Hepatitis C Virus RNA by In situ Hybridization and Comparison with Histopathology

    Microsoft Academic Search

    Francesco Negro; Donatella Pacchioni; Yohko Shimizu; Roger H. Miller; Gianni Bussolati; Robert H. Purcell; Ferruccio Bonino

    1992-01-01

    A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and

  14. One Mismatch Detection of DNA Hybridization Using Fluorescence Polarization

    Microsoft Academic Search

    Masafumi Shimizu; Noriko Minamikawa; Makoto Tsuruoka

    2007-01-01

    The potential of fluorescent polarization analysis as a method for detection of mismatch DNA hybridization was investigated. The dependency of DNA hybridization rate on salt concentration was surveyed. In greater than 0.1 M NaCl, the hybridization of probe and target DNA proceeds rapidly and the reaction is complete within 3 min. Furthermore, the hybridization of probe DNA and one mismatch target DNAs

  15. Hybridization chain reaction modulated DNA-hosted silver nanoclusters for fluorescent identification of single nucleotide polymorphisms in the let-7 miRNA family.

    PubMed

    Qiu, Xue; Wang, Pei; Cao, Zhijuan

    2014-10-15

    A simple microRNA (miRNA) detection system based on hybridization chain reaction (HCR) has been developed using highly fluorescent DNA-hosted silver (Ag) nanoclusters. In this assay, a new type of hairpin DNA probe (MB1) containing a poly-cytosine nucleotide loop is designed and used as one of the HCR monomers, which is also demonstrated to be an ideal template for in situ synthesis of highly fluorescent Ag nanoclusters. Correspondingly, another HCR monomer (MB2) contains a poly-guanine nucleotide sticky end. Two monomers are stable to coexist in solution until the introduction of the initiator strand (let-7a) triggers a cascade of hybridization events that yields nicked double helices analogous. By taking advantage of HCR, a small amount of let-7a leads to the conformational change of a large amount of MB1, which results in the decrease of fluorescent signal greatly. Overall, this label-free, enzyme-free method allows the sensitive detection of let-7a with high specificity towards single nucleotide polymorphisms in the let-7 miRNA family. In addition, the simple "mix and measure" assay can be extended to detect other types of targets upon slight modification, and thus provides a tool for the early diagnosis and risk assessment of malignancy. PMID:24836018

  16. A facile heating cell for in situ transmittance and fluorescence X-ray absorption spectroscopy investigations.

    PubMed

    An, Pengfei; Hong, Caihao; Zhang, Jing; Xu, Wei; Hu, Tiandou

    2014-01-01

    A facile heating cell has been designed for in situ transmittance and fluorescence X-ray absorption spectroscopy (XAS) measurements up to 1273?K under vacuum or an inert atmosphere. These high temperatures are achieved using a tantalum heating element by ohmic heating. Because of the small specific heat capacity, the temperature can be changed in a matter of minutes from room temperature to high temperature. Furthermore, a commercial power controller was adapted to provide stable temperature control. The construction of the heat shielding system provides a novel approach to reducing the beam's path length and the cell's size. The cell is inexpensive and easy to build. Its performance was evaluated by in situ XAS measurements of the temperature-dependent structure of ceria nanocrystals. Some preliminary results for the structural mechanism in ceria nanocrystal redox applications are given. PMID:24365932

  17. In situ preparation of fluorescent CdTe quantum dots with small thiols and hyperbranched polymers as co-stabilizers

    NASA Astrophysics Data System (ADS)

    Shi, Yunfeng; Ma, Zhimin; Cui, Ningning; Liu, Yanli; Hou, Xiaoyu; Du, Weimin; Liu, Lin; Gangsheng, Tong

    2014-03-01

    A new strategy for in situ preparation of highly fluorescent CdTe quantum dots (QDs) with 3-mercaptopropionic acid (MPA) and hyperbranched poly(amidoamine)s (HPAMAM) as co-stabilizers was proposed in this paper. MPA and HPAMAM were added in turn to coordinate Cd2+. After adding NaHTe and further microwave irradiation, fluorescent CdTe QDs stabilized by MPA and HPAMAM were obtained. Such a strategy avoids the aftertreatment of thiol-stabilized QDs in their bioapplication and provides an opportunity for direct biomedical use of QDs due to the existence of biocompatible HPAMAM. The resulting CdTe QDs combine the mechanical, biocompatibility properties of HPAMAM and the optical, electrical properties of CdTe QDs together.

  18. In situ preparation of fluorescent CdTe quantum dots with small thiols and hyperbranched polymers as co-stabilizers

    PubMed Central

    2014-01-01

    A new strategy for in situ preparation of highly fluorescent CdTe quantum dots (QDs) with 3-mercaptopropionic acid (MPA) and hyperbranched poly(amidoamine)s (HPAMAM) as co-stabilizers was proposed in this paper. MPA and HPAMAM were added in turn to coordinate Cd2+. After adding NaHTe and further microwave irradiation, fluorescent CdTe QDs stabilized by MPA and HPAMAM were obtained. Such a strategy avoids the aftertreatment of thiol-stabilized QDs in their bioapplication and provides an opportunity for direct biomedical use of QDs due to the existence of biocompatible HPAMAM. The resulting CdTe QDs combine the mechanical, biocompatibility properties of HPAMAM and the optical, electrical properties of CdTe QDs together. PMID:24636234

  19. In situ preparation of fluorescent CdTe quantum dots with small thiols and hyperbranched polymers as co-stabilizers.

    PubMed

    Shi, Yunfeng; Ma, Zhimin; Cui, Ningning; Liu, Yanli; Hou, Xiaoyu; Du, Weimin; Liu, Lin; Gangsheng, Tong

    2014-01-01

    A new strategy for in situ preparation of highly fluorescent CdTe quantum dots (QDs) with 3-mercaptopropionic acid (MPA) and hyperbranched poly(amidoamine)s (HPAMAM) as co-stabilizers was proposed in this paper. MPA and HPAMAM were added in turn to coordinate Cd2+. After adding NaHTe and further microwave irradiation, fluorescent CdTe QDs stabilized by MPA and HPAMAM were obtained. Such a strategy avoids the aftertreatment of thiol-stabilized QDs in their bioapplication and provides an opportunity for direct biomedical use of QDs due to the existence of biocompatible HPAMAM. The resulting CdTe QDs combine the mechanical, biocompatibility properties of HPAMAM and the optical, electrical properties of CdTe QDs together. PMID:24636234

  20. Mapping Bronchial Carcinoma In Situ Lung Cancer Lesions By Combined Imaging Fluorescence Bronchoscopy And Ratioing Fluorometer Probe

    NASA Astrophysics Data System (ADS)

    Balchum, Oscar J.; Profio, A. Edward; Razum, Nickolas J.

    1988-06-01

    A two-component system of instrumentation and methods, IFB and RFP, when used in combination employing hematoporphyrin derivates, DHE, had highly satisfactory sensitivity (95%) and specificity (100%) for localizing carcinoma in situ lesions in the bronchi of individuals with early stage lung cancer, having a normal chest x-ray and detected by a positive sputum cytology test. The more detailed mapping of additional subjects may increase the specificity of RFP (Ratioing Fluorometer Probe) by itself to an adequate level. Digital computer subs traction of background antofluorescence may increase contrast to enhance the specificity of IFB (Imaging Fluorescence Bronchoscopy).

  1. Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization.

    PubMed

    Al-Hussinee, L; Lumsden, J S

    2011-05-24

    Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge. PMID:21797039

  2. In-situ tryptophan-like fluorescence: A real-time indicator of faecal contamination in drinking water supplies.

    PubMed

    Sorensen, J P R; Lapworth, D J; Marchant, B P; Nkhuwa, D C W; Pedley, S; Stuart, M E; Bell, R A; Chirwa, M; Kabika, J; Liemisa, M; Chibesa, M

    2015-09-15

    Enteric pathogens are typically inferred from the presence of surrogate indicator organisms such as thermotolerant (faecal) coliforms (TTCs). The analysis of TTCs requires time-consuming incubation in suitable laboratories, which can limit sampling resolution, particularly during critical pollution events. Here, we demonstrate the use of in-situ fluorimeters targeting tryptophan-like compounds as a rapid, reagentless indicator of TTCs in groundwater-derived potable water supplies in Africa. A range of other common indicators of TTCs were also determined including nitrate, turbidity, and sanitary risk survey scores. Sampling was conducted during both the dry and wet seasons to investigate seasonality. Tryptophan-like fluorescence was the most effective predictor of both presence/absence and number of TTCs during both seasons. Seasonal changes in tryptophan-like fluorescence in deeper supplies suggest it is transported more efficiently through the aquifer than TTCs. Moreover, the perennial elevated concentrations in some wells suggest it is more resilient than TTCs in groundwater. Therefore tryptophan-like fluorescence could also be a better indicator of some smaller, more easily transported, and long-lived, pathogenic enteric viruses. These sensors have the potential to be included in real-time pollution alert systems for drinking water supplies throughout the world, as well as for mapping enteric pathogen risks in developing regions. PMID:26026711

  3. Controllable in situ synthesis of magnetite coated silica-core water-dispersible hybrid nanomaterials.

    PubMed

    Qu, Haiou; Tong, Sheng; Song, Kejing; Ma, Hui; Bao, Gang; Pincus, Seth; Zhou, Weilie; O'Connor, Charles

    2013-08-20

    Magnetite nanoparticle coated silica (Fe3O4@SiO2) hybrid nanomaterials hold an important position in the fields of cell imaging and drug delivery. Here we report a large scale synthetic procedure that allows attachment of magnetite nanoparticles onto a silica surface in situ. Many different silica nanomaterials such as Stöber silica nanospheres, mesoporous silica nanoparticles, and hollow silica nanotubes have been coated with a high density layer of water-dispersible magnetite nanoparticles. The size and attachment efficiency of the magnetite nanoparticle can be well tuned by adjusting the precursor concentration and reflux time. The functionalization of Fe3O4@SiO2 nanoparticles with dye molecules and biocompatible polymers impart optical imaging modality and good colloidal stability in either buffer solution or serum. The functionalized materials also exhibited strong potential as negative contrast agents in T2 weighted magnetic resonance imaging. PMID:23889037

  4. Controllable In-Situ Synthesis of Magnetite Coated Silica-Core Water-Dispersible Hybrid Nanomaterials

    PubMed Central

    Qu, Haiou; Tong, Sheng; Song, Kejing; Ma, Hui; Bao, Gang; Pincus, Seth; Zhou, Weilie; O'Connor, Charles

    2013-01-01

    Magnetite nanoparticle coated silica (Fe3O4@SiO2) hybrid nanomaterials hold an important position in the fields of cell imaging and drug delivery. Here we report a large scale synthetic procedure that allows attachment of magnetite nanoparticles onto a silica surface in-situ. Many different silica nanomaterials such as Stöber silica nanospheres, mesoporous silica nanoparticles, and hollow silica nanotube have been coated with a high density layer of water-dispersible magnetite nanoparticles. The size and attachment efficiency of the magnetite nanoparticle can be well tuned by adjusting the precursor concentration and reflux time. The functionalization of Fe3O4@SiO2 nanoparticles with dye molecules and biocompatible polymers impart optical imaging modality and good colloidal stability in either buffer solution or serum. The functionalized materials also exhibited strong potential as negative contrast agents in T2 weighted magnetic resonance imaging. PMID:23889037

  5. Genomic in situ hybridization identifies parental chromosomes in hybrid scallop (Bivalvia, Pectinoida, Pectinidae) between female Chlamys farreri and male Argopecten irradians irradians

    PubMed Central

    Huang, Xiaoting; Bi, Ke; Lu, Wei; Wang, Shi; Zhang, Lingling; Bao, Zhenmin

    2015-01-01

    Abstract Interspecific crossing was artificially carried out between Chlamys farreri (Jones & Preston, 1904) ? and Argopecten irradians irradians (Lamarck, 1819) ?, two of the dominant cultivated scallop species in China. Genomic in situ hybridization (GISH) was used to examine the chromosome constitution and variation in hybrids at early embryonic stage. The number of chromosomes in 66.38% of the metaphases was 2n = 35 and the karyotype was 2n = 3 m + 5 sm + 16 st + 11 t. After GISH, two parental genomes were clearly distinguished in hybrids, most of which comprised 19 chromosomes derived from their female parent (Chlamys farreri) and 16 chromosomes from their male parent (Argopecten irradians irradians). Some chromosome elimination and fragmentation was also observed in the hybrids.

  6. Largely Enhanced Single-molecule Fluorescence in Plasmonic Nanogaps formed by Hybrid Silver Nanostructures

    PubMed Central

    Zhang, Jian; Lakowicz, Joseph R.

    2013-01-01

    It has been suggested that narrow gaps between metallic nanostructures can be practical for producing large field enhancement. We design a hybrid silver nanostructure geometry in which fluorescent emitters are sandwiched between silver nanoparticles and silver island film (SIF). A desired number of polyelectrolyte layers are deposited on the SIF surface before the self-assembly of a second silver nanoparticle layer. Layer-by-layer configuration provides a well-defined dye position. It allows us to study the photophyical behaviors of fluorophores in the resulting gap at the single molecule level. The enhancement factor of a fluorophore located in the gap is much higher than those on silver surfaces alone and on glass. These effects may be used for increased detectability of single molecules bound to surfaces which contain metallic structures for either biophysical studies or high sensitivity assays. PMID:23373787

  7. Hybrid single nanoreactor for in situ SERS monitoring of plasmon-driven and small Au nanoparticles catalyzed reactions.

    PubMed

    Li, Pan; Ma, Bingbing; Yang, Liangbao; Liu, Jinhuai

    2015-07-01

    A hybrid single nanoreactor composed of small Au nanoparticles (NPs) and a long Ag nanowire (NW) with a length of 10 ?m can be conveniently located by optical microscopy, allowing us to monitor by in situ surface enhanced Raman spectroscopy (SERS) the plasmon-driven and small Au NPs catalyzed reactions with high sensitivity and reproducibility. PMID:26087227

  8. Whole-mount in situ hybridization of sectioned tissues of species hybrids to detect cis-regulatory changes in gene expression pattern.

    PubMed

    Futahashi, Ryo

    2011-01-01

    To distinguish whether differences in gene expression between species or between individuals of the same species are caused by cis-regulatory changes or by distribution differences in trans-regulatory proteins, comparison of species-specific mRNA expression in an F1 hybrid by whole-mount in situ hybridization is a rarely used yet very powerful tool. If asymmetric expression pattern is observed for the two alleles, this implies a cis-regulatory divergence of this gene. Alternatively, if symmetric expression pattern is observed for both alleles, the change in expression of this gene is probably caused by changes in the distribution of trans-regulatory proteins. In this chapter, I describe how to prepare RNA probes, tissue samples and how to detect mRNA expression pattern using in situ hybridization. Although I choose to present here the detection of yellow-related gene (YRG) expression pattern in the larval epidermis of swallowtail butterflies, this protocol can be adapted to other species and tissues. YRG mRNA expression is correlated with interspecific differences of yellow and green larval color pattern such as V-shaped markings in swallowtail butterflies. F1 hybrids show an intermediate color pattern between parental species. In this case, both species-specific YRG mRNA showed a similar expression pattern in F1 hybrids, suggesting that the change in expression of YRG is mainly caused by changes in the distribution of trans-regulatory proteins. PMID:22065447

  9. In situ probing of intracellular pH by fluorescence from inorganic nanoparticles.

    PubMed

    Guo, Junhong; Xiong, Shijie; Wu, Xinglong; Shen, Jiancang; Chu, Paul K

    2013-12-01

    Intracellular pH (pHi) plays a critical role in the physiological processes of cells. Nanoscale sensors based on pH-sensitive fluorescent proteins attached on nanoparticles (NPs) have been designed but inorganic NP-dependent fluorescent nanosensors have not yet been explored. Herein we describe a pH sensitive inorganic semiconductor fluorescent probe based on ultrathin 3C-SiC NPs which can effectively monitor pH in the range of 5.6-7.4 by taking advantage of the linear dependence between the fluorescent intensity ratio of the surface OH(-) and H(+) bonding states to band-to-band recombination and pH. Detection of pHi is demonstrated in living HeLa cells. In particular, pHi measurements during apoptosis confirm the validity and sensitivity of this technique in monitoring real-time changes in the intracellular environment. Toxicity assessment and confocal laser scanning microscopy indicate that the 3C-SiC NPs have low cytotoxicity and are compatible with living cells. PMID:24008041

  10. MARYLAND/VIRGINIA CHESAPEAKE BAY AND TRIBUTARIES IN SITU FLUORESCENCE PROFILES

    EPA Science Inventory

    As part of the Chesapeake Bay Program, surface to bottom fluorescence measurements (vertical profiles) have been made at fixed sampling stations in the upper Chesapeake Bay, Maryland tributaries, and the Potomac River since July 1984. The data through December of 1995 are availab...

  11. Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation.

    PubMed

    Milián-García, Y; Ramos-Targarona, R; Pérez-Fleitas, E; Sosa-Rodríguez, G; Guerra-Manchena, L; Alonso-Tabet, M; Espinosa-López, G; Russello, M A

    2015-03-01

    Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value. PMID:25335559

  12. Photophysical properties of some fluorescent materials containing 3-methoxy-7H-benzo[de]anthracen-7-one embedded in sol-gel silica hybrids

    NASA Astrophysics Data System (ADS)

    Raditoiu, Alina; Raditoiu, Valentin; Culita, Daniela Cristina; Baran, Adriana; Anghel, Dan Florin; Spataru, Catalin Ilie; Amariutei, Viorica; Nicolae, Cristian Andi; Wagner, Luminita Eugenia

    2015-07-01

    Several fluorescent materials were prepared by embedding 3-methoxy-7H-benzo[de]anthracen-7-one in transparent sol-gel silica hybrids, in acid catalysis and at room temperature. The photophysical properties of the materials are studied in relationship with the structure and based on interactions established between the fluorophore, which display high sensitivity to minor changes in the local environments, and hybrid silica networks. Colored materials show marked differences in absorption and fluorescence spectra due to the environments around the dyestuff molecules created in the host matrices by grafting different organic moieties onto silica surface. The fluorescence emission spectrum of the obtained materials reveals a positive solvatochromism due to the intramolecular charge transfer character of the excited states and changes in the fluorophore dipole moment. Variations recorded in the fluorescence parameters of the hybrid materials confirm that some interactions are established between the fluorescent dyestuff and hybrid silica network.

  13. Localization of the neuropeptide APGWamide in gastropod molluscs by in situ hybridization and immunocytochemistry.

    PubMed

    de Lange, R P; van Minnen, J

    1998-02-01

    The amidated tetrapeptide Ala-Pro-Gly-Trp-NH2 (APGWamide) plays a key role in the control of male copulation behavior in the basommatophoran pulmonate freshwater snail Lymnaea stagnalis. The morphological basis for a conserved role of APGWamide in the control of male reproduction in gastropod molluscs is presented. The prosobranch Littorina littorea, the opisthobranch Aplysia californica, the basommatophoran pulmonate Bulinus truncatus, and the stylommatophoran pulmonates Arion ater and Limax maximus have been examined for the presence of APGWamide producing neurons using immunocytochemistry and in situ hybridization. In all species investigated a cluster of APGWamide expressing neurons is present in the anteromedial region of the cerebral ganglia. The asymmetrical distribution which exists in Lymnaea and which coincides with the innervation of the asymmetrically located penial complex is also found in the opisthobranch Aplysia, as well as in the stylommatophoran pulmonate slugs Arion and Limax, in which APGWamide immunoreactive neurons are only found in the mesocerebrum of the right cerebral ganglion. APGWamide immunoreactive varicose fibers innervate muscles of the male accessory sex organs in Bulinus and Aplysia, confirming the hypothesis that APGWamide may be a biochemically and functionally conserved factor in the regulation of gastropod mollusc reproduction. PMID:9473361

  14. Whole-mount in situ hybridization of Hawaiian bobtail squid (Euprymna scolopes) embryos with DIG-labeled riboprobes: II. Embryo preparation, hybridization, washes, and immunohistochemistry.

    PubMed

    Lee, Patricia N; McFall-Ngai, Margaret J; Callaerts, Patrick; de Couet, H Gert

    2009-11-01

    Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody. PMID:20150061

  15. In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

    Microsoft Academic Search

    Koji Yamaguchi; Michael A. Nalesnik; Brian I. Carr

    1993-01-01

    Summary  In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary\\u000a liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas\\u000a no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In\\u000a 53

  16. In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye*

    PubMed Central

    Namkung, Wan; Song, Yuanlin; Mills, Aaron D.; Padmawar, Prashant; Finkbeiner, Walter E.; Verkman, A. S.

    2009-01-01

    The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K+], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K+ ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K+-selective, increasing >4-fold with increasing [K+] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K+] was 20.8 ± 0.3 mm and decreased by inhibition of the Na+/K+ pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTRinh-172), or K+ channels (TEA or XE991). ASL [K+] was increased by forskolin but not affected by Na+/K+/2Cl? cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K+] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K+]. [K+] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K+] is not a factor in CF lung disease. In intact airways, ASL [K+] was also well above extracellular [K+]: 22 ± 1 mm in pig trachea ex vivo and 16 ± 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K+] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K+]. PMID:19364771

  17. Multispectral In-situ Measurements of Organic Matter and Chlorophyll Fluorescence in Seawater: Documenting the Intrusion of the Mississippi River Plume in the West Florida Shelf

    NASA Technical Reports Server (NTRS)

    DelCastillo, Carlos E.; Coble, Paula G.; Conmy, Robyn N.; Mueller-Karger, Frank E.; Vanderbloomen, Lisa; Vargo, Gabriel A.

    2000-01-01

    We performed multispectral in-situ fluorescence measurement of colored dissolved organic matter and chlorophyll in surface water of the West Florida Shelf using West Labs Spectral absorption and Fluorescence Instrument (SAFIre). Continuous measurements underway allowed us to simultaneously map the dispersion of riverine organic material and chlorophyll on the shelf. By using two fluorescence emission ratios we were able to differentiate between riverine and marine CDOM. Our data also showed unusually high concentrations of CDOM offshore. These were attributed to an intrusion of the Mississippi River Plume. We performed limited comparisons between in-situ chlorophyll concentrations measured with SAFIre and chlorophyll values obtained from SeaWiFS satellite data using OC4 and MODIS algorithm. Our results show that, although both algorithms overestimated chlorophyll, MODIS performed better than OC4, particularly in areas with high CDOM concentrations. Analysis of the relationship between chlorophyll and CDOM concentrations within the study area showed regional variability causes by differences in river source.

  18. Analysis of Epstein-Barr viral genomes in lymphoid malignancy using southern blotting, polymerase chain reaction and in situ hybridization

    Microsoft Academic Search

    Kohichi Ohshima; Masahiro Kikuchi; Fuyuki Eguchi; Yuhichi Masuda; Yoshiaki Sumiyoshi; Hidehito Mohtai; Morishige Takeshita; Nobuhiro Kimura

    1990-01-01

    Summary  109 malignant lymphomas were surveyed by Southern blot analysis and polymerase chain reaction (PCR) for Epstein-Barr virus\\u000a (EBV) DNA and compared with 16 examples of non-neoplastic lymphadenopathy and 4 normal thymuses. In specimens positive by\\u000a the method of Southern and PCR, in situ hybridization studies were performed on formalin-fixed, paraffin-embedded sections.\\u000a By Southern blot analysis, two of seven Hodgkin’s disease

  19. Localization of laminin B1 mRNA in retinal ganglion cells by in situ hybridization

    PubMed Central

    1990-01-01

    In the nervous system, neuronal migration and axonal growth are dependent on specific interactions with extracellular matrix proteins. During development of the vertebrate retina, ganglion cell axons extend along the internal limiting (basement) membrane and form the optic nerve. Laminin, a major component of basement membranes, is known to be present in the internal limiting membrane, and might be involved in the growth of ganglion cell axons. The identity of the cells that produce retinal laminin, however, has not been established. In the present study, we have used in situ hybridization to localize the sites of laminin B1 mRNA synthesis in the developing mouse retina. Our results show that there are at least two principal sites of laminin B1 mRNA synthesis: (a) the hyaloid vessels and the lens during the period of major axonal outgrowth, and (b) the retinal ganglion cells at later development stages. Muller (glial) cells, the major class of nonneuronal cells in the retina, do not appear to express laminin B1 mRNA either during development or in the adult retina. In Northern blots, we found a single transcript of approximately 6-kb size that encodes the laminin B1 chain in the retina. Moreover, laminin B1 mRNA level was four- to fivefold higher in the postnatal retina compared to that in the adult. Our results show that in addition to nonneuronal cells, retinal ganglion cells also synthesize laminin. The function of laminin in postnatal retinas, however, remains to be elucidated. Nevertheless, our findings raise the possibility that neurons in other parts of the nervous system might also synthesize extracellular matrix proteins. PMID:2351694

  20. Use of Discriminatory Probes for Strain Typing of Formalin-Fixed, Rabies Virus-Infected Tissues by In Situ Hybridization

    Microsoft Academic Search

    Susan A. Nadin-Davis; Mary Sheen; Alexander I. Wandeler

    2003-01-01

    An in situ